Gerhard Krauss Biochemistry of Signal Transduction and Regulation Fifth, Completely Revised Edition

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Gerhard Krauss

Biochemistry of Signal Transduction and Regulation Related Titles

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Biochemistry of Signal Transduction and Regulation

Fifth, Completely Revised Edition Author All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Gerhard Krauss publisher do not warrant the information contained Universität Bayreuth in these books, including this book, to be free of Biochemisches Laboratorium errors. Readers are advised to keep in mind that Universitätsstraße 30 statements, data, illustrations, procedural details or 95449 Bayreuth other items may inadvertently be inaccurate. Germany Library of Congress Card No.: applied for

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Contents

Preface XXVII

1 Basics of Cell Signaling 1 1.1 Cell Signaling: Why, When, and Where? 1 1.2 Intercellular Signaling 3 1.2.1 Tools for Intercellular Signaling 3 1.2.2 Steps of Intercellular Signaling 5 1.2.2.1 Formation of a Signal in the Signal-Producing Cell as a Result of an External Trigger 5 1.2.2.2 Transport of the Signal to the Target Cell 6 1.2.2.3 Registration of the Signal in the Target Cell 6 1.2.3 Regulation of Intercellular Signaling 7 1.3 Hormones in Intercellular Signaling 8 1.3.1 The Chemical Nature of Hormones 8 1.3.2 Hormone Analogs: Agonists and Antagonists 11 1.3.2.1 Antagonists 11 1.3.2.2 Agonists 12 1.3.3 Endocrine, Paracrine, and Autocrine Signaling 13 1.3.3.1 Endocrine Signaling 14 1.3.3.2 Paracrine Signaling 15 1.3.3.3 Autocrine Signaling 15 1.3.4 Direct Protein Modiﬁcation by Signaling Molecules 15 1.4 Intracellular Signaling: Basics 15 1.4.1 Reception of External Signals 16 1.4.2 Activation and Deactivation of Signaling Proteins 16 1.4.3 Processing of Multiple Signals 18 1.4.4 Variability of Signaling Proteins 18 1.5 Molecular Tools for Intracellular Signaling 18 1.5.1 Receptors 19 1.5.1.1 Receptors Receive External Signals and Trigger Intracellular Signaling 19 1.5.1.2 Membrane-Bound Receptors 20 1.5.1.3 Intracellular Receptors 21 VIj Contents

1.5.1.4 The Interaction Between Hormone and Receptor 21 1.5.1.5 Regulation of Receptor Activity 22 1.5.2 Signaling Enzymes 23 1.5.3 Scaffolding Proteins 24 1.5.4 Diffusible Intracellular Messengers: Second Messengers 25

2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins 27 2.1 Modular Structure of Signaling Proteins 27 2.1.12.1.1.1 Catalytic Domains 28 2.1.1.2 Targeting and Interaction Domains 28 2.1.1.3 Regulatory Domains 28 2.1.1.4 Unstructured, Flexible Sections 30 2.1.1.5 Multivalency 30 2.1.1.6 Differential Use of Modules 31 2.1.1.7 Multiple Inputs, Regulatory Influences, and Outputs 31 2.1.1.8 Subtypes of Signaling Modules 31 2.2 Modular Signaling Complexes 31 2.2.12.2.1.1 Specificity 33 2.2.1.2 Signal-Directed Assembly 33 2.2.1.3 Variability 33 2.2.1.4 Regulation 33 2.3 Regulation of Signaling Enzymes by Effector Binding 34 2.3.12.3.1.1 Allostery in Signaling Enzymes 34 2.3.1.2 Low-Molecular-Weight Effectors 35 2.3.1.3 Inhibitor Proteins 35 2.3.1.4 Activator Proteins 36 2.3.1.5 Metal Ions 36 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 36 2.4.1 Chemical Nature of PTMs 37 2.4.1.1 Allosteric and Conformational Functions of PTMs 38 2.4.1.2 Recognition of PTMs by Interaction Domains 38 2.4.1.3 Dynamic Nature of PTMs 38 2.4.1.4 Examples of Regulatory PTMs 38 2.4.2 Recognition of Protein Modifications by Modification-Specific Interaction Domains 41 2.4.3 Multisite Protein Modification 42 2.4.3.1 PTM Patterns are Used as a “Bar Code” 43 2.4.3.2 Multiple Modifications Often Show Combinatorial Characteristics 43 2.4.4 Binding Properties of Regulatory Interaction Domains 43 2.4.4.1 Versatility and Variability of Interaction Domains 46 2.4.5 How Interaction Domains Read PTM Patterns 47 2.4.5.1 Inducible Interactions 47 2.4.5.2 Cooperative Interactions and Multisite PTMs 47 2.4.5.3 Sequential PTM-Dependent Interactions and Cross-Regulation 47 Contents jVII

2.4.5.4 Mutually Exclusive PTMs and Interactions 48 2.4.5.5 Antagonistic Action of PTMs 49 2.4.5.6 Regulation of Intramolecular Interactions by PTMs 49 2.4.5.7 Convergent Recognition of a PTM 50 2.5 Regulation by Protein Phosphorylation 51 2.5.1 General Aspects of Protein Phosphorylation 51 2.5.1.1 Properties and Interactions of Phosphorylated Proteins 52 2.5.2 Allosteric Functions of Protein Phosphorylation 53 2.5.3 Organization of Signaling Pathways by Protein Phosphorylation 54 2.5.3.1 Tyr-Phosphorylation 54 2.5.3.2 Ser/Thr Phosphorylation 54 2.6 Regulation by Protein Lysine Acetylation 55 2.6.1 General Aspects of Protein Lysine Acetylation 55 2.6.2 Enzymes of Protein Lysine Acetylation 56 2.6.2.1 HATs 56 2.6.2.2 HDACs 56 2.6.2.3 Cleavage Mechanism 57 2.6.3 Regulatory Functions of Lysine Acetylation 57 2.7 Regulation by Protein Methylation 58 2.7.1 Protein Lysine Methylation 58 2.7.1.1 Lysine Methyl Transferases 59 2.7.1.2 Lysine Demethylases, KDMs 59 2.7.1.3 Interaction Domains for Methylated Lysine 61 2.7.2 Protein Arginine Methylation 61 2.7.2.1 Arginine Demethylation/Citrullination 61 2.7.2.2 Interaction Domains 62 2.8 Ubiquitin Modiﬁcation of Proteins 62 2.8.1 Pathways of Protein Degradation 63 2.8.1.1 Lysosomal Pathway 64 2.8.1.2 Autophagosomal Pathway 64 2.8.1.3 Proteasomal Pathway 64 2.8.2 Basics of Ubiquitin Modiﬁcation 65 2.8.2.1 The Ub-Conjugation Reactions: E1, E2, and E3 Enzymes 66 2.8.2.2 E1: Activation of Ubiquitin 68 2.8.2.3 E2: Transacylation to the Ubiquitin-Conjugating Enzyme E2 68 2.8.2.4 E3: Ubiquitin Transfer to the Target 68 2.8.3 Structure and Regulation of Ub-Protein Ligases 69 2.8.3.1 Hect Domain E3 Enzymes 69 2.8.3.2 RING Domain E3 Enzymes 71 2.8.3.3 Structure and Regulation of Cullin-RING Ligases 73 2.8.3.4 Substrate Recognition by Cullin-RING Ligases 74 2.8.3.5 N-End Rule 74 2.8.3.6 Examples of Cullin-RINGE3 Ligases 75 2.8.3.7 SCF Complex 75 2.8.3.8 Anaphase-Promoting Complex (APC) 76 VIIIj Contents

2.8.3.9 Cbl Proteins 77 2.8.4 Degradation in the Proteasome 78 2.8.4.1 The 20S Proteasome 78 2.8.4.2 The 19S Activator 80 2.8.5 Nonproteolytic Functions of Ubiquitin Conjugation 81 2.8.5.1 Deubiquitination 81 2.8.5.2 Multiplicity of Ub Conjugation 81 2.8.5.3 Ub-Binding Domains and Ub Receptors 84 2.8.5.4 Ub-Conjugation in Regulation of the NFkB Pathway 85 2.8.6 Ubiquitin-Like Proteins: Sumo-Modiﬁcation 88 2.9 Lipidation of Signaling Proteins 90 2.9.1 Myristoylation 91 2.9.2 Palmitoylation 92 2.9.2.1 Functions of Palmitoylation 93 2.9.3 Farnesylation and Geranylation 93 2.9.4 Dual Lipidation 95 2.9.5 Cholesterol Membrane Anchor 95 2.9.6 The Switch Function of Lipid Anchors 96 2.9.7 The Glycosyl-Phosphatidyl-Inositol (GPI) Anchor 98 Questions 99 References 100

3 Organization of Signaling 103 3.1 Scaffold Proteins 103 3.1.1 General Aspects of Scaffold Proteins 103 3.1.2 Scaffolds as Organizers of Signaling Circuits 105 3.1.2.1 Organization of Sequential Signaling and Signaling Circuits 106 3.1.2.2 Scaffolds Organize Signaling Complexes and are Targets of Regulation 107 3.1.2.3 Scaffolds Organize Feedback Loops 107 3.1.2.4 Scaffolds as Signaling Enzymes and Allosteric Regulators 108 3.2 Signal Processing in Signaling Paths and Signaling Networks 108 3.2.1 Speciﬁcity of Signaling 109 3.2.2 PTM-Induced Formation of Signaling Complexes 109 3.2.3 Signaling through Preassembled Multiprotein Complexes 110 3.2.4 Signaling in Dependence of Subcellular Localization: Spatial Organization 111 3.2.4.1 Spatial Control by Scaffolding 111 3.2.4.2 Spatial Control by Phosphorylation 112 3.2.4.3 Lipid Anchors 112 3.2.4.4 Clustering of Signaling Proteins on the Nanoscale 112 3.2.5 Temporal Control of Signaling 113 3.3 Architecture of Signaling Pathways 113 3.3.1 Linearity, Branching, Crosstalk, and Networks 114 Contents jIX

3.3.1.1 Linearity 114 3.3.1.2 Branching 114 3.3.1.3 Crosstalk 114 3.3.1.4 Networks 115 3.3.2 Regulatory Circuits and Responses in Biological Networks 116 3.3.2.1 Circuits and Cascades 116 3.3.2.2 Feedback Loops 117 3.3.2.3 Negative Feedback 117 3.3.2.4 Positive Feedback 119 3.3.2.5 Bistability 120 3.3.2.6 Dynamic Behavior of Responses 121 3.3.3 Network Structures 121 3.3.3.1 Interaction Networks: Hubs 122 3.3.3.2 Layered Networks 124 3.3.3.3 Modularity 126 3.3.3.4 Redundancy and Robustness 126 Questions 127 References 127

4 The Regulation of Gene Expression 129 4.1 The Basic Steps of Gene Expression 129 4.1.1 Regulation of Transcription 129 4.1.1.1 Conversion of the pre-mRNA into the Mature mRNA 131 4.1.1.2 Regulation at the Level of mRNA and Translation 131 4.1.1.3 Nature of the Regulatory Signals 131 4.2 The Components of the Eukaryotic Transcription Machinery 131 4.2.1 The Basic Features of Eukaryotic Transcription 132 4.2.2 Elementary Steps of Eukaryotic Transcription 135 4.2.3 The Eukaryotic RNA Polymerases 135 4.2.4 The Core Promoter and Structure of the Transcription Start Site 137 4.2.5 General Transcription Factors and the Basal Transcription Apparatus 139 4.2.5.1 TFII D 139 4.2.5.2 TFIIH 141 4.2.6 The Mediator Complex 143 4.2.7 C-Terminal Domain (CTD) of RNA Polymerase II and the Onset of Transcription 146 4.3 The Principles of Transcription Regulation 149 4.3.1 Elements of Transcription Regulation 149 4.3.2 Regulation of Eukaryotic Transcription by Speciﬁc Transcription Factors 151 4.3.2.1 Activation and Repression of Transcription 152 4.3.3 Coregulators of Transcription 153 4.3.4 DNA-Binding of Speciﬁc Transcription Factors 153 Xj Contents

4.3.4.1 DNA-Binding Domains 154 4.3.5 Structure of the Recognition Sequence and Quaternary Structure of DNA-Binding Proteins 155 4.3.5.1 Palindromic Arrangement 155 4.3.5.2 Direct Repeats of the Recognition Sequence 155 4.3.5.3 Identity of a RE 156 4.3.5.4 Homodimers and Heterodimers 156 4.3.6 Communication with the Transcription Apparatus: Transactivation Domains 157 4.3.7 Clustering of REs and the Enhanceosome 159 4.3.8 DNA Recognition and Selectivity of Transcription Activation 161 4.3.8.1 Sequence of the RE 161 4.3.8.2 Allostery in Transcription Factor–RE Interactions 162 4.3.8.3 Influence of Neighboring Sequences and Chromatin Surrounding 162 4.3.9 Repression of Transcription 163 4.4 The Control of Transcription Factors 165 4.4.1 Classification of Transcription Factors by their Function in Signal Transduction Networks 165 4.4.1.1 Constitutively Active Transcription Factors 166 4.4.1.2 Regulatory Transcription Factors 166 4.4.2 Mechanisms for the Control of the Activity of DNA-Binding Proteins 167 4.4.2.1 Changes in the Concentration of Regulatory DNA-Binding Proteins 169 4.4.2.2 Regulation by Binding of Effector Molecules 169 4.4.3 PTM of Transcription Regulators 170 4.4.3.1 Regulation by Phosphorylation 171 4.4.3.2 Regulation by Acetylation 175 4.5 Chromatin Structure and Transcription Regulation 175 4.5.1 Chromatin Architecture at Promoters 177 4.5.1.1 Histone Variants 180 4.5.1.2 Chromatin Remodeling 180 4.5.1.3 Chromatin Modification 182 4.5.2 Histone Acetylation 182 4.5.2.1 Histone Acetyltransferases 184 4.5.2.2 Histone Deacetylation 185 4.5.3 Histone Methylation 186 4.5.3.1 Enzymes of Histone Lysine Methylation 188 4.5.3.2 Enzymes of Histone Lysine Demethylation 189 4.5.3.3 Histone Arginine Methylation 189 4.5.4 Histone Phosphorylation 190 4.5.5 Histone Ubiquitination 192 4.5.6 Recognition of Histone Modifications by Protein Domains 192 4.5.7 Histone Modification Crosstalk 193 Contents jXI

4.5.7.1 Crosstalk Mechanisms 194 4.5.7.2 Is There a Histone Modiﬁcation “Code”? 196 4.5.8 Histone Modiﬁcation and Epigenetics 197 4.5.9 DNA Methylation 197 4.5.9.1 DNA Methyltransferases 200 4.5.9.2 Coupling DNA Methylation to Gene Repression 200 4.5.9.3 Linking DNA Methylation and Histone Methylation 201 4.5.9.4 Biological Functions of DNA Methylation 202 4.5.10 Summary of the Regulatory Steps in Transcription 203 Questions 205 References 206

5 RNA Processing, Translational Regulation, and RNA Interference 209 5.1 Pre-mRNA Processing 209 5.1.1 Capping and Polyadenylation 210 5.1.1.1 Capping 210 5.1.1.2 Polyadenylation 210 5.1.2 Alternative Splicing 210 5.1.3 Regulation of Alternative Splicing 211 5.1.3.1 SR Proteins 213 5.1.3.2 hnRNPs 214 5.1.4 Chromatin Structure and Splicing 214 5.1.5 Coupling of pre-mRNA Processing, Transcription, and Translation 215 5.1.5.1 RNA Pol II Poised for Transcription 215 5.1.5.2 Formation of 50 Cap 215 5.1.5.3 Chromatin Modification and Start of Productive Elongation 215 5.1.5.4 Transcription and Nuclear Pores 216 5.1.5.5 Splicing During Transcription Elongation 216 5.1.5.6 RNA Pol II Termination 216 5.1.5.7 Linkage to Cytoplasmic Events 216 5.2 Regulation at the Level of Translation 217 5.2.1 Overview of Translation Initiation 217 5.2.2 The General Mechanisms of Translational Control 218 5.2.2.1 mRNA-Specific Control of Translation 219 5.2.2.2 Global Control of Translation 219 5.2.3 mRNA-Specific Regulation by 50-Sequences: Control of Ferritin mRNA Translation by Iron 220 5.2.4 mRNA-Specific Translational Regulation by Protein Binding to 30-UTRs 222 5.2.5 Global Translational Regulation of mRNAs by Targeting eIF-4E 222 5.2.5.1 4E-BP1 Phosphorylation 224 5.2.5.2 S6 Kinase Phosphorylation 226 5.2.6 Regulation of Translation via eIF-2 226 5.3 Regulation by RNA Silencing 229 XIIj Contents

5.3.1 Basics of RNA Silencing 230 5.3.2 The miRNAs 233 5.3.2.1 miRNA Biogenesis 233 5.3.2.2 Formation of miRISC 235 5.3.2.3 Posttranscriptional Repression and mRNA Decay 235 5.3.2.4 Regulation of miRNAs 238 5.3.2.5 Regulatory Functions of miRNAs 240 5.3.2.6 miRNAs and Cancer 243 5.3.3 siRNAs 243 5.3.3.1 Sources and Processing of siRNAs 243 5.3.3.2 Posttranscriptional Silencing by siRNAs 246 5.3.3.3 Functions and Applications of siRNAs 246 Questions 247 References 248

6 Signaling by Nuclear Receptors 251 6.1 Ligands of Nuclear Receptors (NRs) 252 6.2 Principles of Signaling by Nuclear Receptors (NRs) 254 6.3 Structure of Nuclear Receptors (NRs) 257 6.3.1 DNA-Binding Elements of NRs: HREs 258 6.3.2 The DNA-Binding Domain of NRs 260 6.3.3 Ligand-Binding Domain 261 6.3.3.1 Ligand Binding 262 6.3.3.2 Ligand Binding and AF-2 Function 263 6.3.3.3 Switch Function of Ligand Binding 264 6.3.3.4 Promiscuous Ligand Binding 265 6.3.4 Transactivation Functions of the NRs 265 6.3.5 Structure of an Intact NR Complex on DNA 266 6.3.6 Orphan Nuclear Receptors 268 6.4 Transcriptional Regulation by NRs 268 6.4.1 Coregulators in NR Function 269 6.4.2 NR Coactivators 271 6.4.2.1 SRC/p160 Family of Coactivators 271 6.4.2.2 TRAP Complex, Mediator 273 6.4.2.3 Variability of Coactivator Recruitment 273 6.4.3 Corepressors of NRs 274 6.5 Regulation of Signaling by Nuclear Receptors 274 6.5.1 Regulation at the Level of Ligand Concentration 275 6.5.2 Regulation by PTM 276 6.5.2.1 Regulation by Phosphorylation 276 6.5.3 Ubiquitination and Sumoylation of NRs 278 6.5.4 Composite DNA Elements, Interaction with Other Transcription Factors, and Long-Range Effects 278 6.5.4.1 Crosstalk with Other Transcription Factors 279 6.5.4.2 Long-Range Actions of NRs 279 Contents jXIII

6.5.5 Determinants of Cell- and Gene-Speciﬁcity of NR Action 280 6.6 Subcellular Localization of NRs 280 6.6.1 Nuclear and Cytoplasmic Pools 281 6.6.2 Other Subcellular Pools 281 6.6.3 Ligand-Dependent Translocation of Nuclear Receptors 281 6.6.4 Nuclear Translocation 283 6.7 Nongenomic Functions of NRs and their Ligands 284 6.7.1 Nuclear Receptor Functions Outside of the Nucleus 284 6.7.1.1 Cytoplasmic Functions 284 6.7.1.2 ER Actions at the Cell Membrane 285 6.7.1.3 NR Action at the Mitochondrion 288 6.7.2 Hormone Binding to Other Receptor Types 288 Questions 289 References 289

7 G Protein-Coupled Signal Transmission Pathways 291 7.1 Transmembrane Receptors: General Structure and Classiﬁcation 291 7.1.1 Signaling via Transmembrane Receptors 291 7.1.2 Signaling via Ligand- or Voltage-Gated Ion Channels 292 7.2 Structural Principles of Transmembrane Receptors 294 7.2.1 The Extracellular Domain of Transmembrane Receptors 294 7.2.2 The Transmembrane Domain 296 7.2.2.1 Structure of Transmembrane Elements 296 7.2.3 The Intracellular Domain of Membrane Receptors 298 7.2.3.1 Recruitment of Downstream Signaling Proteins 298 7.2.3.2 Triggering of Enzyme Activity 299 7.2.4 Regulation of Receptor Activity 300 7.2.4.1 Receptor Phosphorylation and Receptor Recycling 300 7.2.4.2 Ubiquitination and Degradation 301 7.3 G Protein-Coupled Receptors 301 7.3.1 Classiﬁcation of GPCRs 304 7.3.2 Structure of G Protein-Coupled Receptors 305 7.3.2.1 Overall Structure 307 7.3.2.2 Ligand Binding and Effector Activation 309 7.3.2.3 Binding of Activated GPCR to G Protein 310 7.3.2.4 Ligand-selective GPCR Signaling 311 7.3.3 Regulation of GPCRs 312 7.3.3.1 Phosphorylation of GPCRs 314 7.3.3.2 Desensitization and Downregulation of GPCRs 315 7.3.3.3 GPCR Phosphorylation by GRKs 316 7.3.3.4 Binding of Arrestin 317 7.3.3.5 Signal Switching 319 7.3.3.6 Coupling to other Signaling Pathways 319 7.3.4 Oligomerization of GPCRs 319 XIVj Contents

7.4 Regulatory GTPases 320 7.4.1 The GTPase Superfamily: General Functions 321 7.4.2 Switch Functions of GTPases and the GTPase Cycle 321 7.4.2.1 Modulation and Regulation of the Switch Function 323 7.4.3 Inhibition of GTPases by GTP Analogs 324 7.4.4 The G-Domain as a Common Structural Element of the GTPases 325 7.4.5 The GTPase Families 326 7.5 The Heterotrimeric G Proteins 327 7.5.1 Classiﬁcation of the Heterotrimeric G Proteins 328 331 7.5.1.1 Gs Subfamily 331 7.5.1.2 Gi Subfamily 332 7.5.1.3 Gq Subfamily 332 7.5.1.4 G12 Subfamily 7.5.2 Toxins as Tools in the Characterization of Heterotrimeric G Proteins 332 7.5.3 The Functional Cycle of Heterotrimeric G Proteins 334 7.5.3.1 Inactive Ground State 334 7.5.3.2 Activation 334 7.5.3.3 Transmission of the Signal 336 7.5.3.4 Termination of the Signal and GTPase-Activating Proteins 336 7.5.3.5 Heterotrimeric G Proteins in Supramolecular Complexes 337 7.5.4 Structural and Mechanistic Aspects of the Switch Function of G Proteins 337 7.5.4.1 Coupling of the Activated Receptor to the G Protein 337

7.5.4.2 Structure of the Ga-subunit 337 7.5.4.3 Structure of the Heterotrimer 339 7.5.4.4 Conformational Changes Upon Activation by GTP 339 7.5.4.5 Mechanism of GTP Hydrolysis 340 7.5.4.6 Acceleration of GTP Hydrolysis by GTPase-Activating Proteins 342

7.5.5 Structure and Function of the Gbg-Complex 342 7.5.5.1 Structure of the Gbg-Complex 343 7.5.5.2 Speciﬁcity of Gbg Signaling 343 7.5.5.3 Signaling by the bg-Complex 344 7.5.6 Membrane Association of the G Proteins 345 7.5.7 Regulators of G Proteins: Phosducin and RGS Proteins 346 7.5.7.1 Phosducin 347 7.5.7.2 RGS Proteins 347 7.5.7.3 Non-G Protein Signaling by RGS Proteins 349 7.6 Receptor-independent Functions of Heterotrimeric G Proteins 350 7.6.1 Novel G Protein Cycle 350 7.7 Effector Molecules of G Proteins 352 7.7.1 Adenylyl Cyclase and cAMP as “Second Messenger” 352 7.7.1.1 Structure of Adenylyl Cyclase 354 7.7.1.2 Regulation of Adenylyl Cyclase 355 Contents jXV

7.7.2 Phospholipase C 357 7.7.2.1 Phospholipase C-b 360 7.7.2.2 Phospholipase C-g 362 7.7.2.3 Phospholipases C-d 362 7.7.2.4 Phospholipase C-e 362 7.8 GPCR Signaling via Arrestin 363 Questions 365 References 366

8 Intracellular Messenger Substances: “Second Messengers” 369 8.1 General Properties of Intracellular Messenger Substances 369 8.2 Cyclic AMP 371 8.2.1 Formation and Degradation of cAMP 371 8.2.1.1 Formation by ACs 371 8.2.1.2 Phosphodiesterases and cAMP Breakdown 372 8.2.2 Targets of cAMP 372 8.2.3 Compartmentalization of cAMP Signaling 374 8.3 cGMP and Guanylyl Cyclases 375 8.3.1 Guanylyl Cyclases 375 8.3.1.1 Guanylyl Cyclase Receptors 376 8.3.1.2 Soluble Guanylyl Cyclases 377 8.3.2 Targets of cGMP 377 8.4 Metabolism of Inositol Phospholipids and Inositol Phosphates 378 8.4.1 Other Inositol Messengers 380 8.4.2 Activation of PLC and Inositol Phosphate Formation 381 þ 8.5 Storage and Release of Ca2 383 þ þ 8.5.1 Release of Ca2 from Ca2 Storage 384 384 8.5.1.1 The InsP3 Receptor 8.5.1.2 The Ryanodine Receptor 387 8.5.1.3 cADP-Ribose and NAADP 387 þ 8.5.1.4 Ca2 Channels and Apoptosis 388 þ 8.5.1.5 Tool Kit for Ca2 Release 389 þ 8.5.2 Inﬂux of Ca2 from the Extracellular Region 390 þ 8.5.3 Removal and Storage of Ca2 390 þ 8.5.4 Temporal and Spatial Changes in Ca2 Concentration 391 8.5.4.1 Calcium Oscillations 392 8.6 Functions of Phosphoinositides 392 393 8.6.1 The Messenger Function of PtdIns(3,4,5)P3 393 8.6.2 Functions of PtIns(4,5)P2 and Other Phosphoinositides þ 8.7 Ca2 as a Signal Molecule 394 þ 8.7.1 The EF Hand: A Ca2 -Binding Module 396 þ 8.7.2 Calmodulin as a Ca2 Sensor 398 þ 8.7.3 Target Proteins of Ca2 /Calmodulin 399 þ 8.7.4 Other Ca2 Sensors 399 XVIj Contents

8.8 Diacylglycerol as a Signal Molecule 401 8.9 Other Lipid Messengers: Ceramide, Sphingosine, and Lysophosphatidic Acid 401 8.9.1 Ceramide 402 8.9.2 Sphingosine 403 8.9.3 Lysophosphatidic Acid (LPA) 404 8.10 The NO Signaling Molecule 404 8.10.1 Reactivity of NO 405 8.10.2 Synthesis of NO 407 8.10.2.1 nNOS 408 8.10.2.2 eNOS 408 8.10.2.3 iNOS 409 8.10.3 Physiological Functions of Nitrosylation 409 8.10.4 Nitrosylation of Metal Centers 409 8.10.4.1 NO-Sensitive Guanylyl Cyclase 409 8.10.4.2 Nitrosylation of Hemoglobin 410 8.10.5 Regulatory Functions of Protein S-Nitrosylation 411 8.10.5.1 Selectivity of Protein S-Nitrosylation 411 8.10.5.2 Transnitrosylation 412 8.10.5.3 Denitrosylation 412 8.10.5.4 Target Proteins for S-Nitrosylation 413 8.10.6 Toxic Action of NO and Nitrosative Stress 413 Questions 414 References 415

9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases 417 9.1 Classification, Structure, and Characteristics of Protein Kinases 417 9.2 Structure and Regulation of Protein Kinases 420 9.2.1 The Protein Kinase Reaction 421 9.2.2 Main Structural Elements of Protein Kinases 422 9.2.3 Substrate Binding and Recognition 425 9.2.4 Control of Protein Kinase Activity 427 9.2.5 Regulation of Protein Phosphorylation by Subcellular Localization and Specific Targeting Subunits 430 9.3 Protein Kinase A 431 9.3.1 Structure and Substrate Specificity of PKA 432 9.3.2 Regulation of PKA 435 9.3.3 A-Kinase Anchor Proteins (AKAPs) 436 9.3.3.1 AKAPs as Multivalent Scaffolds 436 9.4 The PI3 Kinase/Akt Pathway 439 9.4.1 PI3K 440 9.4.1.1 Classification and Properties of PI3K 440 9.4.1.2 Activation of PI3K 441 9.4.2 Akt Kinase (PKB) 442 9.4.2.1 Activation of Akt Kinase 443 Contents jXVII

9.4.2.2 Signaling by Akt Kinase 444 9.4.2.3 Phosphatase and Tensin Homolog PTEN Phosphatase 446 9.5 Protein Kinase C 447 9.5.1 Classification and Structure of PKC 447 9.5.2 The Protein Kinase C Family 447 9.5.2.1 Stimulation by Phorbol Esters 449 9.5.3 Activation of PKC 449 9.5.4 Regulation of Protein Kinase C 450 þ 9.5.4.1 Regulation by Ca2 and DAG 451 9.5.4.2 Regulation by Phosphorylation: PDK1 452 9.5.4.3 Protein Kinase C-Interacting Proteins and Regulation by Localization 453 9.5.5 Receptors for Protein Kinase C, RACK Proteins 453 9.5.6 Functions and Substrates of PKC 454 þ 9.6 Ca2 /Calmodulin-Dependent Protein Kinases, CaM Kinases 455 9.6.1 General Function of CaM Kinases 455 9.6.2 CaM Kinase II 456 9.6.2.1 Structure and Activation of CaMKII 457 9.6.2.2 Memory Function of CaMKII 460 9.7 Ser/Thr-Specific Protein Phosphatases 461 9.7.1 Classification and Structure of Ser/Thr Protein Phosphatases 462 9.7.2 Function and Regulation of Ser/Thr Protein Phosphatases 462 9.7.3 Protein Phosphatase I, PPI 464 9.7.4 Protein Phosphatase 2A, PP2A 465 9.7.5 Protein Phosphatase 2B, Calcineurin 467 Questions 469 References 470

10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity 473 10.1 Structure and Function of RTKs 474 10.1.1 General Structure and Classiﬁcation 475 10.1.2 Ligand Binding and Receptor Dimerization 477 10.1.2.1 Receptor Heterodimerization 481 10.1.3 Structure and Activation of the TK Domain 481 10.1.3.1 Autoinhibition by the Activation Loop: The Insulin Receptor 483 10.1.3.2 Juxtamembrane Autoinhibition 484 10.1.3.3 Autoinhibition by C-Terminal Sequences 484 10.1.3.4 Allosteric Mechanism of Activation 485 10.1.4 RTK Activation and Downstream Signaling 485 10.1.4.1 Nucleation of Signaling Complexes 485 10.1.5 Recruitment of Effectors via Interaction Domains 490 10.1.5.1 SH2 Domains 491 10.1.5.2 P-Tyrosine-Binding (PTB) Domain 493 10.1.5.3 C2 Domains 493 XVIIIj Contents

10.2 Downstream Effector Proteins of RTKs 494 10.2.1 Adapter Proteins in RTK Signaling 494 10.2.1.1 Insulin Receptor Substrate (IRS) 495 10.2.1.2 Tyr-Phosphorylation: Effector Recruitment 495 10.2.1.3 Ser/Thr Phosphorylation: Signal Dampening 496 10.2.2 Adaptor Proteins: FRS, Grb2, Gab, Shc, LAT, and p130 Cas 497 10.2.2.1 Fibroblast Growth Factor Receptor Substrate (FRS) 497 10.2.2.2 Grb2 497 10.2.2.3 Gab 499 10.2.2.4 Shc 499 10.2.2.5 PDZ-Containing Adapter Proteins 500 10.2.2.6 LAT 500 10.2.2.7 p130Cas 500 10.2.3 Downstream Effectors of RTK Signaling 500 10.2.4 RTKs as Part of Signaling Networks 501 10.2.5 Attenuation and Feedback Regulation in RTK Signaling 504 10.2.5.1 Antagonistic Ligands, Heterodimerization 505 10.2.5.2 Inhibition by Inhibitor Proteins 505 10.2.6 Endocytosis and Trafficking of RTKs 506 10.2.6.1 Ubiquitination 506 10.2.7 RTK Dysfunction in Disease 506 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 507 10.3.1 Structure and General Function of Non-RTKs 507 10.3.1.1 Functions of non-RTKs 508 10.3.2 Src Tyrosine Kinase 508 10.3.2.1 Structure of Inactive Src 509 10.3.2.2 Inactive State of Src 511 10.3.2.3 Activation of Src 511 10.3.3 Abl Tyrosine Kinase 513 10.3.3.1 Structure of Abl 513 10.3.3.2 Activation of Abl 515 10.3.3.3 Substrate Selection and Interaction Partners of Abl 515 10.3.3.4 Regulation of Abl 517 10.3.3.5 Cellular Functions of Abl 517 10.3.3.6 Oncogenic Activation of Abl 518 10.4 Protein Tyrosine Phosphatases 519 10.4.1 General Functions of PTPs 519 10.4.2 Classification, Structure, and Mechanism of PTPs 520 10.4.3 Catalytic Mechanism of PTPs 520 10.4.3.1 Receptor Protein Tyrosine Phosphatases (PTPRs) 521 10.4.3.2 Nonreceptor Protein Tyrosine Phosphatases (PTPNs) 523 10.4.3.3 Autoinhibition of PTPN Enzymes 523 10.4.4 Regulation of Cell Signaling by PTPs 525 10.4.4.1 Negative Regulation by PTPs, and Tumor Suppression 526 10.4.4.2 Positive Regulation by PTPs, and Oncogenic Functions 526 Contents jXIX

10.4.5 Regulation of PTP Activity 527 10.4.6 Oxidation of PTPs 528 10.4.7 Subcellular Localization of PTPs 532 Questions 532 References 533

11 Signal Transmission via Ras Proteins 535 11.1 The Ras Superfamily of Monomeric GTPases 535 11.1.1 Crosstalk Among Ras Superfamily Members 538 11.2 GTPase-Activating Proteins (GAPs) of the Monomeric GTPases 539 11.3 Guanine Nucleotide Exchange Factors (GEFs) of the Monomeric GTPases 541 11.3.1 Catalytic Domains 541 11.3.1.1 Mechanism of Nucleotide Exchange 542 11.3.1.2 Multivalency of GEFs 542 11.3.1.3 GEFs Can Activate Two Different GTPases 543 11.3.1.4 GEFs as Effectors of GTPases: Linkage of and Crosstalk between GTPases 543 11.4 Guanine Nucleotide Dissociation Inhibitors (GDIs) 544 11.5 The Ras Family of Monomeric GTPases 545 11.5.1 General Properties of the Ras Protein 545 11.5.2 Structure of the GTP- and GDP-Bound Forms of Ras Protein 547 11.5.3 GTP Hydrolysis Mechanism and Stimulation by GAPs 548 11.5.4 Structure and Biochemical Properties of Transforming Mutants of Ras Protein 550 11.5.5 Membrane Localization of Ras Protein 551 11.5.5.1 Ras Nanoclusters 551 11.5.6 GAPs in Ras Signal Transduction 552 11.5.7 Guanine Nucleotide Exchange Factors (GEFs) in Ras Signal Transduction 553 11.5.7.1 Structure and Activation 553 11.5.7.2 Regulation 554 11.6 Raf Kinase as an Effector of Signal Transduction by Ras Proteins 555 11.6.1 Structure of Raf kinase 557 11.6.2 Mechanism of Activation and Regulation of Raf Kinase 557 11.6.2.1 Formation of Homodimers and Heterodimers 559 11.6.2.2 Negative Feedback by Inhibitory Phosphorylations 560 11.6.3 Oncogenic Activation of Raf 561 11.7 Further Ras Family Members: R-Ras, Ral, and Rap 561 11.8 Reception and Transmission of Multiple Signals by Ras Protein 562 11.8.1 Multiple Input Signals of Ras Protein 562 11.8.2 Multiple Effector Molecules of Ras Proteins 564 11.8.3 The Ras Signaling Network 566 11.9 The Further Branches of the Ras Superfamily 568 11.9.1 The Rho/Rac Family 568 XXj Contents

11.9.2 The Rab Family 569 11.9.3 The Ran Family 570 11.9.4 The Arf Family 570 Questions 570 References 571

12 Intracellular Signal Transduction: The MAP Kinase Pathways 573 12.1 Organization and Components of MAPK Pathways 575 12.1.1 MAPKs 577 12.1.2 MAPK Kinases (MAP2Ks, MEKs) 577 12.1.3 MEK Kinases (MAPKK Kinases, MAP3Ks) 579 12.2 Regulation of MAPK Pathways by Protein Phosphatases and Inhibitor Proteins 579 12.2.1 Feedback Loops in MAPK Regulation 580 12.2.2 Regulation by MAPK Phosphatases 581 12.2.3 MAPK Regulation by Inhibitory Proteins 583 12.3 Scaffolding in MAPK Signaling 583 12.4 The Major MAPK Pathways of Mammals 586 12.4.1 The ERK Pathway 586 12.4.1.1 Input Signals 586 12.4.1.2 Substrates of ERKs 587 12.4.2 The JNK and p38 MAPK Pathways 588 12.4.2.1 Input Signals and Signal Entry Points of the JNK and p38 Pathways 589 12.4.2.2 Substrates of the JNK and p38 Pathways 590 12.4.2.3 The JNK Module 591 12.4.2.4 The p38 Module 591 Questions 592 References 592

13 Membrane Receptors with Associated Tyrosine Kinase Activity 593 13.1 Cytokines and Cytokine Receptors 593 13.1.1 Cytokines 594 13.1.1.1 Cytokine Structure 595 13.1.1.2 General Functions of Cytokines 595 13.1.2 Structure and Activation of Cytokine Receptors 596 13.1.2.1 Classiﬁcation and General Features 596 13.1.2.2 Structural Domains 597 13.1.2.3 Oligomeric Structure 598 13.1.2.4 Cytokine Binding and Activation 599 13.1.2.5 Gp130 Receptors 601 13.1.2.6 Shared gc-Chain Receptors 603 13.1.2.7 Shared b-Chain Receptors 603 13.1.3 Activation of Cytoplasmic Tyrosine Kinases 604 13.2 The Jak-STAT Pathway 608 Contents jXXI

13.2.1 The Janus Kinases 609 13.2.1.1 Kinase Regulation 609 13.2.1.2 Coupling to Other Receptor Types 610 13.2.1.3 Nuclear Functions of Jaks 610 13.2.2 The Stat Proteins 611 13.2.2.1 Structure of Stats 612 13.2.2.2 Activation of Stats 612 13.2.2.3 Signaling Function of STATs 614 13.2.2.4 Signaling by Unphosphorylated STATs 615 13.2.2.5 Acetylation of STATs 615 13.2.3 Regulation of Cytokine Receptor Signaling 615 13.2.3.1 Protein Tyrosine Phosphatases 617 13.2.3.2 SOCS Proteins 617 13.2.3.3 PIAS (Protein Inhibitors of Activated Stats) 617 13.3 T- and B-Cell Receptors 618 13.3.1 Immunoreceptor Structure 618 13.3.1.1 T-Cell Receptor Structure 618 13.3.1.2 B-Cell Receptor Structure 619 13.3.2 Activation and Signaling of the T-Cell Antigen Receptors 621 13.3.2.1 Initiation of Signaling 621 13.3.2.2 Zap70 621 13.3.2.3 LAT (Linker for Activation of T Lymphocytes) 623 13.4 Signal Transduction via Integrins 623 13.4.113.4.1.1 Talin and Kindlin as Key Activators 625 13.4.1.2 Downstream Signaling 626 13.4.1.3 Focal Adhesion Kinase (FAK) 627 Questions 628 References 629

14 Other Transmembrane Receptor Classes: Signaling by TGF-b Receptors, TNF Receptors, Toll Receptors, and Notch 631 14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling 631 14.1.1 The Family of TGF-b Cytokines 633 14.1.2 TGF-b Receptor 634 14.1.2.1 TGF-b Receptor Structure 635 14.1.2.2 TGF-b Receptor Activation 635 14.1.2.3 Non-Smad Signaling Pathways 636 14.1.3 Smad Proteins 636 14.1.3.1 Smad Activation 638 14.1.3.2 DNA Binding and Transcriptional Regulation by Smads 640 14.1.4 Regulation of TGF-b and Smad Signaling 640 14.1.4.1 Inhibition by I-Smads 641 14.1.4.2 Ubiquitination and Sumoylation 641 14.1.4.3 Phosphorylation 641 XXIIj Contents

14.1.4.4 Activation of Smad Expression 641 14.1.4.5 Growth Regulatory Effects of TGF-b Signaling 642 14.2 Receptor Regulation by Intramembrane Proteolysis: The Notch Receptor 642 14.2.1 Notch: General Function, Structure, and Ligands 643 14.2.1.1 Regulated Intramembrane Proteolysis 643 14.2.1.2 Domain Organization of Notch 644 14.2.1.3 Notch Ligands 644 14.2.2 Processing and Activation of Notch 644 14.2.2.1 Transcription Activation 648 14.3 Tumor Necrosis Factor Receptor (TNFR) Superfamily 648 14.3.1 Biological Functions of TNFR Signaling 650 14.3.2 TNFR Structure 650 14.3.3 TNF Cytokines 651 14.3.4 Receptor Activation and Downstream Signaling 652 14.3.4.1 Downstream Signaling 652 14.3.4.2 Recruitment of Adapter Proteins and Activation of NFkB Pathways 652 14.4 Toll-Like Receptor Signaling 653 14.4.1 TLR Structure 654 14.4.1.1 Ligand Binding and Activation 655 14.4.1.2 TLR Adaptors and Further Signaling 656 14.4.1.3 Adapter-Mediated Downstream Signaling 657 Questions 658 References 659

15 Cell-Cycle Control by External Signaling Pathways 661 15.1 Principles of Cell-Cycle Control 661 15.1.1 Intrinsic Control Mechanisms 663 15.1.2 External Cell Cycle Control 664 15.1.2.1 Mitogenic Signals 664 15.1.2.2 Antimitogenic Signals during Cell–Cell Communication 665 15.1.3 Cell-Cycle Checkpoints 665 15.2 Key Elements of the Cell-Cycle Apparatus 666 15.2.1 Cyclin-Dependent Protein Kinases (CDKs) 666 15.2.1.1 CDKs Involved in Cell-Cycle Regulation 667 15.2.1.2 Other CDKs 667 15.2.2 Cyclins 667 15.2.2.1 Regulation of Cyclin Concentration 670 15.2.3 Inhibitors of CDKs: The CKIs 671 15.2.3.1 Regulation and Function of CKIs 672 15.2.3.2 The CIP/KIP Family 672 15.2.3.3 INK4 Proteins 674 15.2.4 Structural Basis of CDK Regulation 674 15.2.4.1 CDK–Cyclin Complexes 674 Contents jXXIII

15.2.4.2 Phosphorylation of CDKs 676 15.2.4.3 Structural Basis of Inhibition by p27KIP1 677 15.2.5 Multiple Regulation of CDKs 678 15.2.6 Substrates of CDKs 679 679 15.2.6.1 Substrates in G1/S Phase 15.2.6.2 Substrates in G2/M Phase 680 15.3 Regulation of the Cell Cycle by Proteolysis 681 15.3.1 Proteolysis Mediated by the SCF Complex 682 15.3.2 Proteolysis Mediated by the APC/Cyclosome 684 684 15.4 G1 Progression and S Phase Entry 15.4.1 CDK4/6 and the D-Type Cyclins 685 15.4.1.1 Regulation of CDK4–cyclin D1 685 15.4.2 Function of CDK2–Cyclin E in S Phase Entry 687 15.4.3 Function of the Retinoblastoma protein (Rb) in the Cell Cycle 687 15.4.3.1 Domain Structure of Rb 689 15.4.3.2 Control of Rb by Phosphorylation 690 15.4.4 The E2F Transcription Factors and their Control by Rb 691 15.4.4.1 Control of E2Fs by Rb 692 694 15.4.5 Summary of G1 Progression 694 15.4.6 Mitogenic Signals Regulating G1 Entry and Progression 15.4.6.1 Transcriptional Control of Cyclin D1 695 15.4.6.2 Control of Cyclin D1 Translation and Stability 696 697 15.4.7 Negative Regulation of the G1/S Transition 15.4.8 Integration of Mitogenic Signals for Control of Cell Proliferation, Cell Growth, and Survival 697 15.5 Transit Through S Phase and M Phase 699 15.5.1 DNA Replication During S Phase 699 700 15.5.2 G2/M Transition and Progression Through M Phase 700 15.5.2.1 CDK1 Control at G2/M 15.5.2.2 Progression Through M Phase 702 15.6 DNA Damage and DNA Replication Checkpoints 702 15.6.1 Components and Organization of DNA Damage Checkpoints 703 15.6.1.1 Recognition of DNA Damage or Sensing of Replication Stress 707 15.6.1.2 Functions of ATM 708 15.6.1.3 Functions of ATR 709 15.6.1.4 Chk1, Chk2, and Downregulation of CDC25 Phosphatase 709 710 15.6.2 The Mammalian G1DNA Damage Checkpoint 712 15.6.3 The G2/M Checkpoint Questions 712 References 713

16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes 715 16.1 Basic Characteristics of Tumor Cells 715 16.2 Mutations in Cancer Cells 715 XXIVj Contents

16.2.1 Genetic Changes in Cancer Cells 716 16.2.1.1 Small-Scale Changes 717 16.2.1.2 Large-Scale Changes and Genetic Instability 717 16.2.2 Epigenetic Changes in Tumor Cells 717 16.2.2.1 Altered DNA Methylation 718 16.2.2.2 Altered Histone Modiﬁcations in Cancer 720 16.2.2.3 Micro RNAs and Cancer 720 16.2.3 Cancer Genes: Drivers and Passengers 721 16.2.3.1 Oncogenes Versus Tumor Suppressor Genes 722 16.2.4 Carcinogenesis as an Evolutionary Process 722 16.3 Common Physiologic Changes in Tumor Cells: The Hallmarks of Cancer 725 16.3.1 Self-Sufﬁciency in Growth Signals 727 16.3.2 Insensitivity to Antigrowth Signals 728 16.3.3 Evasion of Programmed Cell Death (Apoptosis) 729 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 729 16.4.1 Mechanisms of Oncogene Activation 730 16.4.1.1 Oncogenic Activation by Structural Changes 730 16.4.1.2 Oncogenic Fusion Proteins 731 16.4.1.3 Activation by Protein Concentration Increase 732 16.4.2 Examples of the Functions of Oncogenes 733 16.4.2.1 Oncogenic Receptor Tyrosine Kinases: The ErbB2/neu Receptor 734 16.4.2.2 Oncogenic Nonreceptor Tyrosine Kinases 735 16.4.2.3 Oncogenic Activation of Ras Signaling Pathways 737 16.4.2.4 Oncogenic Activation of Cyclins 737 16.4.2.5 Oncogenic Transcription Factors: Myc 737 16.5 Tumor Suppressor Genes: General Functions 741 16.6 Tumor Suppressors: Rb and ARF Proteins 743 16.6.1 Rb in Cancer 743 16.6.1.1 Rb and Apoptosis 744 16.6.2 The p16INK4a Gene Locus and ARF 746 16.7 Tumor Suppressor Protein p53 747 16.7.1 Overview of p53 Function 749 16.7.1.1 Cousins of p53 751 16.7.2 Structure and Biochemical Properties of the p53 Protein 751 16.7.3 Structure of p53 752 16.7.4 PTMs of p53 753 16.7.4.1 Phosphorylation 753 16.7.4.2 Acetylation 755 16.7.4.3 Methylation 755 16.7.4.4 Ubiquitination 756 16.7.4.5 Neddylation, Sumoylation 756 16.7.5 Control of p53 by Mdm2 756 16.7.5.1 Mdm2 is an Oncogene 757 16.7.5.2 Negative Feedback Between p53 and Mdm2 757 Contents jXXV

16.7.5.3 Other Controls of Mdm2 Function 759 16.7.5.4 p53-Independent Functions of Mdm2 759 16.7.6 Genes Regulated by p53 760 16.7.6.1 Selection of Target Genes 760 16.7.7 Pathways Involved in the Activation of p53 762 16.7.7.1 Activation of p53 by DNA Damage 762 16.7.7.2 Activation of p53 by Oncogenic Stress 762 16.7.7.3 Activation of p53 by Nongenotoxic Stress 763 16.7.8 Classiﬁcation of p53 Target Genes 763 16.7.8.1 p53 Targets Activated in Unstressed Cells and Under Low Intrinsic Stress 764 16.7.8.2 p53 Targets Activated by Moderate Stresses 764 16.7.8.3 p53 Targets Activated by Sustained or Severe Stress 766 16.7.8.4 p53-Mediated Repression 767 16.7.9 Metabolic Regulation by p53 767 16.7.10 p53-Mediated Induction of miRNA 768 16.7.11 Summary of Tumor Suppression by p53 768 16.8 Wnt/b-Catenin Signaling and the Tumor Suppressor APC 770 16.8.1 APC 770 16.8.1.1 Downregulation of b-Catenin 772 16.8.1.2 Activation of b-Catenin 772 Questions 773 References 774

17 Apoptosis 777 17.1 Overview of Apoptotic Pathways 778 17.2 Caspases: Death by Proteolysis 779 17.2.1 Initiator and Effector Caspases 780 17.2.1.1 Initiator Caspases 781 17.2.1.2 Effector Caspases 781 17.2.2 Mechanism of Caspases 781 17.2.3 Caspase Activation and Regulation 783 17.2.3.1 Death Platforms 783 17.2.3.2 Activation of Initiator Caspases 785 17.2.3.3 Activation of Effector Caspases 785 17.2.3.4 Control by Inhibitor Proteins 785 17.2.3.5 Caspase Substrates 786 17.3 The Family of Bcl-2 Proteins: Gatekeepers of Apoptosis 786 17.4 The Mitochondrial Pathway of Apoptosis 789 17.4.1 Permeabilization of the Mitochondrial Outer Membrane 789 17.4.2 Formation of the Apoptosome and Triggering of a Caspase Cascade 791 17.4.2.1 Other Apoptogenic Proteins Released from Mitochondria 792 17.5 Death Receptor-Triggered Apoptosis 792 17.5.1 The Fas/CD95 Signaling Pathway 794 XXVIj Contents

17.5.2 Tumor Necrosis Factor-Receptor 1 and Apoptosis 795 17.6 Links of Apoptosis to Cellular Signaling Pathways 795 17.6.1 PI3 Kinase/Akt Kinase and Apoptosis 796 17.6.2 The Protein p53 and Apoptosis 797 17.6.2.1 Apoptotic Genes Activated by p53 798 17.6.2.2 Transcription-Independent Induction of Apoptosis by p53 799 Questions 799 References 799

Index 801 jXXVII

Preface

When the first edition of this book appeared in 1997 it was written in German, but it was then substituted by four English editions that are now followed by the Fifth edition that includes data and references up to 2012. Due to the huge number of publications available on signal transduction, the majority of the citations in the book are in fact reviews, with original articles having been selected on a more-or-less subjective basis. The past two decades have witnessed an explosion in information on signal transduction that has been based mainly on the progress in genome sequencing and on the ability to analyze signaling proteins with respect to their function, structure, chemical modification and subcellular localization. Today, with such huge amounts of data available it is increasingly difficult – if not impossible – to follow all new developments in the “jungle of signal transduction.” Hence, it is the aim of this book to provide advanced students of biology, biochemistry and chemistry, as well as teachers and researchers, with an overview of – and an orientation within – this highly complicated area of research. Perhaps the most impressive progress has been achieved during the past few years in studies of post-translational modification, interaction partners and macromolecular associations of signaling proteins. Indeed, when taken together these data have revealed an extensive interplay and networking of signaling pathways and their components, and attempts have been made to address this aspect in this new edition of the book. The vast progress that has been made recently in the area of signal transduction makes it increasingly necessary to concentrate on the best-studied components and core reactions in cellular signaling, with special emphasis placed on human systems. Due to the species- and cell-type specificity of many signaling paths, and the extensive crosstalk and networking, I have not attempted to describe distinct signaling pathways in a complete way; rather, I have concentrated on the main classes of signaling proteins and on well-characterized core processes. Conse- quently, in an attempt to guide students through this complex research area, I have included chapter introductions and summaries on all important topics. Questions have also been added to nearly all of the chapters to aid the learning process. Another major change is the addition of two new chapters – Chapters 2 and 3 – as an introductory section. Chapter 2 provides an extended overview of the properties XXVIIIj Preface

and organization of signaling proteins, with strong emphasis placed on their posttranslational modiﬁcations and interaction partners. At present, there is no comparable overview of these features available in the literature. In Chapter 3, I have summarized the structural organization of signaling paths, as well as their crosstalk and networking properties. By introducing the basic features of signaling paths and their interactions at the start of the book, these new chapters should facilitate an understanding of the central signaling paths presented in the following chapters. Unfortunately, the addition of these two new chapters has made it necessary to restrict the total volume of the book, and the chapters on ‘Regulation of the Cell Cycle’ and ‘Apoptosis’ have been shortened accordingly such that only the very basic regulatory features of these processes are now presented. I am grateful to all of the people who have encouraged me to complete this extensive update of the book. In particular, I wish to thank Prof. Clemens Steegborn and Prof. Oliver Hobert for reviewing parts of the book, and I am also very grateful to Christine Diederichs, Dr Sebastien Moniot, Dr Hannes Krauss and Enno Krauss for their help in producing the ﬁgures.

Bayreuth, January 2014 Gerhard Krauss 1

1 Basics of Cell Signaling

1.1 Cell Signaling: Why, When, and Where?

One characteristic common to all organisms is the dynamic ability to coordinate constantly one’s activities with environmental changes. The function of communicating with the environment is achieved through a number of pathways that receive and process signals originating from the external environment, from other cells within the organism, and also from different regions within the cell. In addition to adopting the function of an organism to environmental changes in a signal-directed way, other essential features of multicellular organisms also require the coordinated control of cellular functions. The formation and maintenance of the specialized tissues of multicellular organisms depend on the coordinated regulation of cell number, cell morphology, cell location, and the expression of differentiated functions. Such coordination results from a complex network of communication between cells in which signals produced affect target cells where they are transduced into intracellular biochemical reactions that dictate the physiological function of the target cell (Figure 1.1). The basis for the coordination of the physiological functions within a multicellular organism is intercellular signaling (or intercellular communication), which allows a single cell to influence the behavior of other cells in a specific manner. As compared to single-cell organisms, where all cells behave similarly within a broad frame, multicellular organisms contain specialized cells forming distinct tissues and organs with specific functions. Therefore, the higher organisms have to coordinate a large number of physiological activities such as:

Intermediary metabolism Response to external signals Cell growth Cell division activity Differentiation and development: coordination of expression programs Cell motility Cell morphology.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 2 1 Basics of Cell Signaling

Figure 1.1 Intercellular and intracellular diverse signals. The extracellular signals are signaling. The major method of intercellular transduced into intracellular signaling chains communication employs messenger that control many of the biochemical activities substances (hormones) that are secreted by of a cell and can also trigger the formation of signal-producing cells and registered by target further extracellular signals. cells. All cells produce and receive multiple,

& Intercellular signaling: — Communication between cells. Intracellular signaling: — Signaling chains within the cell, responding to extracellular and intracellular stimuli.

Signals generated during intercellular communication must be received and processed in the target cells to trigger the many intracellular biochemical reactions that underlie the various physiological functions of an organism. Typically, a large number of steps is involved in the processing of the signal within the cell, which is broadly described as intracellular signaling. Signal transduction within the target cell must be coordinated, fine-tuned and channeled within a network of intracellular signaling paths that finally trigger distinct biochemical reactions and thus determine the specific functions of a cell. Importantly, both intercellular and intracellular signaling are subjected to regulatory mechanism that allow the coordination of cellular functions in a developmental and tissue-specific manner. 1.2 Intercellular Signaling 3

1.2 Intercellular Signaling

Intercellular signal transduction inﬂuences nearly every physiological reaction. It ensures that all cells of a particular type receive and transform a signal. In this manner, cells of the same type react synchronously to a signal. A further function of intercellular communication is the coordination of metabolite ﬂuxes between cells of various tissues. In higher organisms, intercellular signaling pathways have the important task of coordinating and regulating cell division. The pathways ensure that cells divide synchronously and, if necessary, arrest cell division and enter a resting state.

& Intercellular signaling: — Processes sensory information — Controls Metabolic fluxes Cell division Growth Differentiation Development.

Cellular communication assumes great importance in the differentiation and development of an organism. The development of an organism is based on genetic programs that always utilize inter- and intracellular signaling pathways. Signal molecules produced by one cell inﬂuence and change the function and morphology of other cells in the organism. Intercellular signaling pathways are also critical for the processing of sensory information. External stimuli, such as optical and acoustic signals, stress, gradients of nutrients, and so on, are registered in sensory cells and are transmitted to other cells of the organism via intercellular signaling pathways.

1.2.1 Tools for Intercellular Signaling

Various forms of communication between cells are currently known (Figure 1.2):

Extracellular messengers: Cells send out signals in the form of speciﬁc messenger molecules that the target cell transmits into a biochemical reaction. Signaling cells can simultaneously inﬂuence many cells by messenger molecules so as to enable a temporally coordinated reaction in an organism. Gap junctions: Communication between bordering cells is possible via direct contact in the form of “gap junctions.” Gap junctions are channels that connect two neighboring cells to allow a direct exchange of metabolites and signaling molecules between the cells. 4 1 Basics of Cell Signaling

Figure 1.2 The principal mechanisms between cells. Gap junctions are coated by of intercellular communication. proteins (shown as circles in the figure) that (a) Communication via intercellular can have a regulatory influence on the messengers; (b) Communication via gap transport; (c) Communication via surface junctions, which provide direct connections proteins.

Cell–cell interaction via cell-surface proteins: Another form of direct communication between cells occurs with the help of surface proteins. In this process, a cell-surface protein of one cell binds a speciﬁc complementary protein on another cell. As a consequence of the complex formation, an intracellular signal chain is activated which initiates speciﬁc biochemical reactions in the participating cells. Communication is then only possible upon direct contact between the target cell and the surface protein of the partner cell. Electrical signaling: A further intercellular communication mechanism relies on electrical processes. The conduction of electrical impulses by nerve cells is based on changes in the membrane potential. The nerve cell uses these changes to 1.2 Intercellular Signaling 5

communicate with other cells at specialized nerve endings, the synapses. It is central to this type of intercellular communication that electrical signals can be transformed into chemical signals. This type of communication will not be discussed in this book.

& Cells communicate via: — Messenger substances — Gap junctions — Surface proteins — Electrical signals.

In the following, the main emphasis will be on the intercellular communication via extracellular messengers, the hormones.

1.2.2 Steps of Intercellular Signaling

In the communication between cells of an organism, the signals (messengers such as hormones) are produced in specialized cells. The signal-producing function of these cells is itself regulated, so that the signal is only produced upon a particular stimulus. In this way, signaling pathways can be coupled to one another and coordinated. The following steps are involved in intercellular communication (Figure 1.3).

1.2.2.1 Formation of a Signal in the Signal-Producing Cell as a Result of an External Trigger Most extracellular messengers are produced in response to external triggers and are released by exocytosis. Physical stimuli such as electrical signals, changes in ion concentration or, most frequently, other extracellular signaling molecules, serve as a trigger to increase the amount of the messenger available for extracellular communication. The mechanisms by which the external trigger signals increase the amount of extracellular messenger are diverse, and include stimulation of the biosynthesis of the messenger, an increased production of the mature messenger from precursors, and the release of the messenger from a stored form. The latter mechanism is used extensively in the release of hormones of the neural system (neurotransmitters) in response to electrical signals for example, at synapses.

& Steps of intercellular signaling: 1) Trigger signal induces release of stored messenger or stimulates its biosynthesis 2) Transport to target cell 3) Receipt of signal by the target cell 4) Conversion of signal into intracellular signal chain in the target cell. 6 1 Basics of Cell Signaling

Figure 1.3 The individual steps of intercellular the target cell, where the signal is registered, communication. On receipt of a triggering transmitted further, and finally converted into a stimulus, the signal is transformed into a biochemical reaction. Processes of termination chemical messenger within the signaling cell. or the regulation of communication, which can The messenger is secreted and transported to act at any of the above steps, are not shown.

1.2.2.2 Transport of the Signal to the Target Cell The extracellular signal produced may be distributed via the circulation, or it may reach the target cell simply by diffusion. In long-range signaling via the circulation, the extracellular messenger is often bound to speciﬁc carrier proteins or incorporated into larger protein complexes. Furthermore, the processing or metabolism of a messenger during transport may convert it from an inactive form to an active form.

1.2.2.3 Registration of the Signal in the Target Cell A target cell that receives a signal within the framework of intercellular communication transmits the signal in intracellular pathways that trigger distinct 1.2 Intercellular Signaling 7 biochemical activities in a cell type-speciﬁc manner and determine the response of the target cell. Specialized proteins, termed receptors, are utilized for the reception of signals in the target cell. Only those cells that carry the appropriate receptor will be activated for further transduction of the signal into the interior of the cell. The reception of the signals by the receptor is equivalent to the binding of messenger substance on the receptor or the transmission of physical stimuli into a structural change in the receptor which activates the receptor for further signal transduction. There are two principal ways by which target cells can process incoming signals:

Cell-surface receptors receive the signal (e.g., a messenger substance) at the outside of the cell, become activated, and initiate signaling events in the interior of the cell. In such signaling pathways, the membrane-bound receptor transduces the signal at the cell membrane so that it is not necessary for the signal to actually enter the cell. The messenger enters into the target cell and binds and activates the receptor localized in the cytosol or nucleus.

1.2.3 Regulation of Intercellular Signaling

The result of communication between the signaling and receiving cells is the triggering of multiple biochemical reactions in the target cell. The nature and extent of these reactions depends on many individual reactions that participate either directly or indirectly in signal transduction.

& Hormone signaling is mainly regulated via: — External trigger signals — Feedback loops — Degradation — Modification — Amount of receptors — Activity of receptor.

Beginning with the hormone-producing cell, the following processes are all contributing factors for hormonal signal transduction in higher organisms (Figure 1.3):

Biosynthesis of the hormone: The enzymes involved in biosynthesis of a hormone can, for example, be controlled by other signal transduction pathways. Often, feedback mechanisms exist that couple the activity of the biosynthetic enzymes to the concentration of the circulating hormone. Degradation and modiﬁcation of the hormone: The active hormone may be inactivated by the metabolism, or inactive hormone precursors may be converted into the active hormone by enzymatic transformation. 8 1 Basics of Cell Signaling

þ Storage and secretion of the hormone: There are signals (electrical signals, Ca2 - signals) to trigger the secretion of stored hormones. Transport of the hormone to the target cell: The distribution of a hormone via the circulation contributes to the accessibility of that hormone at a particular location of an organism. Reception of the signal by the hormone receptor: The hormone receptors are primarily responsible for the registration of the signal and the further transduction of the signal in intracellular signaling paths. Therefore, the amount, speciﬁcity, and activity of receptors at a target cell is subjected to multiple regulatory mechanisms. For example, the receptor may be downregulated in response to the amount of circulating hormone, or intracellular signaling paths may control receptor activity from inside the cell (see Section 6.2.4).

All of the above steps are subjected to regulation. A precise control of these steps is at the heart of all developmental programs, and most of the information available on the control of intercellular communication has been gained from developmental studies and from the failure of the control mechanisms, either artiﬁcially induced or inborn. The mechanisms for the control of hormone and receptor concentration are mostly based on feedback regulation. Negative and/or positive feedback loops (see Section 3.3.2) are used to adjust the intercellular communication to the development and function of the whole organism. The feedback controls operate mainly at the level of the enzymes involved in hormone biosynthesis, storage or degradation, and via the amount of receptor available for conversion of the extracellular signal into an intracellular response.

1.3 Hormones in Intercellular Signaling

Signaling molecules for the communication between cells are known as hormones, while hormones that are proteins and regulate cell proliferation are known as growth factors. Hormones are either produced in specialized hormone-producing cells, or they may be introduced into the organism as inactive precursors (e.g., vitamins) that require metabolic activation to generate the active form. Examples of the latter type include vitamin D and retinoic acid. Typically, the hormone- producing cells contain biosynthetic pathways that are responsible for production of the hormone; furthermore, hormones may be speciﬁcally inactivated by modifying enzymes. Details on the metabolism of hormones are beyond the scope of this book.

1.3.1 The Chemical Nature of Hormones

The chemical nature of hormones is extremely variable. 1.3 Hormones in Intercellular Signaling 9

& Hormones can be: Amino acids and amino acid Derivatives of fatty acids derivatives Peptides Steroids Proteins Retinoids Nucleotides Small inorganic molecules, for example, NO

Examples of important hormones are listed in Tables 1.1–1.3.

Table 1.1 Examples for hormones that bind to nuclear receptors.

Hormone Biochemical and/or physiological function

Steroids Progesterone preparation of the uterus for implantation of the embryo, maintenance of early H3C pregnancy C=O

O Estradiol OH preparation of the uterus to receive the blastocyst, control of uterine contraction, generation of secretory system of breasts during pregnancy

HO Testosterone differentiation and growth of the male OH reproductive tract, stimulation of male secondary sex characteristics, skeletal muscle growth

O Cortisol metabolism of carbohydrates, lipids and ﬂ CH2OH proteins, antiin ammatory, immunsup- C=O pressive, induction of Tyr aminotransferase and Trp cyclooxygenase OH OH

O (continued) 10 1 Basics of Cell Signaling

Table 1.1 (Continued)

Hormone Biochemical and/or physiological function

AldosteroneCH2OH water and ion balance, backresorption of OH ions in the kidney C O O CH

Steroid-related hormones þ 1,25-Dihydroxycholecalciferol metabolism of Ca2 and phosphate, bone 2þ (from vitamin D3) mineralization, resorption of Ca and phosphate in the intestine

HO OH

Other hormones 0 3,5,3 -Triiodothyronine (T3 hormone) increased oxygen consumption and I I increased heat formation, stimulation of NH2 glycolysis and protein biosynthesis

HO O CH2 CH COOH

Retinoids All-trans-retinoic acid formed from all-trans-retinal, broad effect on differentiation and morphogenesis COOH

9-cis-retinoic acid

COOH 1.3 Hormones in Intercellular Signaling 11

Table 1.2 Examples of hormones that bind to TM receptors.

Hormone Biochemical and/or physiological function

Epinephrine raises blood pressure, contraction of smooth muscles, HO glycogen breakdown in liver, lipid breakdown in adipose tissue HO CH CH2 NH2

OH CH3 Norepinephrine contraction of arteries HO

HO CH CH2 NH3 OH Histamine relaxation of blood vessels CH2 CH2 NH3 N N H Derivatives of arachidonic acid Prostaglandin E2 contraction of smooth muscles O COOH

HO OH

1.3.2 Hormone Analogs: Agonists and Antagonists

The modiﬁcation of hormones can lead to compounds that are known as agonists or antagonists.

& Hormone antagonists: — Bind to a receptor and suppress signaling. Hormone agonists: — Bind to a receptor and trigger a physiological response.

1.3.2.1 Antagonists Hormone derivatives that bind to a receptor but suppress signal transduction are termed antagonists. Hormone antagonists find broad pharmaceutical and medical application since they specifically interfere with certain signal transduction pathways in the case of hormonal dysregulation. Antagonists with a much higher affinity for a receptor than the unmodified hormone are medically very interesting. 12 1 Basics of Cell Signaling

Table 1.3 Peptide hormones and protein hormones.

Hormone Biochemical and/or physiological function

Glucagon (polypeptide: 29 aa) glycogenolysis in liver, release of fatty acids from triglycerides in adipose tissue Insulin (polypeptide, a-chain 21 aa; stimulation of glucose uptake in muscle and b-chain 30 aa) adipose tissue, catabolism of carbohydrates, storage of triglycerides in adipose tissue, protein synthesis, cell proliferation; inhibition of glycogenolysis Gastrin (polypeptide: 17 aa) secretion of HCl and pepsin in stomach Secretin (polypeptide: 27 aa) stimulation of secretion of pancreatic proteases Adrenocorticotropin (polypeptide: 39 aa) biosynthesis in anterior pituitary, stimulation of formation of corticosteroids in adrenal cortex, release of fatty acids from adipose tissue Follicle-stimulating hormone (FSH) (poly- stimulation of growth of oocytes and follicle peptide: a-chain 92 aa; b-chain 118 aa)

Thyrotropic hormone (TSH) (polypeptide: release of thyroxine (T4 hormone) and of T3 in a-chain 92 aa; b-chain 112 aa) thyroid gland TSH releasing hormone (peptide: 3 aa) formation in hypothalamus, stimulates synthesis and release of TSH in anterior pituitary Vasopressin (peptide: 9 aa) formation in posterior pituitary, backresorption of water in the kidney, contraction of small blood vessels þ Parathyroid hormone (polypeptide: 84 aa) Formation in parathyroid gland, increase of Ca2 þ in the blood, mobilization of Ca2 from the bone

aa ¼ amino acids.

Such high-affinity antagonists require very low dosages in therapeutic applications. A few important antagonists and agonists of adrenaline are shown in Figure 1.4. Propranolol, which is an example of a medically important hormone antagonist, binds with an affinity that is three orders of magnitude greater than its physiological counterpart, adrenaline, on the b-adrenergic receptor, such that very effective blockade of the adrenaline receptor is possible. Some antagonists have been classified as “neutral” (see Section 7.3.2.2); these have no effect on signaling activity but can prevent other ligands from binding to the receptor.

1.3.2.2 Agonists Hormone analogs that bind specifically to a receptor and initiate the signal transduction pathway in the same manner as the genuine hormone are termed agonists. By their influence on the receptor, agonists have been classified further into subcategories (see also Section 7.3.2.2):

Full agonists are capable of maximal receptor stimulation. Partial agonists are unable to elicit full activity even at saturating concentrations. 1.3 Hormones in Intercellular Signaling 13

Figure 1.4 Structure of important agonists and antagonists of adrenaline, and their affinity for the b-adrenergic receptor.

Inverse agonists reduce the level of basal or constitutive activity below that of the unliganded receptor.

The ability of hormone derivatives to function as an agonist or antagonist may depend on the cell type under investigation. A notable example is the synthetic estrogen analog tamoxifen, which in some tissues functions as an agonist and in other tissues as an antagonist of the estrogen receptor (see Section 6.4.1).

1.3.3 Endocrine, Paracrine, and Autocrine Signaling

Various forms of intercellular communication by hormones can be discerned based on the range of the signal transmission (Figure 1.5). 14 1 Basics of Cell Signaling

Figure 1.5 (a) Endocrine signal transduction, hormone reaches the target cell, which is in which the hormone is formed in the found in close juxtaposition to the hormone- specialized endocrine tissue, released into the producing cell, via diffusion; (c) Autocrine extracellular medium, and transported via the signal transduction, in which the hormone acts circulatory system to the target cells; on the same cell type as that in which it is (b) Paracrine signal transduction, in which the produced.

1.3.3.1 Endocrine Signaling In endocrine signaling, the hormone messenger is synthesized in speciﬁc signaling (or endocrine) cells and exported via exocytosis into the extracellular medium (e.g., blood or lymphatic ﬂuid in animals). The hormone is then distributed throughout the entire body via the circulatory system so that remote regions of an organism can be reached. Only those cells or tissues that contain the appropriate receptor for the hormone elicit a hormonal response.

& Endocrine signaling: — Production of hormone in endocrine cells — Transport of hormone to target cell via circulation. 1.4 Intracellular Signaling: Basics 15

Paracrine signaling: — Hormone reaches target cell by diffusion — Close neighborhood of signaling cell and target cell. Autocrine signaling: — Hormone-producing cell and target cell are of the same cell type.

1.3.3.2 Paracrine Signaling Paracrine signal transduction occurs over medium range. The hormone reaches the target cells from the hormone-producing cell by passive diffusion. The producing cell must be found in the vicinity of the receiving cells for this type of communication. The signaling is rather local, and the participating signaling molecules are sometimes termed tissue hormones or local mediators. One special case of paracrine signal transduction is that of synaptic neurotransmission, in which a nerve cell communicates with either another nerve cell or with a muscle cell.

1.3.3.3 Autocrine Signaling In autocrine signaling, cells of the same type communicate with one another. The hormone produced by the signaling cell affects a cell of the same type by binding to receptors on these cells, initiating an intracellular signal cascade. If an autocrine hormone is secreted simultaneously by many cells, then a strong response is triggered. Autocrine mechanisms are of particular importance in the immune response.

1.3.4 Direct Protein Modification by Signaling Molecules

A special case of intercellular signaling is represented by a class of small, reactive signaling molecules, such as nitric oxide (NO; see Section 8.10). NO is synthesized in a cell in response to an external signal and is delivered to the extracellular ﬂuid. NO reaches neighboring cells either by diffusion or in a protein-bound form, and a modiﬁcation of the target enzymes ensues which results in a change in the activity of these enzymes. NO is characterized as a mediator that lacks a receptor in the classical sense.

1.4 Intracellular Signaling: Basics

External signals such as hormones, sensory signals or electrical signals are speciﬁcally recognized by receptors that transduce the external signal into an intracellular signaling chain. The intracellular signaling paths control all functions of the cell such as intermediary metabolism, cell division activity, morphology, and the transcription program. 16 1 Basics of Cell Signaling

1.4.1 Reception of External Signals

Cells employ two principal methods of transducing external signals into intracellular signaling paths. In the ﬁrst method, signal receipt and signal transduction occur at the cell membrane by transmembrane receptors that register the signal at the cell membrane. In a second method, the messenger passes the cell membrane and binds to the receptor that is localized in the cytosol or in the nucleus (see Section 1.5.1). Upon receiving a signal, the receptor becomes activated to transmit the signal further. The activated receptor passes the signal on to components (usually proteins) further downstream in the intracellular signaling pathway; these components then become activated themselves for further signal transmission. Depending on the nature of the external stimulus, distinct signaling paths are activated and a multitude of biochemical processes are triggered in the cell.

& Receipt of external signals occurs by: — Transmembrane receptors — Cytosolic or nuclear-localized receptors.

1.4.2 Activation and Deactivation of Signaling Proteins

Intracellular signal transduction occurs in network involving many signaling components that communicate with each other. The key functions in intracellular signaling are performed by proteins that have the ability to speciﬁcally recognize, process, and transduce signals. The major signal transducers are:

Receptors Signaling enzymes Regulatory GTPases.

In the absence of a signal, the signal transducers exist in an inactive or less-active ground state, but upon receipt of the signal the signal transducers become activated and transit into the active state. Only if they are in the active state is transmission of the signal further to the next signaling component possible. The active state is then terminated after some time by deactivation processes, and the signal transducer transited back into the inactive state from which it may start another round of activation and deactivation (Figure 1.6). A multitude of mechanisms are used to activate the signaling proteins, such as:

Binding of other signaling molecules Conformational transitions Covalent modiﬁcations Membrane targeting Compartmentalization. 1.4 Intracellular Signaling: Basics 17

Figure 1.6 Activation and deactivation of on to the next signaling component. signaling proteins. Activating signals trigger a Deactivating or regulatory signals limit the transition from the inactive ground state into lifetime of the activated state and induce a the active state, from which signals are passed return to the ground state.

These regulatory mechanisms and their functions in signaling organization and networking will be presented in more detail in Chapters 2 and 3. Following activation, the signaling protein must be deactivated in order to terminate or attenuate signaling. By restraining the lifetime of the activated state with the help of specific deactivation mechanisms, the signal flow can be controlled and fine-tuned, and it can also be coordinated with signaling through other signaling paths. The mechanisms for deactivation are variable.

& Mechanisms for the activation of signaling proteins: — Binding of activators (e.g., hormones) — Signal-induced conformational transitions — Covalent modifications — Membrane association — Removal of inhibitors.

& Mechanisms for the inactivation of signaling proteins: — Binding of inhibitors — Inhibitory modifications — Removal of activating modifications. 18 1 Basics of Cell Signaling

The deactivation mechanism may be intrinsic to the signaling protein and may be enhanced by specific accessory proteins (see GTPases; Chapters 7 and 11). Other deactivation mechanisms use signal-directed inhibitory modifications of the signaling protein, such as phosphorylation. The removal of activating modifications by specific enzyme systems represents another means of terminating signaling. The many methods of activating and inactivating signaling proteins are best illustrated with the example of protein kinases (see Chapter 9).

1.4.3 Processing of Multiple Signals

A signaling protein often must receive several signals simultaneously in order to become fully activated. The ability to process multiple input signals at the same time is based on the modular structureof signaling proteins, many of which are composed of several signaling domains each capable of recognizing a different signal (see Section 2.1). This property allows for the processing of different signals, the ﬁne-tuning and regulation of signaling, and for formation of large interaction networks (see Chapter 3).

1.4.4 Variability of Signaling Proteins

& Isoforms of signaling proteins: — Increase variability of signaling — Have similar, but not identical, signaling properties — Are encoded by specific genes — Arise by alternative splicing.

One striking feature of the signaling paths in higher vertebrates is their variability and multiplicity. Different cell types may harbor variants of signaling pathways that control different biochemical reactions. This variability is to a large part due to the existence of subtypes or isoforms of signaling proteins. Families of signaling proteins exist whose members have in common a core activity but differ in the details of substrate recognition and regulation. For nearly all signaling proteins, genes encoding the isoforms of a particular signaling protein are found in the genome. In addition, alternative splicing contributes a great deal to the occurrence of multiple forms of signaling proteins (see Section 5.1.2).

1.5 Molecular Tools for Intracellular Signaling

The main tools for intracellular signal transduction comprise the receptors, signaling enzymes, second messengers, and adapter or scaffolding proteins (Figure 1.7). The various signaling components cooperate to trigger speciﬁc biochemical activities that underlie the many physiological functions of an 1.5 Molecular Tools for Intracellular Signaling 19

Figure 1.7 Components of intracellular signal downstream effector is usually a membrane- transduction. The receipt of an extracellular associated process. The example shown in this signal by a membrane receptor (shown here as figure is only to be construed as an example for the binding of a hormone to its receptor) the composition of a generic signaling activates the receptor for further signal pathway. The structure of the intracellular transduction. The activated receptor R passes signaling pathways of a cell are highly variable. the signal onto downstream effector proteins, Some signal transduction pathways are much E. Adapter proteins may be involved in the simpler than that represented in the figure, but pathways between effector proteins. The others involve many more components and transduction of a signal from the receptor to its are much more complicated. organism. In the following subsections, only the basic properties of signaling components will be discussed. The cooperation of signaling molecules and the formation of signaling networks will be detailed in Chapters 2 and 3.

1.5.1 Receptors

1.5.1.1 Receptors Receive External Signals and Trigger Intracellular Signaling The first step in processing external signals involves receptors that specifically recognize the signal and initiate intracellular signaling. Signals in the form of hormones are usually produced by specialized cells and initiate a reaction in only a certain cell type. Only those cells that possess a cognate protein – the receptor of the hormone – can act as target cells. Hormone receptors specifically recognize and bind the cognate hormone based on their chemical nature. The binding of the hormone to the receptor in the target cell induces an intracellular cascade of reactions at the end of which lies a defined biochemical response. 20 1 Basics of Cell Signaling

Figure 1.8 Principles of signal transduction by binds the receptor either in the cytosol (R) or transmembrane receptors and nuclear nucleus (R0). Nuclear receptors act as nuclear receptors. Upper part: Transmembrane transcription factors that bind specific DNA receptors receive the signal on the cell surface elements (e.g., HRE: hormone-responsive and convert it into an intracellular signal that element) found in the promoter region of can be passed on until it reaches the nucleus. regulated genes to control their Lower part: In signal transduction via nuclear transcription rate. receptors, the hormone enters the cell and

In the same way, physical stimuli such as light or pressure can be registered only by those cells that possess the appropriate receptors. An example is rhodopsin in the vision process, where excitation of the receptor by the physical stimulus triggers a conformational change in the receptor that is used for further signal transduction. The receptors of the target cell can be divided into two classes: (i) membrane- bound receptors; and (ii) soluble cytoplasmic or nuclear-localized receptors (Figure 1.8).

1.5.1.2 Membrane-Bound Receptors Membrane-bound receptors represent the largest receptor class, and are actually transmembrane proteins, in that they display an extracellular domain linked to an intracellular domain by a transmembrane domain. All transmembrane receptors function as oligomers (dimers or higher oligomers) composed of identical or different subunits. The binding of a hormone to the extracellular side of the 1.5 Molecular Tools for Intracellular Signaling 21 receptor induces a speciﬁc reaction on the cytosolic domain, which then triggers further reactions in the target cell. The mechanisms of signal transmission over the membrane are diverse and will be discussed in more detail in Chapters 7, 10, 13, and 14. One characteristic of signal transduction via membrane-bound receptors is that the signaling molecule does not need to enter the target cell to activate the intracellular signal chain.

& Transmembrane receptors: — Receive signals at the extracellular side and transmit the signal into the cytosol — Structural parts: Extracellular domain Transmembrane domain Cytosolic domain.

1.5.1.3 Intracellular Receptors The most prominent class of intracellular or cytoplasmic receptors comprises the nuclear receptors that are found in the cytosol and/or in the nucleus (see Chapter 6). In order to activate the nuclear receptors, the hormone must penetrate the target cell by passive diffusion.

& Intracellular receptors: — Are nuclear- and/or cytoplasm-localized — Function as ligand-controlled transcriptional activators.

The nuclear receptors can be classiﬁed as ligand-controlled transcription activators. The hormone acts as the activating ligand, while the activated receptor stimulates the transcriptional activity of genes which carry DNA elements speciﬁc for the receptor.

1.5.1.4 The Interaction Between Hormone and Receptor Receptors are the specific binding partners for signaling molecules; the former are able to recognize and specifically bind the latter based on their chemical structure. Such binding and recognition are governed by the same principles and the same noncovalent interactions as those for the binding of a substrate to an enzyme, namely H-bonds, electrostatic interactions (including dipole–dipole interactions), van der Waals interactions, and hydrophobic interactions. In most cases, receptors bind their cognate signaling molecule with an affinity greater than that usually observed for an enzyme and substrate. The binding of a hormone to a receptor can in most cases be described by the simple reaction scheme:

½H½R ½þH ½ÐR ½HR ; with K ¼ D ½HR 22 1 Basics of Cell Signaling

where [H] is the concentration of free hormone, [R] is the concentration of the free receptor, and [HR] is the concentration of hormone–receptor complex. The value 6 for the equilibrium constant of dissociation, KD, usually lies in the range of 10 to 10 12 M. This simple formalism is applicable only to cytoplasmic receptors; for membrane-bound receptors a quantitative treatment of the binding equilibrium is much more difﬁcult.

& Hormone–receptor interaction: — Reversible complex formation due to noncovalent interactions — Regulatory input signal is the concentration of hormone.

A decisive factor for the intensity of signal transmission is the concentration of the hormone–receptor complex, as activation of the signal pathway requires this complex to be formed. The concentration of the complex depends on the concentration of the available hormone, the afﬁnity of the hormone for the receptor, and the concentration of the receptor. All three parameters represent – at least in principle – control points for signal transduction pathways. The variable signal – a change in which is registered to thereby activate a signal transmission – is in most cases the concentration of the freely circulating hormone. An increase in the concentration of freely circulating hormone, triggered by an external signal, leads to an increase in the concentration of the hormone–receptor complex, which in turn results in an increased activation of subsequent reactions in the cell. A major switch for the activation of an intracellular signaling pathway is therefore a signal-directed increase in the concentration of the freely circulating hormone ligand.

& Receptor signaling depends on: — Hormone concentration — Receptor concentration — Receptor activity and modification.

1.5.1.5 Regulation of Receptor Activity The activity of receptors is tightly regulated in order to adapt signaling to the intensity and duration of the extracelluar signals. In addition, regulatory mechanisms initiated by intracellular signaling pathways modulate the flow of information through the receptors. The modulation and regulation of signaling through receptors is achieved by multiple mechanisms. The major receptor controls operate at the level of receptor concentration and by receptor modification with concomitant changes in receptor affinity. The amount of receptor present on the cell surface may be regulated via receptor expression, by targeted degradation, and by internalization, all of which processes affect the intensity of the signal transduction on a long time scale. The regulatory receptor modifications are mostly found as phosphorylations introduced in response to signals originating from the same or other signaling pathways. 1.5 Molecular Tools for Intracellular Signaling 23

1.5.2 Signaling Enzymes

Signaling enzymes are at the heart of intracellular signaling. By modifying other enzymes or proteins, signaling enzymes will either carry the signal on or terminate signaling. Much like classical enzymes, signaling enzymes can be regulated by allosteric transitions in response to the binding of effector molecules, by covalent modifications such as phosphorylation, or by membrane targeting. These mechanisms serve to induce the transition of enzymes from an inactive or low active state into the active state, which makes enzymes the ideal instrument for the receipt and transmission of signals. The most prominent signaling enzymes are the protein kinases and protein phosphatases (Chapter 9) involved in the synthesis and degradation of second messengers (Chapter 8), and the regulatory GTPases (Chapters 7 and 11). The most important tool for control signaling processes is the covalent modification of signaling proteins. Nearly all signaling proteins of higher eukaryotes are modified by posttranslational modifications (PTMs), of which many are known (see Section 2.4). The covalent modification of signaling proteins regulates cellular signaling mainly in the following ways:

PTMs can directly regulate of the activity and function of a signaling protein. PTMs often provide attachment points for interaction modules located on partner proteins; in this way, multiprotein signaling complexes may form. PTMs may serve to regulate the subcellular distribution of signaling proteins.

The most frequently used tool for signal transmission in a cell is the reversible modification of proteins by phosphorylation or acetylation that serves to either activate or inactivate signaling proteins. The modification status of a protein is controlled by the activity of modifying enzymes (e.g., protein kinases) and demodifying enzymes (e.g., protein phosphatases) (see Chapter 9). Both classes of enzymes are elementary components of signaling pathways, and their activity is subject to manifold regulation. The importance of the protein kinases for cellular functions is illustrated by the large number (almost 500) of these enzymes encoded in the human genome (Chapter 9). An alternative and frequently used modification of signaling enzymes is ubiquitination, which serves primarily to reduce the amount of enzyme available (see Section 2.8). Details of the multitude of regulatory protein modifications, and their functions, are presented in Section 2.4.

& Signaling enzymes: — Activate or inactivate other signaling proteins — Receive and transmit signals — Produce low-molecular-weight messengers substances, the second messengers — Switch between active and inactive states. 24 1 Basics of Cell Signaling

The following general functions can be attributed to signaling enzymes:

Signaling enzymes catalyze the covalent modiﬁcation of signaling proteins to regulate their activity and subcellular location. Signaling enzymes catalyze the formation, degradation, or release of small molecule effectors, the second messengers. The enzymes involved in the formation or degradation of second messengers, such as the phospholipases or the adenylyl cyclases, are major components of signaling pathways (see Chapter 8) and are reversibly activated and inactivated during signal transduction. Regulatory GTPases switch between active and inactive conformations, depending on the binding of GDP or GTP. The regulatory GTPases (see Chapters 7 and 11) function as switches that can exist in an active, GTP-bound state or the inactive, GDP-bound state. In the active state, the GTPases can transmit signals to downstream components in the signaling chain. In the inactive state, signal transmission is repressed and an activating upstream signal in the form of exchange of bound GDP by GTP is required in order to activate the GTPase for further signal transmission.

1.5.3 Scaffolding Proteins

Scaffolding proteins do not harbor enzyme activities; rather, adapter proteins mediate signal transmission between the proteins of a signaling chain by bringing these proteins together, where they function as clamps to colocalize proteins for effective and speciﬁc signaling.

& Adapter proteins: — Do not carry enzyme activity — Provide docking sites for other signaling proteins — Help to organize multiprotein signaling complexes — Carry regulatory modifications.

Furthermore, adapter proteins help to target signaling proteins to specific subcellular sites and to recruit signaling molecules into multiprotein signaling complexes. In the latter case, the adapter proteins may function as a scaffold or docking site for assembling different signaling molecules at distinct sites; the proteins are then also termed docking or scaffolding proteins. Typically, scaffolding proteins contain several binding domains with distinct binding specificities for complementary sites on the target proteins. Furthermore, adapter proteins are often subjected to regulatory modifications, such as phosphorylations, that provide signal-directed docking sites for signaling proteins. The multiple functions of adapter proteins are presented in more detail in Section 3.1. 1.5 Molecular Tools for Intracellular Signaling 25

1.5.4 Diffusible Intracellular Messengers: Second Messengers

The intracellular activation of enzymes in a signaling chain can lead to the formation of diffusible small signaling molecules in the cell. These intracellular signaling molecules, termed “second messengers” (see Chapter 8), activate and recruit cognate enzymes for further signal transduction. The following properties are important for the function of diffusible intracellular messengers: Second messengers may be rapidly formed from precursors by enzymatic reactions. Typically, enzymes involved in the formation of second messengers are parts of signaling pathways and are activated during signaling to produce the second messenger in a regulated manner. Often, these enzymes have high turnover numbers and can form a large amount of second messenger, leading to high local concentrations. Second messengers may be rapidly released from intracellular stores.For þ example, the second messenger Ca2 is stored in specific compartments and is released from storage upon a regulatory signal. This mechanism provides for the fast and locally controlled production of the second messenger. Second messengers may be rapidly inactivated or stored in specific compartments. To allow for a termination of the second messenger function, the messengers are degraded by specific enzymes, or are removed by storage or transport into the extracellular medium (see Section 8.5). Second messengers may activate different effector proteins. Binding sites for a þ particular second messenger (Ca2 , cAMP) may occur on different signaling proteins. This property allows a given second messenger to regulate multiple target proteins, which leads to a diversification and variability of second messenger signaling. Second messengers allow the amplification of signals. The enzymatic production of large amounts of a messenger makes an important contribution to the amplification of signals.

& Second messengers: — May be formed and inactivated by enzymatic reactions — May be released from stores — Are cytosolic or membrane-localized — Activate signaling enzymes — Allow signal amplification — Are produced and become active in a timely and locally controlled way.

Currently, two types of second messengers are known:

Cytosolic messengers bind to target proteins in the course of signal transduction functioning as an effector that activates or modulates signaling through the 26 1 Basics of Cell Signaling

target protein. The most frequent targets of second messengers are the protein kinases. Membrane-associated messengers interact with their target protein at the inner side of the cell membrane; in this case the target proteins may also be membrane- associated, or the targets may be recruited to the membrane on binding the second messengers. 27

2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Proteins that function as signal transducers must be able to receive and convey signals in a highly specific and dynamic way. The incoming signals lead to the activation of the signaling protein such that it can pass the signal on to downstream targets, and in doing this signaling proteins will interact with upstream and downstream partners in a specific manner. Whereas, classical enzymes are designed to modify large numbers of substrate molecules, signaling proteins such as protein kinases generally affect small numbers of target molecules and have often separated their catalytic function from the binding regions for the upstream and downstream partners. A major tool for shaping the activity of signal transducers in time and space are posttranslational modifications (PTMs). Another distinguishing feature of signaling proteins is their modular construction that allows signaling proteins to interact with a variety of upstream and downstream partners in a regulated fashion.

2.1 Modular Structure of Signaling Proteins

Summary Signaling proteins are constructed from multiple signaling modules or domains that may act either independently or in cooperation. By function, signaling modules may be classified as catalytic modules, interaction or targeting modules, and regulatory modules. Furthermore, unstructured regions can be used for regulatory purposes. The modular construction allows signaling proteins to interact with multiple upstream and downstream partners in a regulated fashion, and this makes signaling proteins multivalent with respect to input and output signals. This also allows for a high versatility and flexibility in signaling. For most signaling modules, subtypes exist with distinct functional and regulatory properties.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 28 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

The multifunctionality of signaling proteins is based on their construction from multiple signaling modules or domains that may act independently or in cooperation, and serve distinct functions in signaling (Figure 2.1a). Several signaling modules are present in hundreds of copies in the human proteome, and these are used repeatedly to regulate distinct processes in a cell type-speciﬁc manner. The cell therefore uses a limited set of signaling modules which are joined together in diverse combinations, to direct the actions of regulatory systems. A vast number of module combinations are found in the proteome, and module or domain shufﬂing is assumed to be a major source of the evolutionary innovation in signaling behavior. The many signaling modules include interaction domains for downstream and upstream partners, sites for PTM and catalytic domains, and these modules may be used to compartmentalize molecular components, and to activate enzymes and direct them to their targets. Based on their function(s), signaling modules may be allocated to several classes, as described in the following subsections.

2.1.1.1 Catalytic Domains The catalytic domains directly transmit signaling information, for example, through phosphorylation. In the absence of the signal, the activity of the catalytic domain is low due to, for example, autoinhibition, inhibitory modiﬁcations, or the binding of inhibitors. Activation then occurs in a signal-directed fashion via the relief of any inhibitory constraints.

2.1.1.2 Targeting and Interaction Domains Signaling molecules must be targeted to their upstream and downstream partners in order to be able to receive and transmit the signal. To this end, distinct domains mediate interaction with substrates, other signaling proteins, and membranes. Often, PTMs are recognized by interaction domains in processes of substrate recruitment, in the assembly of larger signaling complexes, and in subcellular compartmentalization. Of outstanding importance here is the targeting of signaling proteins to the inner side of the cell membrane as many signaling events occur in close association with the cell membrane, and speciﬁc membrane targeting domains are used to bring signaling proteins to the membrane (see Sections 2.9 and 3.2.3).

& Typical domains of signaling proteins: — Catalytic domains — Regulatory domains — Protein interaction domains — Membrane-targeting domains.

2.1.1.3 Regulatory Domains The receipt of regulatory signals is often mediated by domains that have a positive or negative inﬂuence on the enzymatic domain. The regulatory domains may bind 2.1 Modular Structure of Signaling Proteins 29

Figure 2.1 (a) Modular structure and function (b) Functional variability of modular signaling of signaling proteins. The major functional proteins. Modular signaling proteins typically modules of signaling proteins are shown in harbor many PTMs and undergo multiple arbitrary sequence. The modules receive interactions with upstream and downstream multiple signals from upstream effectors, partners. As a result, multiple inputs and transmit multiple signals to downstream outputs shape signal transmission, and the targets, and communicate with each other by differential use of modules allows for cell type- allosteric interactions (blue arrows). Major specific signaling. A further diversification of tools for storing and conveying signaling signaling is based on the existence of signaling information are the posttranslational protein subtypes that may be expressed in a modifications (PTMs) that are attached in a cell type-specific manner and differ slightly in reversible way to the signaling protein; signaling properties. 30 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

low- or high-molecular-weight regulators to control the conformation and activity of the catalytic domain by allosteric mechanisms.

2.1.1.4 Unstructured, Flexible Sections An important mechanism for storing and conveying signaling information uses unstructured or loosely structured regions of proteins (for reviews, see Refs [1,2]). The unstructured parts are often found on the N- or C-termini of signaling proteins, or they may connect structured domains forming ﬂexible hinges. The signaling function of unstructured sections can be based on two mechanisms:

The binding of an effector or substrate to other parts of the protein can induce a transition from an unstructured state to a structured one which may be essential for allosteric activation of the signaling protein. Unstructured sections of signaling proteins often harbor multiple sites for PTMs that are introduced in a signal-directed fashion and may direct the assembly of distinct signaling complexes. An outstanding example is the tumor suppressor protein p53 (see Chapter 16) that harbors a large number of sites for PTM on its unstructured N- and C-terminal parts.

An important function of signaling domains is their ability to assemble functional signaling complexes. Thereby, the signaling cassettes of the signaling protein cooperate in promoting the assembly of larger signaling complexes in a regulated and location-speciﬁc manner. For example, the Tyr-modiﬁcation sites of receptor tyrosine kinases serve as attachment points for interaction modules of downstream effector proteins, which in turn may be phosphorylated by the kinase activity of the receptor for further signal transduction and for the association of further signaling proteins (see Chapter 10; Figure 10.11). The construction from multiple signaling modules endows signaling proteins with the following characteristics (see Figure 2.1b).

2.1.1.5 Multivalency The presence of multiple modules makes signaling proteins multivalent with respect to interaction partners, regulatory inputs and subcellular localization, and it allows for the association of large signaling complexes. For instance, the platelet- derived growth factor (PDGF) receptor harbors multiple Tyr-phosphorylation sites that direct the attachment of distinct downstream effectors (see Chapter 10; Figure 10.11).

& Modules in signaling proteins: — Can be differentially used in a time- and location-specific manner — Allow for multiple inputs, regulatory influences and outputs — Provide for multivalency. 2.2 Modular Signaling Complexes 31

2.1.1.6 Differential Use of Modules Modules in signaling proteins may be used simultaneously, in sequential order or in distinct subcellular locations only, allowing for a high versatility and flexibility in signaling. Often, signaling proteins go through cycles of function. In doing this, the modules of the signaling protein may be used in a differential fashion, and the use of one module or modification may influence the use of other modules or modifications within the same protein. Furthermore, modules may be engaged in a cell type-specific and tissue-specific manner, a feature that is central to cell type- and tissue-specific signaling.

2.1.1.7 Multiple Inputs, Regulatory Influences, and Outputs The construction from multiple modules allows signaling proteins to receive multiple signals and to respond to multiple controlling inﬂuences. These inputs may be integrated and converted into differential outputs, depending on the cellular environment.

2.1.1.8 Subtypes of Signaling Modules The functional multiplicity of signaling modules is strongly increased by the presence of genome-encoded subtypes or by splice variants. Most modules carry a core functional ability that is ﬁne-tuned in module variants that show slightly different enzymatic activities, different regulatory properties, or a different binding selectivity. Typically, a protein interaction domain recognizes a core determinant, with ﬂanking or noncontiguous residues providing additional contacts and an element of selectivity. For example, more than 100 different SH2 domains are known that bind to Tyr-phosphates on target proteins. Each of these variants recognizes a Tyr-phosphate on the target site, but with different requirements for the neighboring sequences.

2.2 Modular Signaling Complexes

Summary Most signaling events are mediated by multiprotein complexes that are of a modular structure comprising different signaling proteins. The recruitment of multiple signaling components into signaling complexes occurs signal-directed and is mediated by PTMs and interaction domains present on the complex subunits. The use of signaling complexes allows for the efficient and rapid transmission of signals, and also provides for high variability as signaling proteins may associate in a dynamic manner.

Signaling proteins often assemble in signaling complexes that form in response to signal input in dynamic way. A large number of proteins may participate in the formation of the signaling complexes. 32 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

& Signaling complexes: — Regulate major cellular functions — Harbor multiple signaling proteins — Are of a modular structure — Allow for coordination of multiple regulatory influences.

For example, the N-methyl-D-aspartate (NMDA)-type glutamate receptor has been reported to associate with more than 50 different signaling proteins. However, which of these many proteins will be bound to the receptor at any given time point will depend on the modification status of receptor and its interaction partner, on the subcellular location, and on the cell type. By the reiterated use of interaction domains, complex machines are built that regulate for example, targeted proteolysis, endocytosis, protein- and vesicle trafficking, cell polarity, cell division, and gene expression. The large complexes allow an efficient and rapid transmission of signals from one signaling component to the other. The interactions involved are illustrated in Figure 2.2, based on the example of the insulin receptor signaling complex. The organization of signaling proteins in signaling complexes has several distinct advantages, as follows.

Figure 2.2 Insulin signaling complexes. residues on IRS-1 serve to assemble the PI3 Binding of insulin (Ins) to the extracellular kinase into the signaling complex. PI3 kinase subunit of the insulin receptor (InsR) triggers becomes activated and synthesizes the second autophosphorylation at tyrosine residues on messenger PtdInsP3 that mediates the the cytoplasmic part of the receptor. The P-Tyr membrane recruitment and activation of two residues serve as attachment points for the further protein kinases, the Akt kinase and PTB domain of the adapter protein IRS-1 that PDK1 (see Section 9.4). also becomes Tyr-phosphorylated. The P-Tyr 2.2 Modular Signaling Complexes 33

2.2.1.1 Specificity Within a signaling complex, signals can be transduced from one component to the other in a highly efﬁcient way, leading to the generation of robust signals. Signal transduction within a complex is rapid because it does not require diffusion of the reactants. The assembly of several components of a signaling path into a multiprotein complex ensures a tight and speciﬁc coupling of the various reactions and prevents any unwanted dissipation of the signal and side-reactions (see e.g., Function of scaffolds in signaling; Section 3.1).

& Signaling complexes: — Provide for specificity — Provide for variability — Assemble in a signal-directed way — Allow tight regulation.

2.2.1.2 Signal-Directed Assembly The recruitment of signaling proteins into larger complexes is often mediated by PTM of one or more of its components. As these modiﬁcations may be introduced in a signal-directed fashion, the formation and composition of signaling complexes may be triggered in a signal-directed manner (see Figure 2.2).

2.2.1.3 Variability Signaling components of a signaling complex may be replaced by isoforms that differ in the details of regulation and activity. Such an exchange of signaling components can lead to distinct changes in the output signal. The exchange of components in regulatory complexes appears to be used intensively in gene regulatory complexes (e.g., Mediator complexes; see Section 4.2.6) that may associate distinct coactivators, corepressors or chromatin remodeling enzymes, depending on the input signals to the system.

2.2.1.4 Regulation The components of signaling complexes are often themselves of a modular structure which allows the receipt of multiple input and regulatory signals in a sequential order, or at the same time. Due to the close proximity of the signaling components, regulatory signals can become effective in a rapid and efﬁcient manner. For example, signaling complexes are known that contain both protein kinase and protein phosphatase activity. The presence of two opposing enzyme activities within the same complex is an important tool for the downregulation and termination of signaling events. 34 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.3 Regulation of Signaling Enzymes by Effector Binding

Summary The activity of signaling enzymes is controlled in a signal-directed way through allosteric mechanisms that become active in the modular-constructed enzymes. The binding of upstream effectors and/or the attachment of PTMs serves to regulate the enzymatic activity in response to input signals. Effectors may comprise second messengers, inhibitor or activator proteins, targeting proteins, and metal ions.

Typical signaling enzymes are of a modular structure with a catalytic domain coupled to regulatory and targeting domains. The catalytic activity is strictly regulated by a combination of various mechanisms, such that an incoming signal triggers activation of the catalytic activity and signal propagation. Among the many regulatory inﬂuences, regulation by effector binding and regulation by PTMs are the most dominant. Both of these systems involve allosteric conformational changes of the catalytic domain that follow similar basic principles as applied to classical enzymes.

& Signaling enzymes are regulated: — by effectors; — by PTMs; or — via allosteric mechanisms.

2.3.1.1 Allostery in Signaling Enzymes The term allostery, as applied to an enzyme, means that the enzyme can exist in various conformations which differ in activity and substrate or ligand binding. An incoming, activating signal will shift the conformational state towards a more active one, whereas inhibitory signals will lead to a preferential population of the low- activity state. In contrast to what is suggested by the linear representation of modular signaling enzymes in Figure 2.1, the modules are assumed to be densely packed one to another to form a compact signaling entity with many allosteric interactions. Due to the ﬂexible linkage of the modules, many different ways exist by which the modules can inﬂuence the activity of the catalytic domain. For example, an incoming signal may change the packing of the modules due to the interaction of one module with a complementary site on a target protein, or by effector binding (e.g., Src kinase; Section 10.3.2). The rearrangement induced in this way may stabilize an active conformation of the catalytic domain, allowing further signal transmission. 2.3 Regulation of Signaling Enzymes by Effector Binding 35

Of the two major mechanisms employed for the regulation of signaling enzymes – effector binding and PTM – the regulation by effectors will be discussed first. The vast field of PTMs will be detailed in Sections 2.4–2.9. Regulation of the activity of enzymes by the binding of effector molecules is a ubiquitous and general principle for the fine-tuning and control of metabolic activity and other physiological functions. Effector molecules are often low- molecular-weight organic compounds.

& Effectors of signaling enzymes include: — Low-molecular-weight compounds: second messengers, metabolites — Proteins: activators, inhibitors — Metal ions.

Proteins and metal ions can also exercise the function of effectors. The latter molecules bind speciﬁcally to distinct interaction modules or to small target sequences of the signaling enzymes; such binding will result in the inhibition or stimulation of enzymatic activity, or in changes of the subcellular localization.

2.3.1.2 Low-Molecular-Weight Effectors For the regulation of metabolic pathways, metabolites are often used which are a product of that pathway. The basic strategy for the regulation is exemplified in the feedback mechanisms employed in the biosynthetic and degradation pathways of amino acids, purines, and pyrimidines, as well as in glycolysis. In most cases a metabolite (or similar molecule) of the pathway is utilized as the effector for the activation or inhibition of enzymes in that pathway. In many signal transduction pathways, second messengers function as effectors that, by binding to target proteins (see Chapter 8), regulate the flow of information through the pathways. Well-characterized examples include the cyclic nucleotides cAMP and cGMP that function as allosteric regulators of protein kinases (see Chapter 9). Enzyme-specific inhibitor and activator proteins represent other major types of effector molecule.

2.3.1.3 Inhibitor Proteins There are numerous examples of inhibitor proteins that speciﬁcally bind a particular enzyme so as to block its activity.

& Major mechanisms of enzyme inhibitors: — Competition with substrate binding — Deformation of active site and substrate binding site.

Inhibitors can employ a variety of mechanisms to control enzyme activity:

Binding to the substrate binding site. Inhibitors may be related structurally to the substrate without possessing the chemical groups that are necessary for being 36 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

turned over. Because of the structural similarity of the substrate, these inhibitors can bind speciﬁcally to the substrate binding site and compete with the substrate for the enzyme. Well-studied examples include the protease inhibitors (see textbooks for details). Deformation of the active site and/or the substrate binding site. Binding of the protein inhibitor to the enzyme may alter the orientation of the catalytic center in a way that does not allow efﬁcient catalysis and/or strong binding of the substrate. Examples are the inhibitors of the cyclin-dependent protein kinases (CDKs) of the cell cycle (see Section 15.2.4).

Inhibitor proteins themselves are subject to a variety of regulation mechanisms. The function of an inhibitor protein can be regulated, for example, by protein phosphorylation (see Section 9.7.2), by degradation, or by de novo synthesis (e.g., CDK inhibitors; Section 15.2.3).

2.3.1.4 Activator Proteins Examples of the reversible association of activator proteins with an enzyme are the þ Ca2 -calmodulin-dependent enzymes (see Sections 8.7.1 and 8.5); other examples of activating proteins are the cyclins (see Section 15.2.2). Activator proteins themselves can be bound in regulatory networks, as shown in the example of the cyclins. The function of an activator protein can be regulated at the level of gene expression, degradation, or PTM (e.g., phosphorylation; Section 15.3.1).

2.3.1.5 Metal Ions Of primary importance is Ca2, the availability of which is a key control element in þ cellular regulation (see Chapter 8). The regulatory function of Ca2 is based on the þ presence of distinct Ca2 -binding modules in signaling proteins. Examples include protein kinase C (PKC; Section 9.5) and phospolipase C (PLC; Section 7.7.2). PKC þ harbors a Ca2 -binding domain that promotes the membrane recruitment of PKC þ in the presence of appropriate Ca2 levels.

2.4 Posttranslational Modifications (PTMs) in Cellular Signaling

Summary PTM by the covalent attachment of chemical groups to the side chains of amino acids represents a major mechanism by which protein function is regulated in eukaryotes. PTMs may be categorized as either stable or transient. Transients PTMs can be considered as signals that are introduced and removed by modifying enzymes to regulate the activity of signaling proteins, and protein– protein and protein–membrane interactions. In addition to regulating enzyme activity directly, PTMs are recognized by interaction domains located on 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 37

upstream or downstream protein partners. Interaction domains bind to PTMs in a specific fashion to direct the recruitment of substrates, upstream- or downstream-effectors, and the formation of signaling complexes. For most interaction domains, multiple subtypes are known with varying specificity for a particular modification, and its neighboring sequence which largely expands the versatility of PTM-interaction domain recognition.

In cell signaling, reversible regulatory modifications of signaling proteins play a key role in creating, transducing, and fine-tuning signals. In addition, protein modification is at the heart of the transcription program of the cell. Most eukaryotic proteins are modified posttranslationally in one or another form, and over 200 protein modifications have been identified [3].

2.4.1 Chemical Nature of PTMs

PTMs may be divided into two classes:

Stable PTMs, such as disulﬁde formation, glycosylation, lipidation (Section 2.9) and biotinylation, are present in the target proteins for a longer time and are not subject to rapid turnover. These modiﬁcations are often essential for vital functions of mature proteins, such as compartmentalization, transport, and secretion. Transient PTMs are of a dynamic nature and are introduced into proteins for regulatory purposes, above all.

& Classes of PTMs: — Stable PTMs, for example, lipid anchors — Transient PTMs: Introduced by “writers” Removed by “erasers.”

Many different PTMs may be deposited on a target protein in a signal-directed fashion to store signaling information, and the dynamic and combinatorial use of PTMs allows cells to produce a vast spectrum of different biological responses. Thus, the PTMs can be considered as signals that are transmitted to the target protein by modifying enzymes. A crucial issue in cellular regulation is to deﬁne how these signals are interpreted, and how they are integrated into cellular signaling paths and networks. Such modiﬁcation signals control the activity, macromolecular assembly and location of signaling proteins, and as such are major tools for shaping and linking signaling pathways. 38 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

& Regulatory modifications of signaling proteins: — Are introduced in a signal-controlled way — May induce activity changes by allosteric mechanisms — Are recognized by interaction domains — May provide attachments points for target proteins — Are of a dynamic nature — Are used in a combinatorial fashion.

Based on their function, the regulatory modiﬁcations may be divided into two categories, as follows.

2.4.1.1 Allosteric and Conformational Functions of PTMs PTMs can serve to modulate and regulate the activity of the signaling protein by conformational and allosteric mechanisms. As an example, the phosphorylation of protein kinases in the activation loop increases their activity by stabilizing an active conformation (see Section 9.2.3). However, this is a more specialized use of PTMs, because it involves elaborate interactions and conﬁgurations of the signaling protein and this cannot be used easily in a combinatorial manner in cellular signaling.

2.4.1.2 Recognition of PTMs by Interaction Domains A general function of PTMs is to serve as attachment points for the binding of upstream or downstream effectors in signaling pathways, and for the assembly of larger protein complexes. The PTMs are recognized by interaction modules or domains on partner proteins, with different domains being dedicated to the selective recognition of distinct PTMs. By this strategy, the interaction domains serve as a broad device to decode the functional state of a protein during cellular signal transduction.

2.4.1.3 Dynamic Nature of PTMs An important aspect of regulatory protein modifications is their reversibility. To serve as a regulatory tool, the modifications are introduced in a signal-directed way by modifying enzymes functioning as “writers,” while they are removed upon demand by proteins functioning as “erasers” (Figure 2.3a). Typically, the formation and removal of the adducts by “writers” and “erasers” occurs in a signal-directed manner. The enzymes responsible for introducing and removing the modifications are therefore also essential elements of signaling paths.

2.4.1.4 Examples of Regulatory PTMs The most important regulatory protein modiﬁcations are:

Ser/Thr phosphorylation (Section 2.5) Tyr Phosphorylation (Section 2.5; Chapter 9) Lysine acetylation (Section 2.6) 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 39

Figure 2.3 (a) Writers, erasers and readers of their respective ligands (red). The structures PTMs. Modifying enzymes (“writers”) attach were obtained from the Protein Data Bank PTMs to target proteins, while demodifying (accession codes 1JYR, 1Q3L, 1E6I and 1LM8 enzymes (“erasers”) remove the PTMs. The for parts i---iv, respectively). SAM, S-adenosyl- information stored as a PTM is “read” by methionine; SAH, S-adenosyl-homocysteine; interaction domains on upstream or Shc, Grb2 (Section 10.5.7); HP1, downstream effector proteins; (b) Examples chromodomain (Section 4.5.3.1); GCN5, (i---iv) of modifications of proteins by PTMs bromodomain (Section 4.5.2.1); ubiquitin and structures of interaction domains with (Section 2.8); Vps27, a protein required for their respective ligand. The “readers” and endosomal sorting of proteins, contains a “erasers” of the PTMs are indicated. The ubiquitin interaction motif (UIM; Section structures at far right show examples of 2.8.5.2). protein-interaction domains in complex with

Lysine, arginine methylation (Section 2.7.2) ADP-ribosylation (Section 7.5.2) Proline hydroxylation Cysteine oxidation (Section 10.4.3) Cysteine nitrosylation (Section 8.10) Ubiquitination, neddylation, and sumoylation (Section 2.8).

The modifications may be of small size, as for example phosphate or methyl groups, or they may comprise complete small proteins such as ubiquitin. In most cases, specific modifying and demodifying enzymes exist that introduce or remove the modification in a regulated manner.

& Major protein modifications: — Ser, Thr, Tyr phosphorylation — Lys, Arg methylation — Lys acetylation — Cys oxidation — Cys nitrosylation — Lys ubiquitination — Lys sumoylation. 40 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.3 (Continued) 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 41

Some modifications as for example, nitrosylation or cysteine oxidation do not require specific enzymes for the transfer of these groups to the target enzyme. Here, the intrinsic chemical reactivity of the modifying group is often the major determinant for formation of the covalent adduct. However, specific accessory proteins may be necessary to direct the modification to distinct target sites in these cases. The structures of the major PTMs and their cognate interaction domains in complex with the respective ligand are shown in Figure 2.3b. In the following, some general principles of PTM function and recognition will be summarized. The major modifications of signaling enzymes, namely phosphorylation, acetylation, ubiquitination and lipidation, are detailed separately in Sections 2.5–2.9.

2.4.2 Recognition of Protein Modifications by Modification-Specific Interaction Domains

A major function of protein modifications is to provide attachment points for upstream or downstream effector proteins in signaling pathways, or to guide the assembly of large protein complexes. The protein partners recognize the PTMs via interaction domains that are able to specifically detect and bind a particular modification. The human genome harbors many classes of interaction domains, and these are used as cassettes in a combinatorial fashion to recognize a large number of different PTMS. Each domain class is found on tens to hundreds of signaling proteins, which allows for a huge diversification of signaling properties.

& Modification-specific interaction domains: — Are often found as independently folding domains — Exist as multiple subtypes — Bind to distinct protein modifications.

Interaction domains often recognize short peptide motifs that are embedded in target proteins, but do not bind stably until the peptide has acquired an appropriate PTM. Such domains usually recognize the chemical nature of the PTM via a conserved binding pocket for the modified residue. A more variable surface then selectively engages the flanking amino acids, and this allows the interaction domains to distinguish between different peptide motifs with the same PTM. Importantly, subtypes exist for most classes of interaction domains that share the same selectivity for the modified residue but differ in their requirements for neighboring amino acids, a property that serves to create a high diversity of interaction domains. Families of interaction domains can be identified that require the same chemical modification but recognize different neighboring sequences. For example, more than 100 SH2 domains recognizing P-Tyr residues are encoded by the human genome, each of them differing in the details of the sequence requirements of the neighboring amino acids. 42 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.4.3 Multisite Protein Modification

Signaling proteins in higher eukaryotes only rarely carry just a single modification. Indeed, many proteins involved in cellular regulation are modified at multiple sites, a phenomenon referred to as multisite modification. The multiplicity of modification sites on a protein often correlates with its biological importance and the complexity of the corresponding organism. Examples of such proteins include the Cdc25 phosphatases (Section 15.6), receptor tyrosine kinases (Chapter 10), PKC (Section 9.5), and the histones (Section 4.3.6). Furthermore, many transcriptional regulators of vertebrates are subjected to multisite modification. The complexity of multisite modifications is illustrated best by the example of the tumor suppressor protein p53, which functions as a transcription factor. The DNA-binding domain and the tetramerization domain of p53 are phosphorylated, acetylated, sumoylated and ubiquitinated on many sites (Section 16.7.4; Figure 2.4), and these modifications serve to regulate the transcription-activating functions of p53. The following characteristics of multisite modification are important for intracellular signaling:

The same modification may occur on several sites of a signaling protein. Most signaling enzymes carry multiple sites for a particular modification such as Ser/ Thr phosphorylation. Each of the sites may be modified by distinct members of an enzyme class (e.g., distinct Ser/Thr-specific protein kinases), and each modification may serve a different function (see e.g., PDGF receptor; Figure 10.11). The

Figure 2.4 Modification pattern of the tumor domain of p53. The PTMs are introduced in a suppressor protein p53. The figure illustrates differential fashion by distinct regulatory inputs the multiplicity of PTMs attached to the (see Section 16.7.4). For modifications in other tetramerization domain and DNA binding regions of p53, see Figure 16.13. 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 43 neighboring amino acids then specify the functional role of a modification by binding to isoforms of the cognate modification-specific interaction module. The same amino acid can be subject to different modifications. For example, lysine residues can be modified by acetylation, methylation (Section 4.5), ubiquitination, neddylation, and sumoylation (Section 2.5). The different lysine modifications function in a competitive manner, with one modification excluding the other (Section 2.4.4).

& Multisite modification of signaling proteins: — The same modification can occur on several sites — Different modifications can occur on the same protein — The same amino acid can be differently modified — Can be considered a “bar code” — Can show combinatorial characteristics.

2.4.3.1 PTM Patterns are Used as a “Bar Code” Thepresenceofmultiplemodifications of the same or different type can be considered as a dynamic “bar code” that specifies a distinct function of the signaling protein in time and in space. The “bar code” controls the catalytic activity of the signaling protein, the association of interaction modules, and its subcellular localization. One important aspect of multisite modification is the reversibility and the dynamic nature of the modifications. The modification patterns formed change with time and subcellular location, which provides a controloffunctionintimeandspace.

2.4.3.2 Multiple Modifications Often Show Combinatorial Characteristics The effect of a given modification may be context-dependent such that, for example, the presence of one modification prevents the modification at another site. Multisite modification events frequently interplay with each other, and a cooperative effect of modification events may be observed.

2.4.4 Binding Properties of Regulatory Interaction Domains

& Protein interaction domains: — Often fold independently — Exist as multiple subtypes — Assemble signaling complexes — Control enzyme activity.

The interaction domains of signaling proteins bind modified amino acid side chains, peptides, proteins or phospholipids (for a review, see Ref. [4]). The interaction domains may be classified by the characteristics of their ligands (Figure 2.5) and by sequence comparison. Classification by the nature of ligands reveals the following types of interaction domain: 44 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.5 Classification of interaction Section 2.7; (b) Domain---peptide interactions: domains by the nature of the binding For PDZ, see Section 10.2.1.2. EVH1: Ena- substrates. Interaction domains bind modified Vasp homology 1; (c) Domain---domain amino acid side chains, short peptide interactions: For PDZ, see Section 9.2.1.2. For sequences, other protein domains, or DD, DED, CARD, PyD, see Chapter 17; BRCT: phospholipids. (a) Domain-modified peptide BRCA1 C-terminal domain; SAM: sterile a interactions. For SH2, PTB, C2, see Section motif; PB1: phox and Bem1p domain; (d) 10.1.5; for 14-3-3, see Section 2.5.3; FHA, Domain---lipid interactions: For C1, see Section forkhead-associated domain; for MH2, see 9.5; for PH, FYVE, C2, see Section 8.6.1. Section 14.1.3; for Chromo, see Section 2.7. FERM: four point one, ezrin, radixin, moesin. For further Me-Lys binding domains, see ENTH: epsin NH2-terminal homology. 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 45

Figure 2.5 (Continued)

Protein modiﬁcations (Figure 2.5a): As outlined above (see Section 2.4.1), a large number of PTMs of signaling proteins exist that serve as attachment points for partner proteins during formation of signaling complexes. PTMs frequently complete binding sites for interaction domains in protein–protein assemblies and make a major contribution to the speciﬁcity of the interaction. Thus, the interaction domains serve as detectors of protein PTMs formed during the course of signaling events.

& Protein interaction domains bind to: Distinct PTMs Sequence motifs Other protein domains Membrane-bound phosphatidyl-inositides

Peptide motifs (Figure 2.5b): This class of interaction domains binds to short peptide motifs that are exposed on the ligand surface. As an example, the SH3 domain binds to proline-rich motifs of protein ligands, and by this property regulates many cellular functions (Section 10.2.1.2). The speciﬁcity of binding may be quite low, and the biological activities that are regulated by this type of interaction are diverse. 46 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Protein domains (Figure 2.5c): A number of modular domains undergo homo- or heterotypic domain–domain interactions rather than binding short peptide motifs. Such domains frequently identify proteins involved in a common signaling process and then direct their coassembly into functional oligomeric complexes. Compo- nents of apoptotic or inflammatory signaling pathways are characterized by death domains (DDs) or close structural relatives thereof that form heteromeric structures required for caspase dimerization and activation (see Chapters 14 and 17). The distinction between domains that bind peptide motifs and those that interact with other folded domain structures is by no means absolute. PDZ domains, for example, generally recognize short peptide motifs of about four residues at the extreme C termini of their binding partners, but they can also mediate specific heterotypic PDZ–PDZ domain interactions. Examples of PDZ-containing proteins include PSD-95 and the InaD protein (see Section 10.2.1.2). Phospholipids (Figure 2.5d): Many signaling processes are intimately linked to the cell membrane, and the recruitment of signaling proteins to the cell membrane is frequently an essential step in signaling (Section 3.2.3). One mechanism for the attachment of signaling proteins to the cell membrane uses membrane phospholipids that are recognized by phospholipid-binding interaction modules on signaling proteins. The specificity of the phospholipid-binding domains is not well characterized and appears to be rather broad. Some phospholipid-binding domains have also been reported to bind to peptide motifs. Further details on the function and properties of phospholipids and the cognate interaction domains are available in Section 8.6.1.

2.4.4.1 Versatility and Variability of Interaction Domains Classiﬁcation by sequence comparison shows that interaction domains can be used in a highly ﬂexible manner to organize signaling events. The complexity and variability of interaction domain function has been found to be based on:

i) A particular domain class may be able to recognize distinct motifs: Interaction domains are remarkably versatile in their binding properties. An individual domain can engage several distinct ligands, either simultaneously or at successive stages of signaling. For example, the MH2 domain of the Smad proteins (Section 14.1.3) harbors an extended binding surface that is able to interact with phosphoserine motifs, with the scaffolding protein Sara, and with components of the transcription apparatus. ii) Separate ligand-binding sites may occur within an individual domain. iii) Ligand conformation may inﬂuence domain recognition. iv) The same class of interaction domain often occurs twice in a signaling protein. As examples, the signaling protein Ras-GAP (see Section 11.5.6) harbors two SH2 domains, while the protein phosphatase SHP2 (Section 10.4.1.2) contains two SH3 domains. 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 47

v) Interaction domains can be assembled from repeated copies (up to 50) of small peptide motifs, yielding a large interaction surface with multifaceted binding properties. Such repeats include ankyrin repeats, Armadillo repeats, and leucine-rich repeats, among others.

2.4.5 How Interaction Domains Read PTM Patterns

Several mechanisms have been identiﬁed by which interaction domains read PTMs and transmit modiﬁcation patterns into distinct biological responses, as follows (Figure 2.6; for a review, see Ref. [5]).

2.4.5.1 Inducible Interactions Typically, the presence of a PTM is a prerequisite for binding of the cognate interaction domain.

2.4.5.2 Cooperative Interactions and Multisite PTMs PTM-dependent interactions can be cooperative, such that a signal is only generated after two or more sites on the same protein have been modified. This can be achieved in several ways. First, a doubly modified motif can be recognized in an obligatory fashion by two tandem interaction domains. Second, a single domain can possess two binding pockets for the modified residues. Third, a domain with a single binding pocket can bind specifically to a protein that carries several modifications. For example, the WD40-repeat domain of the Saccharomyces cerevisiae protein Cdc4 only binds to its target, the kinase inhibitor Sic1, when the target has been phosphorylated during the G1 phase of the cell cycle on at least six Ser/Thr residues.

& PTMS can function in: — A cooperative manner — A sequential manner — A mutually exclusive manner — An antagonistic manner — Via intramolecular regulation.

2.4.5.3 Sequential PTM-Dependent Interactions and Cross-Regulation One PTM-dependent protein–protein interaction can be required for a subsequent modiﬁcation and interaction. The protein phosphorylation of Cyclin E, for example, creates a binding site for the substrate-targeting domain of an E3 ubiquitin ligase, targeting Cyclin E for degradation in the proteasome (see Section 15.4.2). 48 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

(a) Cooperative (b) Multi-site switch

E3 SH2 P Tyr (WD40)8 F-box E2

Cdc4 P P P SH2 P Tyr P P Sic1 Ub Ub Ub Ub

Protein Kinase PPP PP Sic1 Sic1 degradation

Chr HP1 Chr HP1

1 Cbl SH2 Tyr

2 MeMe MeMe P Me P Me S10 Auro-B S10 K9 K14 K9 K14 3 kinase Ub UIM Lys Histone H3

Figure 2.6 Regulatory properties of PTMs and through an interaction between its SH2 their interaction domains. (a) Cooperative domain and a P-Tyr in the C-terminal tail of the interactions. Tandem SH2 domains in ZAP-70 protein (“off” state). The release of this bind in an obligatory fashion to two P-Tyr sites intramolecular interaction by in ITAMs (see Section 13.3.2); (b) Multi-site dephosphorylation of the tail, or through the switch. Sic1 (substrate inhibitor of cyclin- presence of a combination of activating dependent protein kinase-1) requires ligands (such as Pro-rich or P-Tyr ligands), phosphorylation on at least six sites before it results in an active kinase (“on” state) (see can bind to the SCF (see Section 2.8.3.4) E3 Section 10.3.2); (d) Antagonistic Ub-ligase subunit Cdc4, which is a WD40- modifications. Trimethylated Lys9 of histone repeat-containing protein. This switch-like H3 binds to the HP1 chromodomain, and this interaction leads to Sic1 ubiquitination and interaction is antagonized by the controls cell-cycle progression in phosphorylation of the neighboring Ser10 Saccharomyces cerevisiae; (c) Intramolecular residue by a protein kinase (see Section 4.5.7). interactions. Src kinase is autoinhibited

2.4.5.4 Mutually Exclusive PTMs and Interactions PTMs, and the creation of binding sites for protein-interaction domains, can be mutually exclusive. Some amino acid side residues such as lysine and serine residues can carry different PTMs, and the creation of binding sites for protein- interaction domains can be mutually exclusive (Figure 2.7). Serine residues can be phosphorylated and acetylated, while Lys residues can be modiﬁed by phosphorylation, acetylation, methylation, ubiquitination, neddylation, and sumoylation. Thus, 2.4 Posttranslational Modifications (PTMs) in Cellular Signaling 49

Figure 2.7 Multiplicity of lysine modification. di-, or trimethylated lysine, and each methyl The e-amino group of lysine residues can be modification may have a different biological modified by acetylation and other function. For details of ubiquitination, modifications. Acetylation of lysine precludes SUMOylation and NEDDylation, see Section further modifications by others, and vice versa. 2.8.6. Attachment of methyl groups can yield mono-,

the binding properties of the Lys site and the functions of the signaling protein will vary depending on the type of modiﬁcation that it receives. For example, acetylation of the tumor suppressor p53 at a particular Lys residue enhances its activity, whereas ubiquitination of this residue promotes nuclear export and proteasomal degradation (see Section 16.7.4).

2.4.5.5 Antagonistic Action of PTMs A PTM that is attached to one amino acid can antagonize the ability of an adjacent modified residue to recruit a binding partner. For example, the acetylation of a lysine residue excludes the other lysine modifications. Many examples for such antagonistic modifications are found among the modification of histones (Figure 2.6d; see also Figure 3.23 and Section 4.5.1).

2.4.5.6 Regulation of Intramolecular Interactions by PTMs A protein that sustains a PTM can undergo an intramolecular interaction if it also contains a protein-interaction domain that binds to the modiﬁed site (see Src kinase; Section 10.3.2.2 and Figure 9.22). This, in turn, can block the 50 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

ability of the protein-interaction domain to engage an exogenous ligand, and it can also elicit a conformational change that can inhibit the activity of a linked catalytic domain.

2.4.5.7 Convergent Recognition of a PTM Different types of interaction domain can engage a PTM through distinct structural mechanisms, which indicates that they have evolved separately to recognize the same modification. For example, there are numerous domain classes with entirely different folds that selectively recognize pSer/pThr-containing motifs. In these cases, discrimination between the modified sites is achieved on the basis of the flanking sequence (Table 2.1). Domain families are indicated, together with examples of consensus phosphoserine/phosphothreonine (pSer/pThr) motifs that specific family members recognize, as well as cellular processes that are regulated by these interactions. In the case of 14-3-3, the entire protein is composed of the pSer/pThr-binding fold. Some families are largely dedicated to pSer/pThr recognition (e.g., 14-3-3 proteins), whereas others have only a few members that are known to be involved in phosphopeptide binding (e.g., WW and WD40 domains). In the case of WD40- domain- and armadillo (Arm)-repeat-containing proteins, the phosphopeptide- binding domains are composed of several peptide repeats. b-arrestin (see Section 7.8) binds preferentially to activated G-protein-coupled receptors (GPCRs) following multisite phosphorylation in the GPCR C-terminal tail.

Table 2.1 The multiplicity of Phospho-Ser/Phospho-Thr binding domains.

Domain/Pro- Consensus-binding Functions tein motif

14-3-3 R-S-X-pS-X-P Survival, cell cycle, cytoskeleton, metabolism, signaling TPR repeat pS-Q Nonsense-mediated decay FHA pT-X-X-D Cell cycle, DNA repair, transport MH2 pS-X-pS TGFb signaling WW pS-P Cis---trans prolyl isomerization WD40 L-P-pT-P Cell cycle, ubiquitination BRCT pS-X-X-Y DNA-damage response, cell cycle Polo box S-pS-P Cell cycle b-arrestin pS/pT . . . pS/pT . . . GPCR downregulation and signaling pS/pT Arm repeat pS-pS-L-pS-A-L-pS b-catenin signaling

BRCT, breast cancer-susceptibility protein-1 C-terminal; FHA, forkhead-associated; MH2, MAD- homology-2 (Section 14.1.3); pS, phosphoserine; pT, phosphothreonine; TGFb, transforming growth factor-b (Section 14.1); X, any amino acid. 2.5 Regulation by Protein Phosphorylation 51

2.5 Regulation by Protein Phosphorylation

Summary Protein phosphorylation catalyzed by protein kinases is the most prominent PTM on eukaryotic proteins. Ser/Thr residues and Tyr residues are the main phosphorylation sites. The regulatory function of protein phosphorylation is based on two features: (i) the special ionic properties of the phosphate group mediate conformational and allosteric changes in proteins; and (ii) the phosphate groups are used as attachment points for cognate interaction domains to recruit signaling proteins into signaling complexes and signaling paths.

The phosphorylation of enzymes by speciﬁc protein kinases is the most widespread mechanism for the regulation of enzyme activity in general, and for signaling proteins in particular.

& Protein phosphorylation: — Catalyzed by protein kinases — Uses ATP as P-donor. Phosphorylation on: — Ser/Thr — Tyr — Asp/Glu — His.

Protein phosphorylation represents a ﬂexible and reversible means of regulation, and plays a central role in signal transduction chains in eukaryotes.

2.5.1 General Aspects of Protein Phosphorylation

Proteins are phosphorylated mainly on Ser/Thr residues and on Tyr residues, although occasionally Asp or His residues are phosphorylated, the latter especially in prokaryotic signal transduction pathways. For the regulation of enzyme activity the phosphorylation of Ser and Thr residues is most significant. Apart from the regulation of Tyr kinases, Tyr phosphorylation serves the function of creating specific attachment sites for proteins. Both functions are discussed in greater detail in Chapter 10. Protein phosphorylation is a specific enzymatic reaction in which one protein serves as a substrate for a protein kinase (Chapter 9). The protein 52 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

kinases are phosphotransferases that catalyze the transfer of a phosphate group from ATP to an acceptor amino acid in the substrate protein (see Figure 2.3) and, as a consequence, the activity and/or structure of the substrate protein is changed. The human genome harbors about 500 different protein kinases, which illustrates the central role of protein phosphorylation in cellular functions. The classiﬁcation, properties and regulation of protein kinases are presented in detail in Chapter 9.

& Phosphate groups in proteins: — Carry two negative charges — Can engage in H-bond networks — Mainly interact with Arg — Are stable against hydrolysis — Are removed by phosphatases.

A general switch function can be ascribed to protein phosphorylation, and this is based on different mechanisms that may operate either alone or in combination. Phosphorylation by protein kinases inﬂuences the function, activity and subcellular location of the protein substrate, especially in the following ways:

The induction of conformational changes by allosteric mechanisms (Section 2.4.2). The most prominent example of this regulatory function is the phosphorylation of protein kinases in the activation loop (see Chapter 9). The creation of binding sites for cognate interaction domains to mediate the formation of signaling complexes (Section 2.4.3).

One characteristic feature of protein phosphorylation is its dynamic nature. Typically, the phosphate residues are introduced transiently by the protein kinases in a signal-directed fashion, and are removed on demand by protein phosphatases.

2.5.1.1 Properties and Interactions of Phosphorylated Proteins The response of a protein upon phosphorylation is dictated by the special

properties of the phosphate group, which has a pKa of about 6.7 and carries two negative charges at neutral pH. Therefore, two negative charges are generated in a substrate protein upon the phosphorylation of an uncharged amino acid side chain. This fact, and the presence of four oxygen atoms, allows the phosphate group to form an extensive network of H-bonds which can link the different parts of a polypeptide chain. Similar twofold negatively charged groups do not occur in other structural elements of proteins. These electrostatic interactions and the network of H-bonds are therefore of special importance for the control of protein functions by phosphorylation. Analyses of protein–protein interactions in existing structures have shown that phosphate groups most commonly interact with the main-chain nitrogens at the 2.5 Regulation by Protein Phosphorylation 53 start of a helix, where glycine is often located. In nonhelix interactions, the phosphate groups most commonly contact residues of arginine, the guanidinium group of which is well suited for interactions with phosphate because of its planar structure and its ability to form multiple hydrogen bonds. The electrostatic interaction between arginine residues and the phosphate group provides tight binding sites that often function as organizers of both short-range and long-range conformational changes. The phosphate esters of Ser, Thr, or Tyr residues are quite stable at room temperature and neutral pH, and the rate of their spontaneous hydrolysis is very low. Therefore, in order to remove the phosphate residue the cell will utilize specific enzymes – the protein phosphatases – which, based on substrate specificity, can be classified as either Ser/Thr- or Tyr-specific phosphatases (see Chapters 9 and 10).

2.5.2 Allosteric Functions of Protein Phosphorylation

The molecular basis of the control function of protein phosphorylation has been elaborated for many proteins. A wide range of different mechanisms has been identiﬁed, ranging from large-scale allosteric conformational changes (e.g., as in glycogen phosphorylase; see below) to small-scale conformational changes induced upon phosphorylation. Phosphorylations are even known that affect enzyme activity solely by electrostatic effects, without any apparent change in enzyme conformation. For example, isocitrate dehydrogenase from Escherichia coli is phosphorylated directly in the substrate-binding site with only minimal conformational changes resulting [6].

& Protein phosphorylation: — May induce large- and small-scale conformational transitions — May function directly by electrostatic effects.

One of the best characterized examples of phosphorylation-induced allosteric transitions is the regulation of glycogen phosphorylase by phosphorylation. Rabbit muscle glycogen phosphorylase is phosphorylated at Ser14 on the N- terminal tail, and this phosphorylation activates the enzyme. As a consequence of Ser-phosphorylation, the N-terminal tail undergoes a large conformational change of about 50 A, which then induces further conformational changes that propagate to the active site, allowing efﬁcient catalysis. For a more detailed account, the reader is referred to the original report [7] and a review by Johnson and O’Reilly [8]. The regulation of protein kinases by phosphorylation represents another example of phosphorylation-induced allosteric transitions. As outlined in Chapters 9 and 15, and illustrated in Figures 8.4 and 14.8, Tyr phosphorylation of the protein kinases in the activation loop induces a reorganization of the active site that results in a 54 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

large increase in activity. Here, the Tyr-phosphate serves as an organization center that optimally orients the catalytic residues at the active site and allows productive substrate binding (see also Figure 9.2).

2.5.3 Organization of Signaling Pathways by Protein Phosphorylation

Many of the basic functions of PTMs and their interactions were ﬁrst elucidated for protein phosphorylation, which is the most widespread tool for cellular regulation in eukaryotes. About one-third of the eukaryotic proteome is phosphorylated, and many signaling proteins carry multiple Ser/Thr- and/or Tyr-phosphorylation sites that perform distinct functions and are recognized by cognate interaction domains.

2.5.3.1 Tyr-Phosphorylation This modiﬁcation is introduced by protein tyrosine kinases that are found as cytoplasmic enzymes or as part of transmembrane receptors (see Chapter 10). Tyrosine kinases activate signaling paths through autophosphorylation, or through the phosphorylation of downstream effector proteins; the Tyr-phosphates are then recognized by interaction domains of which two major classes are known, the SH2 domains and PTB domains. Subtypes of these domains are found on many central signaling proteins (see Figure 9.10). The detailed functions of these domains and their involvement in regulation and integration of signaling paths are described in Section 10.1.5, using the example of receptor tyrosine kinases.

& Tyr-phosphate: — Allosteric function or attachment function — Recognized by SH2, PTB, and C2 domains.

2.5.3.2 Ser/Thr Phosphorylation Many proteins are phosphorylated at several Ser/Thr sites, which potentially produces combinatorial or cooperative effects. For example, the tumor suppressor protein p53 harbors at least 18 different Ser/Thr phosphorylation sites (Section 16.7) that are modiﬁed by different protein kinases in response to distinct signals. Ser/Thr phosphorylation is more prevalent than Tyr phosphorylation, and there is a correspondingly larger array of domains that selectively bind to pSer/pThr sites (Table 2.1). One important protein class with speciﬁcity for Ser/Thr-phosphates is the 14-3-3 proteins.

& Ser/Thr phosphate: — Allosteric function or attachment function — Multiple cognate interaction domains, for example, 14-3-3 proteins. 2.6 Regulation by Protein Lysine Acetylation 55

The 14-3-3 proteins form noncovalent dimers, which can consequently bind to two P-Ser/P-Thr-containing peptides that have an appropriate consensus sequence. As a result, 14-3-3 proteins can regulate the conformation and catalytic activity of multiphosphorylated enzymes, and can control the interactions and localizations of phosphorylated ligands. For example, 14-3-3 proteins are important regulators of Raf kinase (see Section 11.6) since, by binding to Ser-phosphorylated Raf kinase, 14-3-3 proteins can promote the cytoplasmic localization of Raf kinase, keeping the latter enzyme away from its membrane-localized substrates.

2.6 Regulation by Protein Lysine Acetylation

Summary The acetylation of proteins at Lys residues is a widespread regulatory PTM, comparable in its occurrence to phosphorylation. Lys acetylation removes a positive charge on the e-amino group of the Lys side chain, which in turn modulates ionic interactions and leads to allosteric and conformational changes. Furthermore, the Ac-Lys formed is recognized by a cognate interaction domain, the Bromo domain, found on protein partners. This serves to assemble large protein complexes with functions for example, in chromatin modification and remodeling. The attachment of the acetyl group is catalyzed by lysine acetyltransferases (KATs), often named histone acetyltransferases (HATs). The removal of acetyl groups is catalyzed by protein lysine deacetylases (KDACs), also named histone acetyltransferases (HDACs).

The side chain of a lysine residue is the target of several mutually exclusive modifications (Figure 2.7) of which acetylation (Section 2.6), methylation (Section 2.7) and ubiquitination (Section 2.8) are especially prominent. First identified in histones about 40 years ago, lysine acetylation is now known to occur in a large number of proteins at a frequency comparable to that of phosphorylation. Indeed, a global mass spectrometric analysis identified 3600 acetylation sites in the human proteome [9]. Protein acetylation is now known to regulate all major functions of the cell, including intermediary metabolism, cytoskeleton dynamics, endocytosis, transcription, and many intracellular signaling pathways.

2.6.1 General Aspects of Protein Lysine Acetylation

The impact of lysine acetylation on protein function is based primarily on two mechanisms. First, acetylation removes a positive charge from the lysine residue, which leads to a loss of electrostatic interactions and to an impairment of the ability to from hydrogen bonds. As a consequence, the structure, activity and 56 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

properties of the protein may be altered. Second, acetylated lysine is used as a docking site for the association with a cognate interaction domain, the bromodomain that is found on proteins involved in chromatin modiﬁcation and remodeling (see Section 4.5.2).

& Lys acetylation: — Removes a positive charge — Serves as a docking site for Bromo domain — Catalyzed by KATs (HATs) — Reversed by KDACs (HDACs).

2.6.2 Enzymes of Protein Lysine Acetylation

Protein lysine acetylation is a dynamic modification (for a review, see Ref. [10]) whereby attachment of the acetyl group is catalyzed by lysine acetyltransferases (KATs). As this modification was originally identified in histones, the enzymes responsible for histone acetylation are often termed HATs. The removal of acetyl residues is catalyzed by protein lysine deacetylases (KDACs), often termed HDACs. Most of these enzymes have been identified from their function in histone acetylation and chromatin restructuring.

2.6.2.1 HATs Three major groups of HATs have been identiﬁed: GCN5-related N-acetyltransferases (GNATs); p300 (E1A-associated protein of 300 kDa)/CBP (CREB-binding protein); and MYST proteins. Unlike the GNAT and MYST families, the p300/CBP group is unique to metazoans. Members of the GNAT and MYST family are mostly found in multiprotein complexes.

& HAT groups: — GNATs — CBP/p300 — MYST

2.6.2.2 HDACs The known HDACs can be allocated to the Rpd3/Hda1family (comprising 11 members in humans) and the sirtuin family (seven members in humans; for a review, see Ref. [11]). The HDACs have been also divided into class I (HDAC 1, 2, 3, 8), class II (HDAC 4, 5, 6, 7, 9, 10), class III (sirtuins, SirT 1–7), and class IV (HDAC 11). The class I HDACs are nuclear proteins widely expressed in a variety of tissues, whereas class II HDACs have a narrower tissue distribution, are much larger in size, and shuttle between the nucleus and cytoplasm as part of their mode of action. 2.6 Regulation by Protein Lysine Acetylation 57

Figure 2.8 Substrates and products of the sirtuin-catalyzed deacetylation reaction.

& HDAC families: — Rpd3/Hda1 — Sirtuin

2.6.2.3 Cleavage Mechanism Members of the Rpd3/Hda family and the sirtuin families differ in the mechanism þ of deacetylation. Typically, the Rpd3/Hda family members contain Zn2 in the catalytic center and deacetylate substrates via a “classic” hydrolytic mechanism. In addition to deacetylating histones, these “classical” HDACs have been recognized as deacetylating and regulating many cytoplasmic proteins that are involved, for example, in signal transduction, cell cycle, and apoptosis. þ The sirtuins use NAD as cofactor and do not release acetate as a hydrolysis þ product. The deacetylation reaction begins with an amide cleavage of NAD and the formation of nicotinamide and a covalent ADP-ribose (ADPR) peptide–imidate intermediate. The intermediate is resolved to form O-acetyl-ADP-ribose, and the deacetylated substrate is released (Figure 2.8). The sirtuins are present from prokaryotes to humans, and are found in numerous compartments of the cell. SirT1, 6, and 7 are located predominantly in the nucleus, SirT2 is mostly cytoplasmic, and SirT3–5 reside in the mitochondria. The sirtuins perform key roles as metabolic sensors, and mediators of survival under stress [11].

2.6.3 Regulatory Functions of Lysine Acetylation

Global analysis of the human “acetylome” has revealed that acetylation may rival phosphorylation as a regulator of cell function. Acetylation regulates all major enzymes of intermediary metabolism, and it is tightly linked to signaling pathways. Furthermore, acetylation has been found to be prevalent in macromolecular complexes, and to be involved in diverse cellular processes 58 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

such as chromatin remodeling, cell cycle control, splicing, and nuclear transport. When compared to protein phosphorylation, the structural and mechanistic aspects of protein lysine acetylation have been less well characterized, and the mechanisms by which acetylation and deacetylation are orchestrated remain to be determined. However, what has been clearly established is the extensive crosstalk of lysine acetylation with other PTMs. Acetylation not only competes with other PTMs but can also influence the modification of neighboring sites in either a positive or negative fashion. Such a cross-regulation is well established between phosphorylation and acetylation on histones (see Section 4.5.7) and on transcription factors such as FoxO1 and p53 (for details, see Ref. [12]). Phosphorylation is often the first wave of PTMs in response to cellular stimuli, and the crosstalk provides an effective means of converting phosphorylation- based signals into acetylation-based actions.

2.7 Regulation by Protein Methylation

Summary The methylation of proteins occurs at Lys and Arg residues. Notably, Lys can become mono-, di-, or trimethylated, and the different degrees of methylation may serve distinct biological functions. Lys methylation is catalyzed by protein lysine methyltransferases (KMTs), while demethylation is catalyzed by protein lysine demethylases (KDMs). Methylated Lys residues are recognized by cognate interaction domains that are sensitive to the number of methyl groups attached. Arginine can be monomethylated and dimethylated by the action of protein arginine methyltransferases (PRMTs). Arg and Lys methylation are of outstanding importance in histone modification and chromatin remodeling.

Protein methylation by methyltransferases is a posttranslational covalent modiﬁcation that occurs on the side-chain nitrogen atoms of lysine and arginine on proteins. In both cases, methylation is a transient mark that is removed by the action of demethylases.

2.7.1 Protein Lysine Methylation

Lysine can accept one, two, or three methyl groups, resulting in mono-, di-, or trimethylated forms (Figure 2.7). The different stages of methylation on a given Lys residue confer different biological readouts to the modiﬁed residue, such that methylation has a greater combinatorial potential with respect to other modiﬁcations 2.7 Regulation by Protein Methylation 59

& Lysine methylation: — Mono-, di-, and trimethylation — Catalyzed by KMTs — Removed by KDMs — Recognized by: Chromodomain Tudor, MBT, PHD, WD40 domains

2.7.1.1 Lysine Methyl Transferases e Methylation of the -NH2 group of lysine residues is catalyzed by protein lysine methyltransferases (KMTs). These enzymes regulate many of the cellular processes in which histone methylation has been implicated, including transcriptional regulation, the maintenance of genome integrity, and the regulation of epigenetic memory. The KMTs that preferentially methylate histones are also named histone methyltransferases (HMTs). Humans encode several families of KMTs that differ in substrate speciﬁcity, assembly in multiprotein complexes, and the degree of lysine methylation. With almost no exception, the KMTs share a strong homology in a 140-amino acid catalytic domain known as the SET domain (for Su(var), Enhancer of Zeste and Trithorax). Important families in vertebrates are the SET and MLL families of HMTs.

& KMT (HMT) families: — SET — MLL KDM (HDM) families: — Amine oxidase family — JmjC domain family

2.7.1.2 Lysine Demethylases, KDMs The demethylation of methylated lysine is catalyzed by protein lysine demethylases (KDMs). As lysine demethylation was ﬁrst discovered during studies of histone methylation, the erasers of the methylation marks have been also named histone demethylases (HDMs). However, these enzymes are also active towards methylated proteins other than histones (for a review, see Ref. [13]), and both terms are currently in use. Two classes of KDMs have been identiﬁed that differ in their enzymatic mechanism:

Amine oxidase enzymes which use FAD as a cofactor and target removal of the ﬁ Kme1/Kme2 modi cation states. Formaldehyde and H2O2 are produced as byproducts (Figure 2.9a). 60 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.9 Methylation of lysine and arginine on one of the terminal (v) guanidino nitrogen residues. (a) LSD1 (Section 2.7.1) catalyzes the atoms. These two nitrogen atoms are demethylation of dimethylated histone equivalent. The subsequent generation of H3K4Me2 through a FAD-dependent oxidative asymmetric dimethylarginine is catalyzed by reaction. The products are hydrogen peroxide, type I enzymes, while the production of formaldehyde, and mono-methyl H3K4; (b) symmetric dimethylarginine is catalyzed by Types of methylation on Arg residues. Types I, type II enzymes. II and III PRMTs generate monomethylarginine

KDMs which belong to a large family of proteins that contain a Jumonji-C(JmjC) domain as their catalytic core. Of the 27 JmjC members found in humans, 15 have been shown to be active towards methylated histones. The JmjC domain- containing proteins are iron and a-ketoglutarate-dependent oxygenases that target the removal of all three lysine methylation states on histones. 2.7 Regulation by Protein Methylation 61

2.7.1.3 Interaction Domains for Methylated Lysine e The positive charge of the -NH2 group of lysine is not changed upon methylation. Hence, the mechanistic impact of lysine methylation is based on slight changes in H-bonding, the additional volume of the hydrophobic methyl groups and, most importantly, the recognition of the methyl groups by cognate interaction domains on partner proteins. The interaction domains of binding partners bind selectively methylated lysine and are sensitive towards the number of methyl groups attached to a given lysine (reviewed in Ref. [14]). Currently, five types of interaction domain are known with specificity for methylated lysine residues, namely Chromodomain, Tudor, malignant brain tumor (MBT), plant homeodomain (PHD), and WD40 domains. Most methyl-Lys binding domains have been identified on macromolecular assemblies involved in chromatin modifying and remodeling (Section 4.5.3).

2.7.2 Protein Arginine Methylation

Arginine methylation occurs on both nuclear and cytoplasmic proteins, and plays an important role in regulating chromatin function. The methylation of arginine is catalyzed by PRMTs that transfer methyl groups from S-adenosyl-L-methionine (SAM) to the guanidino nitrogens of arginine residues [15]. The mammalian family of PRMTs has nine members that share a conserved catalytic core, but have little similarity outside the core domain. The PRMTs are divided into three classes (Figure 2.9b):

Monomethylation of arginine is catalyzed by type I, II, and III PRMTs. Type I PRMTs catalyze the asymmetric dimethylation of arginine; this family includes PRMT1 that is responsible for the bulk (ca. 85%) of total protein arginine methylation activity. Type II PRMTs catalyze the formation of symmetric dimethylarginine.

& Arginine methylation: — Monomethylation — Dimethylation: Symmetric Asymmetric — Catalyzed by PRMTs — Recognized by Tudor domain.

2.7.2.1 Arginine Demethylation/Citrullination How the methyl mark on arginine residues is removed remains a matter of debate. Whilst enzymes that erase the arginine methyl marks have been elusive, there is one enzyme system – namely, the peptidyl arginine deaminases (PADs) – that 62 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

interferes with arginine methylation. This class of enzyme catalyzes the conversion of arginine or mono-methyl arginine into citrulline. The PAD-catalyzed production of citrullinated proteins prevents methylation of these sites by PRMTs.

2.7.2.2 Interaction Domains The mechanistic basis of regulation by arginine methylation is not well characterized. Currently, only one cognate interaction domain is known, namely the tudor domain that is found on TDRD3, a coactivator of transcription.

2.8 Ubiquitin Modification of Proteins

Summary Covalent attachment of the 76-amino acid protein ubiquitin (Ub) to acceptor proteins is a widely used tool for regulating protein activity in a signal-directed fashion. The modification of proteins with Ub is catalyzed by a set of three enzyme types, named E1, E2 and E3, of which E3 is responsible for substrate selection and ligation of Ub to acceptor lysines on substrate proteins. A large number of E3 ligases exist, either as single subunit enzymes or as multiprotein complexes, and these are subject to manifold regulations including phosphorylation and subcellular compartmentalization. Substrate selection by E3 ligases may depend on PTMs of the substrate, which opens up the possibility to ubiquitinate substrate proteins for example, in response to phosphorylation signals. Depending on the type of E2 and E3 enzymes, single Ub-residues or poly-Ub chains are attached to substrates. In poly-Ub chains, different types of linkages between the Ub moieties are possible, and this allows for the deposition of distinct Ub-signals on substrate proteins, each with a distinct biological function. Removal of Ub-labels is catalyzed by deubiquitinating enzymes, of which a large number is known. The best-characterized function of Ub-modification is the labeling of proteins for degradation in the proteasome. For example, Ub-mediated degradation of proteins is a crucial step in driving the passage of cells through the cell cycle. In addition to determining the life time of proteins in a signal-directed manner, Ub modification is used for recruiting proteins into subcellular complexes involved, for example, in the replication and repair of DNA and assembly of signaling complexes downstream of transmembrane receptors. The multiple functions of Ub-modification are based on the particular type of Ub-linkage in poly-Ub chains that are recognized by Ub-binding domains present on partner proteins. A series of Ub-like proteins such as SUMO that have specific cellular functions has also been identified. The attachment of SUMO to Lys residues on substrate proteins is involved, for example, in transcription regulation and protein trafficking. 2.8 Ubiquitin Modification of Proteins 63

A major tool for controlling the activity and level of signaling proteins is the covalent attachment of small proteins, of which ubiquitin is the most important. The ubiquitination of proteins was ﬁrst discovered as tag that targets proteins for proteolytic destruction in a regulated manner. However, ubiquitination is now recognized as a major regulatory PTM that is also involved in many nonproteolytic cellular processes such as endocytosis and DNA repair. The multiplicity and versatility of signals created by ubiquitination compares well with signaling by phosphorylation, which has been long been considered the major signaling tool in eukaryotic ells. A large number of enzymes is dedicated to the speciﬁc ubiquitination of target proteins, comparable to the many proteins kinases encoded in eukaryotic genomes. In addition, a ubiquitin tag deposited on a target protein is read and interpreted by many different UB-binding domains, and many enzymes are available that remove the Ub-tag, comparable in number to the protein phosphatases.

2.8.1 Pathways of Protein Degradation

How much of a protein is available for cellular functions depends on its rate of gene expression and its rate of proteolytic degradation. The activities of both processes determine the steady-state concentration of a protein, and it is now well established that protein degradation is an important means of protein regulation. The removal of proteins by proteolysis occurs, for example, under stress conditions when partially unfolded or misfolded proteins must be removed, and it is used by cells in a regulated fashion to degrade proteins as part of the normal cellular program, allowing the function of a protein to be temporally restricted and specifically modified. It is mainly the selective removal of proteins by proteolysis that determines the life span of a protein – that is, how long a protein exists in the cell. Different proteins can have very different life spans, which indicates the existence of specific protein degradation mechanisms. Currently, three major protein degradation pathways have been identified in mammalian cells: the proteasomal pathway; the lysosomal pathway; and degradation via autophagosomes. All of these appear to use the same destruction tag, namely a modification of the protein substrate with the small protein ubiquitin (for a review, see Ref. [16]). The routing of a substrate towards a particular degradation pathway is dependent on the specific nature of the ubiquitin tag that can occur in different lengths and linkage types.

& Protein degradation pathways: — Proteasomal — Lysosomal — Autophagosomal. 64 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.8.1.1 Lysosomal Pathway In this pathway, proteins that enter the cell via endocytosis are degraded. The pathway is used to eliminate foreign proteins, and is also a principal means by which cells turn over plasma membrane proteins, such as receptors or ion channels. The substrates of the lysosomal path are endocytosed and ﬁnally directed to lysosomes, where proteolytic degradation occurs under acidic conditions. The use of ubiquitin as a tag for routing proteins towards this pathway has been recognized only recently.

2.8.1.2 Autophagosomal Pathway This pathway allows for the removal of cytosolic entities or organelles by capturing them within a double-membrane compartment – the autophagosome – which finally leads the substrates to proteolytic destruction in lysosomes. Here too, ubiquitination serves as signal to direct the substrates first to the autophagosome, and finally to destruction.

2.8.1.3 Proteasomal Pathway The most signiﬁcant degradation pathway is the ubiquitin–proteasome pathway in which proteins are degraded in a 26S proteasome following their conjugation by multiple ubiquitin molecules. The ubiquitin–proteasome system is the major tool for the selective proteolysis of proteins, and thereby plays a key regulatory role in the cell.

& Ubiquitin–proteasome pathway: — Ubiquitin attachment to target proteins — Proteolytic degradation of Ub-labeled proteins in 26S proteasome. Functions of ubiquitin: — Proteolytic — Nonproteolytic.

Ubiquitin-mediated proteasomal proteolysis serves the following cellular functions:

Degradation of proteins under stress situations Degradation of denatured and damaged proteins Targeted degradation of regulatory proteins as for example: oncoproteins, tumor suppressor proteins, transmembrane receptors, mitotic cyclins, transcription- activating proteins.

In addition to marking proteins for degradation, many nonproteolytic functions of ubiquitin and ubiquitin-like proteins have been discovered. These will be discussed in Sections 2.8.5 and 2.8.6. 2.8 Ubiquitin Modification of Proteins 65

2.8.2 Basics of Ubiquitin Modification

Ubiquitin is a 76-residue protein which is found in nearly all eukaryotes and occurs either in free form or bound to other proteins. All known functions of ubiquitin are transmitted via its covalent linkage with other proteins: an isopeptide bond is formed between the ubiquitin’s terminal glycine and an amino group of the target protein in a multistep reaction. Usually, the amino group is donated by a lysine residue of the target protein, but N-terminal modiﬁcation is also known. Ubiquitin is a highly versatile molecular signal in the cell because of its ability to modify substrate proteins in its monomeric form (¼ monoubiquitination)ortobe conjugated to preceding ubiquitin moieties which leads to polymeric Ub chains (¼ polyubiquitination). The various types of Ub modiﬁcation are linked to distinct physiological functions in the cell. The polyubiquitination of target proteins serves the purpose, among others, of marking the protein for proteolytic degradation.

& Ubiquitin: — 76 amino acids — Highly conserved — Attachment to Lys-residues via C-terminal glycine.

The ubiquitination of proteins is a highly ordered process, which involves three sequential enzymatic reactions performed by three types of enzyme, E1, E2, and E3 (Figure 2.10). The enzymatic conjugating cascade of ubiquitination is organized in

Figure 2.10 Ubiquitination of proteins and reaction catalyzed by the E3 ubiquitin ligase. degradation in the proteasome. Ubiquitin (Ub) Repetition of this series of events results in the is initially activated by an enzyme E1, whereby formation of a polyubiquitin chain on the the C-terminal carboxyl group of ubiquitin target protein. The E3 enzyme binds directly to becomes attached to the SH group of E1 via a the target protein, and therefore plays an thioester bond. The activated ubiquitin is then important role in determining the substrate transferred from E1-Ub to the ubiquitin- specificity of this pathway. The conjugating enzyme, E2. Finally, the ubiquitin polyubiquitinylated protein is then degraded to is covalently attached to the target protein in a peptides in the 26S proteasome. 66 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

a hierarchical fashion: In the human genome, there are two E1s, about 40 E2s, and over 600 E3s. The modification of a target protein by ubiquitination creates a type of “ubiquitin code” (see Section 2.8.5) that is read by a large number of “ubiquitin binding domains” (UBD) embedded in many signaling proteins and multiprotein complexes, as for example, the proteasome. More than 20 different UBDs have been identified in the human genome, and these appear to have distinct binding preferences for certain ubiquitin modifications (Section 2.8.5.2). The binding of a UBD to the “ubiquitin signal” then provides a link to specific downstream events. Ubiquitin is the founding member of a family of proteins that share common structural characteristics and are attached to target proteins by a similar mechanism. The other members of this family are also known as ubiquitin-like proteins (Section 2.8.6).

& Ub-family: — Many members — Common fold: Ub-fold — Diverse cellular functions. Important members: — Ubiquitin — Nedd — Sumo.

2.8.2.1 The Ub-Conjugation Reactions: E1, E2, and E3 Enzymes Ubiquitin and ubiquitin-like proteins are activated and transferred to the target protein by the same overall mechanisms, as discussed below, using E1, E2, and E3 enzymes (Figure 2.11).

& Ubiquitination requires: — ATP — E1 enzyme — E2 enzyme — E3 enzyme.

However, the details of the transfer reaction, the substrate recognition, and the properties and numbers of E1, E2, and E3 enzymes, vary considerably among the ubiquitin family members.

& E1: — Ubiquitin-activating enzyme — Ubiquitin adenylate intermediate — Active site cysteine — Two members in humans. 2.8 Ubiquitin Modification of Proteins 67

Figure 2.11 Reactions involved in ubiquitination. 68 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.8.2.2 E1: Activation of Ubiquitin In an initial reaction, ubiquitin is activated by linking its C-terminal glycine carboxylate to an SH-group of the ubiquitin-activating enzyme E1. The activation reaction requires ATP and consists of two steps. Initially, an ubiquitin adenylate intermediate is formed, after which the activated ubiquitin reacts with an E1 cysteine residue to from an E1–ubiquitin thiol ester.

2.8.2.3 E2: Transacylation to the Ubiquitin-Conjugating Enzyme E2 The next step of the conjugation cascade is transfer of the ubiquitin moiety from E1-Ub to a cysteine-SH within the active site of the ubiquitin-conjugating enzyme E2 to form E2-Ub.

& E2: — Ub-conjugating enzyme — 40 members in humans — Specific interaction with E3.

A large number of E2 enzymes, comprising 11 members in yeast and about 40 in humans, are dedicated to ubiquitin. The individual ubiquitin E2 enzymes dictate specific biological functions of the ubiquitin because the specificity of the E2/E3 interaction limits the final substrates to which the ubiquitin is attached.

2.8.2.4 E3: Ubiquitin Transfer to the Target The third step of ubiquitination – the transfer of ubiquitin to the target protein – is catalyzed by a ubiquitin-protein-ligase or E3 enzyme. In this reaction, ubiquitin e is linked by its C-terminal glycine in an amide isopeptide linkage to an -NH2- group of Lys residues or to an N-terminal NH2-group of the protein substrate.

& E3: — Ub-protein ligase — ca.600 members in humans — Single-polypeptide or multiprotein complex — Confers substrate specificity.

In a subsequent reaction, a lysine residue of ubiquitin attached to the target protein can itself be a substrate for E3-mediated ubiquitination, resulting in the attachment of multiple ubiquitin molecules in a ubiquitin–ubiquitin linkage and the formation of poly-Ub chains on the target protein.

& E3-mediated Ub-attachment: — Monoubiquitination. Polyubiquitination: — Ub–Ub-attachment. 2.8 Ubiquitin Modification of Proteins 69

In this polyubiquitination, the ubiquitin molecules are linked by an isopeptide bond between K48 (or other K residues; see Section 2.8.5.1) and G76. There is considerable variability in the length and linkage type of the poly-Ub chains. The presence of poly-Ub linked via K48 is a major prerequisite for degradation of the target protein in the proteasome.

2.8.3 Structure and Regulation of Ub-Protein Ligases

The E3 enzymes are primarily responsible for conferring speciﬁcity to ubiquitin conjugation. Each of the large number of E3 enzymes (there are more than 600 E3s in humans), together with its cognate E2 enzyme, recognizes a few substrates that share a particular ubiquitination signal, which is usually a primary sequence motif. Subsequently, the substrate is marked with a secondary signal, the poly-ubiquitin chain, that is then recognized by Ub-binding domains present on proteins that function as Ub-receptors. During Ub-mediated proteolytic degradation, protein components of the proteasome recognize only this secondary signal and therefore will induce the degradation of a huge variety of substrates. The selection of substrates for ubiquitin ligation, and thus for proteasome degradation, occurs primarily by the E3 enzymes.

& Substrate recognition: — Mediated by E2/E3 — May be signal-dependent and require PTMs.

& E3 types: — Hect E3 — Ring E3.

The E3 enzymes generally harbor at least two domains: one domain which is responsible for E2 binding, an another protein-interaction domain that recruits a substrate for ubiquitination. The substrate- and E2-binding domains can form part of the same polypeptide chain, or they can be distinct subunits of a multiprotein complex. Two types of E2-interacting domains are known – Hect domains and RING domains (Figure 2.12) – that recruit a thiolester-linked E2-Ub intermediate for transfer of the Ub moiety to the bound substrate protein.

2.8.3.1 Hect Domain E3 Enzymes Hect (homologous to E6-AP C-terminus) domain enzymes comprise a large family of E3 enzymes that directly participate in the transfer of Ub to the substrate. In the transfer reaction of Hect E3 enzymes, ubiquitin is ﬁrst transferred from the E2 carrier to an active site cysteine of the E3 enzyme to from a covalent Hect E3–Ub e intermediate. Subsequently, Ub is ligated to the -NH2-group of an acceptor lysine 70 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

HECT domain E3s

Ub SH SH E2 E2 E2 SH E2-binding SH Ub Ub (HECT) Substrate K K K binding

RING domain E3s

E2-binding SH (RING) E2 Ub E2 Ub

K K Substrate receptor Substrate

Figure 2.12 Major E3 classes. (a) HECT degron; (b) RING-domain E3s are scaffold domain E3s (Homologous to E6AP C- proteins that use the RING domain (red) to terminus) bind cognate E2s via the conserved bind the E2 and a different domain (orange) to HECT domain and transiently accept ubiquitin bind the substrate. In SCF and other at a cysteine residue in this region; a different multisubunit RING-domain E3s, the RING and region of the same polypeptide chain binds the substrate binding domains occur in separate substrate (green) through an element in the polypeptides.

on the substrate protein. The Hect E3s contain an essential active site Cys residue near the C-terminus, and one or several WW domains (see Figure 2.5b).

& Hect E3: — Covalent Ub–E3 intermediate — Involved in p53 degradation upon infection with papillomavirus.

Members of the Hect family of E3 ligases have been implicated in endocytosis and trafﬁcking of plasma membrane proteins through monoubiquitination, as well as the degradation of membrane receptors and many intracellular proteins through polyubiquitination. Importantly, Hect E3 ligases are assumed to play an important role in tumorigenesis (for a review, see Ref. [17]). The discovery of the oncogenic potential of Hect E3 ligases originated from studies on the targeted degradation of the p53 tumor suppressor protein (see Section 15.6). One ubiquitin-mediated degradation pathway of p53 is linked to infection by oncogenic DNA viruses as, for example, the human papillomavirus. Here, the viral protein involved is the oncoprotein E6 of human papilloma virus and a cellular E3 enzyme, termed E6-AP (E6-associated protein), which belongs to the Hect family of E3 enzymes. Recognition of p53 and transfer of ubiquitin occurs in a complex between the viral E6 protein, E6-AP and p53, with the formation of an E6-AP–Ub intermediate 2.8 Ubiquitin Modification of Proteins 71

Figure 2.13 Ubiquitination of the tumor E2 enzyme. An intermediate is formed where suppressor protein p53. The attachment of the ubiquitin is linked via a thioester bond to ubiquitin molecules to p53 is catalyzed by the an active site cysteine of E6---AP. The E6 protein HECT-domain E3 ubiquitin ligase E6---AP, binds both to E6---AP and p53 and confers which receives the activated ubiquitin from an substrate specificity. C, cysteine; K, lysine.

(Figure 2.13). The ubiquitination initiated by the E6 protein and the ensuing degradation of p53 result in a loss of p53 function, thus offering an explanation for the tumorigenic effect of the papillomavirus. In addition to p53, other tumor suppressors and oncoproteins have also been found to be targets of Hect-mediated ubiquitination.

2.8.3.2 RING Domain E3 Enzymes A distinguishing feature of RING domain E3 enzymes is the presence of a fold comprising a series of histidine and cysteine residues with a characteristic spacing that allows for the coordination of two zinc ions in a structure called the Really Interesting New Gene (RING) finger. RING proteins are encoded in all eukaryotic organisms analyzed to date. The human genome alone encodes about 400 proteins with this fold, and structural studies show the RING domain to be a rigid, globular platform for protein–protein interactions. Importantly, the RING finger motif is found in a large number of proteins of recognized regulatory function that hitherto have not been associated with ubiquitin–proteasome-mediated proteolysis. The RING domain E3 enzymes comprise a large family of E3 ligases that do not form a covalent intermediate with ubiquitin but may instead activate the E2 to directly discharge the ubiquitin thioesterified to its active site cysteine onto the lysine of a substrate (see Ref. [18]). The RING finger motif is found in single- subunit E3 enzymes such as the Cbl-protein (see below) or in the form of multisubunit E3 enzymes (SCF, APC; see below) where the RING finger is located in one specific subunit. This motif does not participate directly in the ubiquitin transfer to the target protein, but rather seems to function as a scaffold that binds the E2 enzyme and positions the substrate optimally for ubiquitin transfer (Figure 2.14). 72 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.14 Components and interactions in protein. Each cullin has its own large family of the E3 superfamily of Cullin-RING ligases. The dedicated SRs, which bind a substrate’s largest E3 superfamily consists of the “degron” motif. The RING domain recruits an multisubunit Cullin-RING ligases (CRLs). CRLs E2---ubiquitin intermediate, and promotes are nucleated by an extended cullin scaffold ubiquitin transfer from the E2 active site interacting with a catalytic RING-containing directly to the substrate associated with the SR. protein, such as RBX1. A cullin’s N-terminal Interactions between E2s and cullin/rbx1 may domain (NTD) binds a substrate receptor (SR) be mediated by transfer of a NEDD (Nd) group either directly or indirectly via an adapter from E2 to the cullin/RING moiety.

There is one subfamily of RING E3s, the U-box proteins, that does not contain the His-Cys motif and therefore cannot bind zinc. & RING E3: — No covalent intermediate — His-Cys sequence motif — Functions: Binding of E2 Substrate recognition Transfer of Ub from Ub–E2 to substrate.

The U-box proteins show a similar fold as the RING domain E3 enzymes, and are therefore included in the RING domain class. One member of the U-box proteins, the E3 ligase CHIP, is involved in the recognition and degradation of misfolded proteins. & RING finger motif: — Domain within RING-E3 ligases.

& Cellular functions of RING E3s: — Cell cycle regulation — Cell proliferation — Apoptosis — Secretion — Trafficking. 2.8 Ubiquitin Modification of Proteins 73

The most intensively studied subclass of RING E3 enzymes are those of the cullin-RING ligase superfamily [19]. The human genome encodes about 350 cullin- RING ligases, and this family will be discussed in more detail in the following subsections.

2.8.3.3 Structure and Regulation of Cullin-RING Ligases To date, six different types of cullin-RING ligases have been identified, each of which employs a distinct family of substrate receptors. The Cullin-RING ligases are multisubunit E3 enzymes that are nucleated by a scaffold protein named cullin and carry a catalytic subunit, the E2-binding RING subunit RBX, of which two types are known. The cullin adopts an extended structure, with RBX and the substrate binding site at opposite ends. Substrate binding is mediated by a substrate receptor (SR) subunit either directly or indirectly via an adapter protein (Figure 2.14). Each cullin has its own large family of dedicated SRs, which bind to sequence elements of the substrate, also named “degrons.” The “degron” motifs are highly variable and may include multiple covalent modifications (Figure 2.15). The latter property is important for the regulation of substrate recognition by E3 enzymes, because the modifications may be introduced in response to cellular signals initiating ubiquitination and degradation under specific conditions only.

& Cullin-RING E3 enzymes: — Superfamily of more than 300 members — Subunits: Cullin scaffold Catalytic RING subunit Rbx Substrate receptor (SR).

Figure 2.15 Mechanisms for modulating 15.3.1. For Tyr-phosphorylation of substrates, substrate recognition by the substrate receptor see Section 2.8.3.4; HIF, hypoxia-induced of E3 enzymes. Shown are PTMs and other factor. For deacetylation of p53, see Section mechanisms known to regulate the recognition 16.7.4; for specific protein association, see of cognate substrates by the substrate Figure 2.13; for aminoacylation, see Section receptors of different E3s. For Ser/Thr 2.8.3.3. phosphorylation of substrates, see Section 74 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Structural studies and modeling of cullin-RING ligases have provided the first insights into the mechanism and regulation of E3 ligases [20]. The data obtained suggest that cullin-RING ligases are conformationally very flexible protein complexes that can exist in different conformations and may be regulated in many ways. The cullin has an extended structure, with RBx1 bound near the C-terminus and the substrate receptor placed near the N-terminus. The position of RBX1 appears to be crucial to the activation of ligation activity, as RBX1 has been found in a closed conformation interacting with the cullin, and in an open conformation where RBX1 is free to rotate and interact with Ub–E2 and contact the substrate bound by the substrate receptor. The transition between the closed and open conformations appears to be regulated by various signals, as for example, the covalent modification of the cullin scaffold by attachment of a Ub-like protein, named NEDD8.

2.8.3.4 Substrate Recognition by Cullin-RING Ligases In order to achieve precise control of substrate ubiquitination to E3 ligases, binding of the substrate is regulated and numerous mechanisms by which such regulation is achieved have been identified. Most important for substrate recognition by E3 enzymes is their ability to select substrates in dependence on the presence of PTMs. As illustrated in Figure 2.15, many substrates require specific PTMs in order to become ubiquitinated by E3 enzymes, and this property allows for the signal-directed proteolysis of substrates and links signal transduction with targeted proteolysis. Ubiquitination and ubiquitin-mediated proteolysis is therefore an important component of the signaling network of the cell. Distinct signals such as phosphorylation or oxidation signals can recruit regulatory proteins into the ubiquitin pathways, providing for another regulatory level of their function and activity. One of the best-characterized examples of signal-dependent proteolysis is the phosphorylation-dependent degradation of cell-cycle regulators (see Sec- tion 15.3). Various structural studies of cullin-RING ligases have shown how modifications such as phosphorylation, glycosylation, and prolyl hydroxylation served as “knobs” that fit into complementary “holes” in cognate substrate receptors. There are also many variations on the recognition of posttranslationally modified substrates. For example, recognition can be amplified by SRs preferentially binding doubly phosphorylated degrons, or by SR dimerization.

2.8.3.5 N-End Rule One important signal for ubiquitination relates to the nature and modification of its N-terminal amino acid, as formulated by the N-end rule. Two branches of the N-end rule have been identified that use distinct E3 enzymes for ubiquitination. These two branches differ by the absence or presence of acetylation of the terminal amino group. In one branch, the in vivo half-life of a protein is related to the identity of its unacetylated N-terminal residue [21]. The second branch, as identified in Saccharomyces cerevisiae, uses N-acetylation of the N-terminal amino acid as a degradation signal. The N-terminus of newly synthesized proteins can be 2.8 Ubiquitin Modification of Proteins 75 acetylated by N-acetyltransferases in a cotranslational reaction, and this can serve as specific degradation signal [22]. In human cells, more than 80% of proteins are N-acetylated, and it is thought that this modification makes a significant contribution to protein degradation under stress conditions when the N-terminus may be available due to partial protein unfolding. Other known functions of N-end rule pathways in higher organisms include the control of nuclear translocation, the regulation of apoptosis, and the fidelity of chromosome degradation.

2.8.3.6 Examples of Cullin-RINGE3 Ligases In the following subsections, three selected examples of E3 ligases – namely Cbl, SCF and APC – will be presented. The E3 ligase Cbl is a single-subunit enzyme with important functions in the downregulation of receptor tyrosine kinases (see Chapter 10), whereas the multisubunit E3 ligases SCF and APC play major roles in the regulation of cell division [23].

2.8.3.7 SCF Complex The SCF (Skp1–Cullin–F-box) E3 ubiquitin ligase family comprises multisubunit enzymes (Figure 2.16) composed of an invariant core complex, containing the Skp1 linker protein, the Cul1 scaffold protein, and the RING domain protein Rbx1.

Figure 2.16 Structure and substrates of the Rbx1 mediates binding to the E2 enzyme. Skp1 SCF complex. The SCF complex is composed is an adapter that mediates binding of of a core complex comprising Cul1, the RING substrate receptors named F-box proteins. The finger protein Rbx1, the adapter Skp1, and the human genome encodes 68 different F-box F-box protein. Cul1 has scaffolding functions proteins function that recognize a distinct set for the assembly of the other subunits, while of substrates, as indicated. 76 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

The latter protein uses its Zn-binding motif to recruit and direct ubiquitinated E2 enzymes towards specific substrates, which are recognized by a suite of substrate receptors called F-box proteins that in turn recruit substrates for ubiquitination by the associated E2 enzyme to form K48-linked chains. F-box motifs are widespread and found in a large number of proteins with functions not related to the ubiquitination system. To date, more than 70 F-box proteins have been identified in humans, although only a few of them have been fully characterized. This large number – in combination with the core complex and the E2 enzymes – provides the basis for multiple substrate-specific ubiquitination pathways. Each F-box protein contained in SCF complexes recognizes a distinct set of substrates, as illustrated in Figure 14.12 for the example of cell-cycle regulators (see Section 15.3.1).

& SCF E3 enzymes contain: — Skp1 — Cullin — Rbx1 — F-box protein.

& F-box protein: — Substrate receptor — May require substrate phosphorylation.

2.8.3.8 Anaphase-Promoting Complex (APC) The APC is a high-molecular-weight complex of at least 12 different subunits that degrades proteins containing a speciﬁc recognition sequence, the destruction box (Section 15.7).

& APC: — 12 subunits — Recognizes destruction box — Produces K11-linked poly-Ub chains — Regulated by subunit phosphorylation — Regulatory function in cell cycle.

The core E3 activity of the APC has been shown to reside in a small RING ﬁnger protein of the APC, Apc11p and a cullin-like subunit, Apc2 (reviewed in Ref. [24]). Interestingly, APC-mediated polyubiquitination in humans produces K11-linked chains to direct the substrate for proteasomal degradation [23]. By contrast, APC from S. cerevisiae marks substrates by K-48-linked poly-Ub chains. The substrates of APC are cell-cycle regulators, such as cyclins, kinase inhibitors, and spindle-associated proteins (Section 15.3.2). 2.8 Ubiquitin Modification of Proteins 77

2.8.3.9 Cbl Proteins The Cbl proteins are single-subunit E3 ubiquitin ligases that have multiple regulatory functions in signal transduction and cytoskeletal regulation. Above all, Cbl proteins have emerged as key regulators of immune functions by inducing the ubiquitination of key components of T-cell receptor signaling [25]. One major function of Cbl proteins is the downregulation of tyrosine kinases, including receptor tyrosine kinases and nonreceptor tyrosine kinases. The Cbl proteins interact with phosphotyrosine residues on activated tyrosine kinases via a speciﬁc SH2 domain (see Section 9.1.5), and they contain a RING ﬁnger domain that mediates the binding of E2 enzymes.

& Cbl: — Single subunit E3 — Contains SH2 domain — Ub-labeling of tyrosine kinases — Oncogenic variants known.

In the complex formed, a transfer of ubiquitin to the kinase occurs, and the kinase is thereby targeted for endocytosis and also for degradation (Figure 2.17). The regulation of Cbl protein activity is complex; Cbl seems to be an inactive E3 enzyme until it encounters the activated kinase that it will degrade. On interaction with the active kinase, the E3 activity of Cbl is induced, which leads to ubiquitination and downregulation of the kinase. Although the mechanism of this

Figure 2.17 Cbl-induced ubiquitination of which is a E3 ubiquitin ligase. The RING finger transmembrane receptors. Ligand binding to motif of Cbl mediates the binding of an E2 receptor tyrosine kinases triggers enzyme from which ubiquitin is transferred to autophosphorylation on the cytoplasmic acceptor lysine residues of the receptor, region of the receptor. The Tyr-phosphates are inducing its internalization and proteasomal bound by an SH2 domain of the Cbl protein, degradation. 78 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

activation has not been determined precisely, the interaction between the Cbl protein and its target is crucial to induction of the Cbl E3 activity. The signaling molecules that are downregulated by the Cbl proteins include receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) (Chapter 10) and nonreceptor tyrosine kinases such as Zap 70 (see Section 13.3). The central importance of the Cbl proteins for cellular regulation is highlighted by the observation that oncogenic forms of Cbl have been found in mouse retroviruses, and Cbl is ascribed the function of a tumor suppressor.

2.8.4 Degradation in the Proteasome

The degradation of protein–ubiquitin conjugates occurs in an ATP-dependent reaction within a large protease complex, the 26S proteasome [26]. The substrate protein is degraded to peptides in the 26S proteasome, while the ubiquitin is released and again is available to form protein conjugates.

& 26S Proteasome: — Central proteolytic machinery — 19S regulatory particle — 20S core particle.

A major prerequisite for proteasomal degradation is the presence of poly-Ub chains comprising at least four Ub chains, linked by K48 isopeptide bonds, on the substrate protein. The 26S proteasome is composed of two protein aggregates, a 20S core particle and one or two 19S regulatory particles (Figure 2.18a). The main proteolytic component of the 26S proteasome is the 20S proteasome. The 19S particle functions as an ATP-dependent activator that is necessary for providing substrate access to the 20S proteasome. The 20S proteasome is also known to associate with two other multiprotein complexes, namely the 11S activator and the PA200 activator, neither of which require ATP for their activating function.

2.8.4.1 The 20S Proteasome The catalytic core of the proteasome is the 20S particle, which consists of four stacked heptameric rings, with two a-type rings surrounding two b-type rings, in a b b a an 7 7 7 7 pattern. The four rings form a barrel-like structure with a central chamber where proteolysis takes place, and a surface that promotes substrate unfolding (Figure 2.18b). Although the a and b subunits share structural similarity, there are important functional differences associated with their N-termini. The N-terminal threonine residues of b subunits of the inner rings contribute to the proteolytic activity, whereas the N-termini of the a subunits form a gate at the center of the a ring that prevents substrate entry. 2.8 Ubiquitin Modification of Proteins 79

Figure 2.18 Structure of the proteasome. (a) whereas the two central rings are composed of The 26 S proteasome; (b) The 20S core seven different b subunits (right image, view complex from yeast. The 20S proteasome is from the center). The active sites reside within composed of a stack of four rings, each the central chamber (shadow ring) of the 20S composed of seven subunits. The two outer proteasome at subunits b1/Pre3, b2/Pup1 and rings are incorporates seven different a b5/Pre2 (marked by circles). subunits (left image, view from the top),

& Eukaryotic 20S particle: — 2 7 distinct a subunits — 2 7 distinct b subunits — Proteolytic activity: N-terminal nucleophile Hydrolase Distinct catalytic centers.

In the 20S proteasome from eukaryotes, each of the two outer rings contains seven different a subunits, whereas each of the two inner rings is built up from seven different b subunits. All subunits occupy speciﬁc positions in the 20S particle. The presence of the protease center in the central channel ensures that the proteolysis is compartmentalized and shielded from the surrounding media. 80 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

An N-terminal threonine has been identified as an essential active site residue in three of the seven b subunits. The OH-group of the threonine functions as a nucleophile during hydrolysis of the peptide bond. A similar mechanism of hydrolysis has been shown for other hydrolases which, because of this property, are now included in the family of N-terminal nucleophile hydrolases. A distinct feature of the vertebrate proteasome is the possibility to exchange specific subunits, thereby generating proteasome variants. By exchange of b subunits, different 20S complexes can form which function in specific degradation reactions. In the proteasome that is involved in processing antigenic peptides, the catalytic reactions are performed by b subunits, which is different from the b subunits of the classical 20S proteasome. This specific proteasome is also called the “immunoproteasome.”

2.8.4.2 The 19S Activator The 20S proteasome is an inherently repressed enzyme because of its closed architecture, and it requires the presence of activator complexes to open the gates formed by the N-termini of the a subunits. The most broadly conserved activator complex is the eukaryotic 19S activator [27], that is responsible for the recognition of ubiquitinated protein substrates and their funneling into the proteolytic cavity.

& 19S regulatory particle: — Substrate selection — Recognizes Ub–protein — Removes Ub — ATP-dependent substrate unfolding — Translocates substrate.

The19S activator associates with the top or bottom surface of the 20S proteasome to form the 26S proteasome, and triggers an opening of the gate to allow the access of unfolded protein substrates. The following activities are required for substrate delivery to the proteolytic chamber:

Recognition of ubiquitinated substrates Removal of the ubiquitin chains by deubiquitinating enzymes Unfolding of the substrate protein under consumption of ATP Translocation of the substrate into the 20S particle.

The 19S regulatory particle from yeast, which is the most extensively studied, is composed of about 19 different subunits. Among these are six ATPase subunits, scaffolding proteins, and proteins involved in Ub recognition and removal. The cooperation of the different subunits of the 19S activator in the ATP-dependent substrate channeling is not completely understood, however, and the structure of the 19S activator remains a matter of debate. 2.8 Ubiquitin Modification of Proteins 81

2.8.5 Nonproteolytic Functions of Ubiquitin Conjugation

In addition to marking proteins for degradation, ubiquitin conjugation has been recognized to serve many nonproteolytic functions. The unifying mechanism underlying both the proteolytic and nonproteolytic functions of ubiquitin and ubiquitin chains is that they serve as three-dimensional signals that are recognized by many different sensors embedded in a large variety of signaling and effector proteins. When these sensors are embedded in the proteasome and proteasome-accessory factors, polyubiquitinated proteins are delivered to the proteasome for degradation. However, when these sensors are embedded in components of the endocytic and vesicle-trafﬁcking machineries, the ubiquitinated proteins are delivered to the lysosome for destruction. When the ubiquitin sensors are embedded in components of protein kinase complexes, DNA repair complexes, chromatin-modifying assemblies, or transcription factors, the signals of protein ubiquitination serve as distinct functional outputs that do not involve proteolysis [28]. These properties of the Ub signaling system provide for a regulatory tool that is comparable to protein phosphorylation in many respects, as for example the large number of conjugating enzymes involved, the variety of mechanisms for decoding the signal, and the large enzyme repertoire for removal of the Ub signal.

& Nonproteolytic Ub functions: — Trafficking — Repair, replication — Transcription — Protein biosynthesis.

2.8.5.1 Deubiquitination Most importantly, Ub modiﬁcation is a dynamic and reversible process. Ubiquitin on target proteins and poly-Ub chains can be disassembled by deubiquitination enzymes, of which a large number is known. The human proteome harbors about 90 different deubiquitination enzymes, which compares well with the large number of human protein phosphatases (see Section 10.4.1).

2.8.5.2 Multiplicity of Ub Conjugation The nonproteolytic functions of Ub are speciﬁed by the number of Ub molecules attached – that is, monoubiquitination versus polyubiquitination – and by the length and linkage type of poly-Ub chains. In polyubiquitination, the “classical” linkage type is conjugation through K48-linked Ub chains, which labels proteins for proteasomal degradation. However, in addition to K48, Ub can be conjugated through any of the other six lysine residues on its surface (Lys6, Lys11, Lys27, Lys29, Lys33, and Lys63), forming atypical Ub chains of various lengths and shapes. Furthermore, conjugation of the 82 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

ubiquitin chains can occur through isopeptide bonds between the N-terminus of one ubiquitin and the C-terminus of another (M1 type).

& Ub conjugation: — Mono-Ub — Poly-Ub: Linear Branched Mixed. Poly-Ub linkages: — Typical: K48: proteasome. — Atypical: K11: APC K63: DNA repair M1: NFkB.

Ubiquitination through alternative lysine sites is now considered as a protein modiﬁcation code, which is used to sort different ubiquitination products to different destinations [29]. The following variations in the linkage and structure of Ub-modiﬁcations have been described (Figure 2.19):

Linkage of poly-Ub chains through any of the seven lysines of Ub. Besides the lysine residues of ubiquitin, the N-terminus of ubiquitin can also be used as an attachment point, thereby generating linear or M1-linked polyubiquitin chains. Use of divergent lysines to form mixed-linkage or branched Ub chains containing two different types of linkage. Formation of heterologous chains by linking Ub with Ub-like proteins. Attachment of multiple mono-Ub moieties to a target protein.

Which of the atypical Ub chains is formed depends on the nature of the E2 and E3 enzymes involved, and to some extent also on the structure of the target protein. The various atypical Ub chains adopt distinct conformations and thus form a signal that is recognized by a Ub-binding domain (UBD) located on an Ub receptor that functions as a sensor to decode the signal. A large number of different UBDs has been identified that are embedded in many different Ub receptors, which translate the ubiquitin code to specific cellular outputs. The particular linkage between two ubiquitin moieties through a specific lysine residue of one ubiquitin and the C-terminus of the other ubiquitin creates a unique binding surface that is specifically recognized by specialized UBDs. As summarized below, the various linkage types result in different shapes of the poly-Ub chain (Figure 2.20) and serve distinct functions: 2.8 Ubiquitin Modification of Proteins 83

(a) (b) Mono-ubiquitination Homotypic Poly-Ub Branched Homotypic Poly-Ub

Ub K63 Ub Target Ub Target Ub Target K48 K48 Linkage:

M1, K6, K11, K27, K29, K33, K48, K63

Ub Su Target Su Su Ub Target

Figure 2.19 A schematic model of possible forks; (c) Heterologous chains are formed ubiquitin chain formations on a target protein. between Ub and ubiquitin-like proteins, for (a) Homotypic atypical and typical chains, such example the small ubiquitin-like modifier as lysine 6 (Lys 6)-, Lys 11-, Lys 27-, Lys 29-, Lys (SUMO) 2; (d) Multiple monoubiquitination 33-, Lys 48-, and Lys 63-linked Ub chains; (b) moieties represent a subtype of multivalent Mixed-linkage atypical chains are formed by chain-like Ub signals owing to the spatial the use of different lysines for sequential Ub organization of multiple monoUb molecules conjugation, leading to the formation of attached to the substrate. K: lysine; M1: bifurcated chains, for example, Lys48/Lys29 methionine 1.

K48-linked chains: These chains have a compact shape. It is the classical linkage type and directs proteins to proteasomal degradation. K11-linked chains: This linkage type is also of a more compact structure. It is formed for example, by the APC of higher eukaryotes and serves as a label for proteasomal degradation. K63-linked chains: Ub-conjugation through K63 produces linear poly-Ub chains that serve as a molecular scaffold to assemble multiprotein complexes involved for example, in DNA replication and DNA repair. M1-linked chains: These are linear Ub chains created upon the formation of multiprotein complexes involved in NFkB activation (see Section 2.8.6.3). 84 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.20 The major alternative K-linkages in Poly-Ub-chains. The major alternative K-linkages of linear poly-Ub chains are shown with examples of their function in signaling processes. TNF-R, tumor necrosis factor receptor (see Section 14.3).

Each of the various linkage types is formed by speciﬁc E2/E3 combinations, and they are recognized by cognate UBDs on partner proteins. The spectrum of cellular activities regulated by atypical ubiquitination is large and comprises the following processes:

Endocytosis Recycling of cell surface proteins Regulation of protein kinases Protein trafﬁcking Protein import into cellular organelles Repair and replication of DNA Processing and presentation of antigens Assembly of ribosomes.

2.8.5.3 Ub-Binding Domains and Ub Receptors To date, 11 families of UBDs have been identiﬁed [30], and these show a high variability and complexity in their mode(s) of ubiquitin binding. Some proteins 2.8 Ubiquitin Modification of Proteins 85 functioning as Ub receptors contain two copies of the same UBD and thus can bind two Ub molecules, whereas others have multiple UBDs of different classes. Interestingly, many UBDs are required for ubiquitination of the protein within which they are carried. The number of proteins carrying UBDs is large. Examples of UBD-carrying proteins include components of the 19S regulatory particle of the proteasome, deubiquitinating enzymes, E3 ligases, DNA polymerases, guanine nucleotide exchange factors, ands transcription factors.

& Ubiquitin receptors: — Contain Ub-binding domains (UBDs) — Recognize mono- or poly-Ub chains.

In the following subsection, one selected example of nonproteolytic functions of Ub-conjugation will presented.

2.8.5.4 Ub-Conjugation in Regulation of the NFkB Pathway Atypical poly-Ub chains have been shown to be involved in the activation of protein kinases that are part of membrane-receptor signaling pathways. A prominent example is signaling in the NFkB pathway [28], where the transcription activator NFkB regulates a variety of genes involved in the immune response and the inflammatory process. NFkBisrequiredforthe expression of genes for the light x-chain of immunoglobulins, for interleukin (IL)-2 and 6, and for interferon b (see Chapter 13), and its activation is regulated by signaling pathways induced by a variety of external stimuli such as cytokines, antigens, and viral stress. The NFkB family of transcription factors consists of five members – NFkB1 (p50), NFkB2 (p52), RelA (p65), c- Rel, and RelB – which bind as either homodimers or heterodimers to NFkB sites within the promoters of target genes and regulate transcription through the recruitment of coactivators or corepressors [31]. NFkB1 and NFkB2 are formed by proteolytic processing of precursor proteins of 105 and 100 kDa (p105 and p100), respectively. The function and regulation of NFkB is shown schematically in Figure 2.21. Transcription-activating forms of NFkB are heterodimers consisting of one p50 or p52 subunit and one Rel subunit. NFkB proteins are normally sequestered in the cytoplasm by specific inhibitor proteins, named inhibitor of NFkB(IkB), that mask the nuclear translocation signal of the heterodimers, thus preventing transport into the nucleus.

& NFkB activation involves: — A canonical pathway — A noncanonical pathway. 86 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.21 Schematic model of TNFa- of linear chain binding by NEMO ○3 . Binding of mediated NFkB activation, illustrating the NEMO to linear ubiquitin chains has been involvement of distinct linkages in poly-Ub hypothesized to cause a conformational chains. TNFa-induced trimerization of TNF change that favors autophosphorylation of receptor 1 (TNF-R1) at the cell membrane IKKa and IKKb subunits, leading to kinase results in recruitment of TRADD, RIP1 and activation ○4 . Upon activation, IKK TRAF2 ○1 , which in turn provides a binding and phosphorylates IkB ○5 , which retains NFkB activation platform for the E3 RING ligases composed of p65 and p50 in an inactive state cIAP1 and cIAP2. cIAPs polyubiquitinate in the cytoplasm. Phosphorylated IkBis b components of the TNFR complex, including specifically recognized by the SCF TrCP E3 RIP1, TRADD, TNF-R1 and the cIAPs RING ligase complex as a substrate for Lys48- themselves, through Lys63 and chain linkages, linked polyubiquitination. Subsequent including Lys11, which promotes recruitment proteasomal degradation of IkB releases the of TAK1---TAB1---TAB2, and LUBAC complexes p65---p50 dimer ○6 , which then translocates through their respective ubiquitin-binding into the nucleus and induces expression of domains ○2 . LUBAC linearly polyubiquitinates target genes ○7 . Behrends 2011 [32], figure 3a. NEMO in IKK complexes, which in turn yields Reproduced with permission of Nature further recruitment of IKK complexes by means Publishing Group. 2.8 Ubiquitin Modification of Proteins 87

Poly-Ub linkage types: — K11 — K48 — K63 — M1.

The signal pathway that leads to phosphorylation and subsequent degradation of NFkB has been well characterized for the cytokines IL-1 and for tumor necrosis factor (TNF) [33]. Two types of NFkB signaling have been identiﬁed, namely canonical and noncanonical: The canonical pathways respond to numerous stimuli, including ligands for antigen receptors, cytokine receptors such as tumor necrosis factor receptor (TNFR) and Toll-like receptors (see Chapter 14). The different signals converge to an IkB kinase complex (IKK), composed of catalytic IKKa and IKKb subunits and a regulatory subunit named NEMO. Upon activation, IKK phosphorylates IKKa; this phosphorylation is the signal for the ubiquitination and degradation of IkB. NFkB proteins are thus released from the inhibited state to translocate in the nucleus and activate transcription of target genes. The noncanonical pathway involves different signaling molecules and leads to the predominant activation of p52/RelB heterodimers. This pathway is based on the proteasome-mediated processing of p100 to p52.

The activation of NFkB is a prototype example of the versatility and multiplicity of Ub-modiﬁcation in the organization of signaling complexes as it involves different types of Ub-linkages. The complexity of NFkB activation, using the example of TNFR-mediated NFkB signaling [32], is shown schematically in Figure 2.21. Importantly, the activation of TNFR triggers the activation of TNFR- associated E3 ligases (named cIAP) that ubiquitinate further components of the TNFR signaling complex. In these processes, ubiquitination participates in the activation of NFkB at multiple points, and different types of Ub-linkage are used to specify distinct functions:

K11-linked Ub chains: Components of the TNFR complex, including the adapter Rip1 are polyubiquitinated via K11-linked Ub chains. K63-linked chains: Polyubiquitination of Rip1 via K-63-linked Ub chains leads to activation of the protein kinase TAK1 that phosphorylates and activates IKK. Furthermore, the NEMO component of the IKK complex becomes polyubiquitinated by K63-linked Ub chains, which serves to recruit IKK to the TNFR signaling complex. M1-linear Ub chains: This type of Ub-linkage is found on another component of the TNFR complex, named LUBAC. The linear Ub chains formed are also involved in the activation of IKK. 88 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

K48-linked chains: Activated IKK phosphorylates IkB which leads to K48-linked polyubiquitination and proteasomal degradation of IkB. NFkB proteins are now allowed to translocate to the nucleus for transcription activation. Precursor processing in the noncanonical pathway: The p52 subunit of NFkB results from the proteolytic processing of a 100 kDa precursor protein (p100) in the cytosol. The processing requires the K48 polyubiquitination of p100, mediated by the 26S proteasome.

2.8.6 Ubiquitin-Like Proteins: Sumo-Modification

Ubiquitin has been the founding member of a family of small proteins that are attached to target proteins for regulatory purposes. These proteins, known as Ub-like proteins, function as reversible tags that are attached to their target by a conjugation and deconjugation pathway that is similar to ubiquitination. The Ub-like proteins share a common fold, named the Ub-fold (Figure 2.22), and can be traced back in evolution to bacterial ancestors (for a review, see Ref. [34]). Of the more than ten different Ub-like proteins identiﬁed to date in humans, the small

Figure 2.22 The ubiquitin fold. The structure of members of the ubiquitin protein family: (a) Ubiquitin; (b) Nedd8; (c) Sumo-1; (d) ThiS. 2.8 Ubiquitin Modification of Proteins 89 ubiquitin-related modiﬁer (SUMO) family has received most attention [35]. Another important Ub-like protein is Nedd.

& SUMO: — Ub-like protein — Attached to lysine residues — E1, E2 enzymes.

SUMO regulates: — Transcription — Protein trafficking.

In mammals, three SUMO proteins are known – SUMO-1, SUMO-2 and SUMO-3 – that are employed in a transient and reversible process to generate signals for regulating the activity and function of a large number of proteins. SUMO-1, a 98-amino acid polypeptide, is covalently linked by an isopeptide bond to lysine residues in proteins in a process analogous to, but distinct from, ubiquitination. E1- and E2-like enzymes are responsible for attachment of the SUMO moiety to lysine residues of the target protein. In most cases, SUMO is attached as a monomer to a substrate, although poly-SUMO chains have been also found. When compared to ubiquitination, sumoylation is more sequence-specific and requires a particular amino acid in the neighborhood of the lysine to be modified. Importantly, sumoylation is a reversible process through the action of SUMO proteases that function as isopeptidases to deconjugate SUMO from substrates. The following general mechanisms of SUMO regulation have been identified: (i) induction of conformational changes; (ii) mediation and modulation of protein– protein and protein–DNA interactions; and (iii) competition with other lysine PTMs. SUMO modification shares most of these regulatory mechanisms with Ub modification and with the other Ub-like proteins. The functions of sumoylation are diverse and are thought to affect as many proteins as does ubiquitination. One major role of sumoylation has been ascribed to the regulation of transcription. Many proteins with functions in transcription regulation are reversibly modified by sumoylation, and in most cases the covalent attachment of SUMO to transcription factors such as nuclear receptors will inhibit transcription. However, the mechanisms of transcription repression appear to be diverse, and include for example, the recruitment of transcriptional corepressors such as histone deacetylases (see Section 4.5.2). Here, the SUMO modification serves as a label for the association of proteins that contain domains with binding specificity for the SUMO modification. Other functional consequences of sumoylation include regulation of the trafficking of proteins. For example, SUMO modification of RanGAP1 (Section 10.1.5) regulates its subcelluar localization by targeting it to the nuclear pore 90 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

complex. Many examples are also known where sumoylation competes with other lysine modiﬁcations. For example, SUMO modiﬁcation of the inhibitor IkB (see above and Figure 2.21) occurs at the same Lys-residues that are used for the attachment of ubiquitin molecules, thus precluding ubiquitination. As a result, IkB is stabilized and remains bound to NFkB, thus inhibiting the nuclear translocation of NFkB and transcriptional activation.

2.9 Lipidation of Signaling Proteins

Summary Many signaling processes are intimately linked to the cell membrane, which requires the recruitment of signaling proteins to membranes or membrane compartments. For this purpose, signaling proteins may carry covalently attached lipid anchors that mediate stable and regulatable association with the cell membrane. Examples of lipid anchors include the attachment of myristoic acid, palmitoic acid and farnesyl or geranyl-geranyl moieties. Often, dual lipidation is required for the stable membrane association of signaling proteins.

The signaling function of many signaling proteins depends on their association with the cell membrane. As outlined in Section 3.2.3, cells have available a palette of tools to achieve a stable and regulatable membrane association.

& Lipid anchors: — Fatty acids — Isoprenoids — Cholesterol — Complex phospholipids.

One central and widely used tool for membrane anchoring is the posttranslational attachment of hydrophobic residues, such as fatty acids, isoprenoids (Figure 2.23) or complex glycolipids to speciﬁc amino acid side chains of target proteins. These lipid moieties favor membrane association by increasing the afﬁnity of the protein to the membrane. Because of their hydrophobic nature, the membrane anchors insert into the phospholipid bilayer and thus mediate membrane association of the protein. In order to achieve a strong and stable membrane association, however, more than one lipid anchor are typically used. Whilst the lipid anchors serve several functions in cell signaling, their main purpose is to promote membrane association of signaling proteins. Many signaling events occur in close association with the inner side of the cell membrane, or at the organelle membranes. Lipid anchors target signaling components to the membrane (as is the case for the cytoplasmic protein tyrosine kinases) so that they can 2.9 Lipidation of Signaling Proteins 91

Figure 2.23 Structure of lipid anchors and representative examples for lipid-modified signal proteins. For heterotrimeric G proteins and GRKs, see Chapter 6. For Ras and Rho proteins, see Chapter 10. participate in membrane-associated signaling pathways. Importantly, the lipid anchors can be used in a dynamic fashion to recruit signaling proteins in a regulated manner into signaling pathways. Furthermore, protein lipidation helps to target signaling proteins to membrane subdomains such as lipid rafts, thereby facilitating the formation of large signaling complexes and localizing signaling events to speciﬁc subcellular sites. Other functions of membrane anchors include the vesicular transport of proteins (e.g., in neurons) and the regulation of enzyme activity (see Section 2.9.6).

2.9.1 Myristoylation

Myristoylated proteins contain a saturated acyl group of 14 carbons, myristic acid

(n-tetradecanoic acid) added via an amide bond to the amino group of the NH2- terminal glycine residue. The reaction is catalyzed by the enzyme N-myristoyl- transferase; typically, this occurs cotranslationally after the initiating methionine 92 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

has been cleaved by an aminopeptidase, though it may occur posttranslationally when an internal glycine residue is exposed by proteolytic cleavage.

& Myristic acid anchor: — C14 fatty acid anchor — At N-terminal glycine — Stable modification.

The consensus sequence for N-myristoylation is Gly-X-X-X-Ser/Thr (X is any amino acid), where the residue following the glycine is often a cysteine. A clumping of basic amino acids at the N terminus can serve as an additional signal for myristoylation. N-myristoylation is generally considered a constitutive process and a permanent modiﬁcation that promotes weak and reversible protein–membrane and protein– protein interactions. Typically, myristate acts in concert with other mechanisms to regulate signaling functions at membranes. As shown below, the myristate anchor may also function as a switch during regulated membrane anchoring. Examples of myristoylated proteins include the cytoplasmic protein tyrosine kinases (family of the Src-kinases; see Section 10.3.2) and the a-subunit of the heterotrimeric G proteins (see Section 7.5.6).

2.9.2 Palmitoylation

Palmitoylated proteins contain a long-chain fatty acid, such as palmitic acid (n-hexadecanoic acid) connected to the protein via a labile thioester bond to cysteine residues. Other long-chain fatty acids such as stearate and oleate have also been found to be incorporated in S-acylated proteins. The thioester bond of S-acylated proteins is less stable than the amide bonds of the myristate anchor; such lability conveys a reversible character to the modiﬁcation and thus permits regulation of membrane association [36].

& Palmitic acid anchor: — At internal Cys — Thioester linkage — Reversible modification.

The distribution of signal proteins between the membrane and cytosol can be regulated via a cyclic process of acylation and deacylation, making the reversible S-acetylation of signal proteins an important tool for the modulation or regulation of signaling pathways. The enzymes involved in acylation and deacylation are the protein acyl transferases and the acyl protein thioesterases. There is no well-defined consensus sequence for palmitoylation other than a requirement for cysteine. Interestingly, palmitoylation sites have been identified 2.9 Lipidation of Signaling Proteins 93 within well-characterized protein domains such as the pleckstrin homology (PH) domains of phospholipases D1 and D2. Fatty acid attachment (palmitate, stearate) through oxyester bonding to serine or threonine has also been recognized. Furthermore, several secreted eukaryotic proteins are modified at the e-amino group of lysine with myristate or palmitate through an amide linkage. Removal of palmitate by thioesterases occurs both constitutively and in response to signals.

2.9.2.1 Functions of Palmitoylation Similar to other lipid modifications, palmitoylation promotes the membrane association of otherwise soluble proteins with functions in signaling pathways. In many cases, palmitic acid-mediated membrane attachment is essential for the participation of these proteins in signaling events. The function of palmitoylation, however, ranges beyond that of a simple membrane anchor. Trafficking of lipidated proteins from the early secretory pathway to the plasma membrane is dependent on palmitoylation in many cases. In addition, modification with fatty acids impacts the lateral distribution of proteins on the plasma membrane by targeting them to lipid rafts [37]. The family of proteins modified with thioester-linked palmitate is large and diverse, and includes transmembrane-spanning proteins and cytoplasmic proteins that require membrane-association for their function. Examples of palmitoylated signaling proteins include the Ras and Rho regulatory GTPases (see Chapter 11), nonreceptor tyrosine kinases such as p56lck, the adapter protein PSD-95 (see Chapter 10), the a-subunit of the heterotrimeric G proteins (see Section 7.5.6), the RGS proteins (see Section 7.5.7), and the enzymes phospholipase D1 and D2.

2.9.3 Farnesylation and Geranylation

ﬁ Proteins with an isoprenoid modi cation possess either a C15-farnesyl residue or a C20-geranyl-geranyl residue, both of which are bound via a thioester linkage to a cysteine residue. As with myristoylation, these are constitutive, stable modiﬁcations performed by farnesyl or geranyl transferases. The isoprenylation occurs at the Cys-residue of the consensus sequence Cys-A-A- X-COOH, whereby the nature of the C-terminal X-residue determines whether farnesylation or geranylation occurs (Figure 2.24).

& Prenyl anchors: — Farnesyl (C15) — Geranyl-geranyl (C20) — At C-terminal Cys via thioether bond.

The prenyl groups are donated by the two isoprenoids farnesyl pyrophosphate and geranyl-geranyl pyrophosphate, that are derived from the 94 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.24 Farnesylation at the C-terminus. enzyme is farnesyl transferase. Subsequently, The signal sequence for farnesylation is the C- the three C-terminal amino acids are cleaved terminal sequence CAAX. In the first step, a (A: alanine, X: any amino acid) and the farnesyl moiety is transferred to the cysteine in carboxyl group of the N-terminal Cys-residue the CAAX sequence. The farnesyl donor is becomes methylated. farnesyl pyrophosphate and the responsible

mevalonate pathway (see textbooks for details). Thereafter, the three C-terminal residues are removed by the prenylation-dependent endoprotease Rce-1 and the new COOH-group of the Cys-residue is methylated to increase the hydrophobicity of the C-terminus. The importance of protein prenylation is underscored by the nature of the estimated more than 300 prenylated proteins in the human proteome, many of which participate in a multitude of signaling pathways. The isoprenoid modiﬁca- tion can be found, among others, on the Ras protein and other members of the Ras superfamily (see Chapter 11), as well as with the a-subunit of G proteins (Section 7.5.6). The bc-complex of G proteins is also associated with the membrane via geranylation. A twofold geranylation is found on two Cys residues of the Rab protein (Section 11.9.2). In addition to promoting membrane association, other functional aspects of protein prenylation have been appreciated. Prenylation can also serve to mediate protein–protein interactions, and has a role in protein trafﬁcking. 2.9 Lipidation of Signaling Proteins 95

2.9.4 Dual Lipidation

Many signaling proteins are dually lipidated, showing both myristoylation and palmitoylation, prenylation, or dual palmitoylation. The dual lipidation is explained by the strong membrane binding mediated by two lipid anchors. It is now generally accepted that any single acylation or prenylation is unable to confer a stable membrane association, indicating that an additive or cooperative effect between intrinsic anchoring motifs drives the membrane localization.

& Dual lipidation: — For example, myristoic plus palmitoic anchor — Enforces membrane attachment.

A dynamic model has been proposed that accounts for the speciﬁc association of dually lipidated proteins with the membrane. According to the “kinetic bilayer trapping” hypothesis, proteins with a single lipid anchor only transiently and weakly associate with the membrane at many sites. A singly acylated protein that reaches the membrane will be rapidly palmitoylated by a membrane-associated palmitoyl transferase and will then remain stably attached to the membrane. Only two examples among a long list of dually lipidated proteins will be mentioned here. The lipidation of cytoplasmic protein tyrosine kinases such as p56lck includes both myristoylation and palmitoylation (Section 10.3.1). H-Ras protein (Section 11.5.5) requires, apart from C-terminal farnesylation (see above), a palmitoyl modiﬁcation in order to bind to the plasma membrane. In both examples, the fatty acid anchors play an essential role in the signal transduction.

2.9.5 Cholesterol Membrane Anchor

A cholesterol modiﬁcation has been demonstrated for the Hedgehog (Hh) protein, which is an extracellular signaling protein with functions in a diverse array of patterning events of metazoan tissues. The Hh family of proteins are found on the surface of cells and can be secreted for signaling to distant cells. Several PTMs are required in order to gain full activity of Hh proteins. & Cholesterol anchor: — For example, Hedgehog protein — Ester bond to C-terminal glycine.

In a maturation process Hh protein performs an autocatalytic cleavage, generating an N-terminal polypeptide containing all of the signaling functions (see Figure 2.25). During cleavage, a cholesterol moiety is attached covalently by an ester function to the C-terminal glycine moiety of the signaling domain. The hydrophobicity of the cholesterol–Hh conjugate is further increased by the addition 96 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

Figure 2.25 Autoprocessing and cholesterol-modification of Hedgehog protein.

of a palmitic acid residue to the N-terminus of the cleavage product. The presence of the cholesterol moiety confers a very stable membrane association to Hh, and it is assumed that the cholesterol modiﬁcation serves to target Hh to distinct subcellular compartments [38]. The cellular mechanisms used for the handling and delivery of cholesterol-modiﬁed proteins are largely unknown.

2.9.6 The Switch Function of Lipid Anchors

Lipid anchors can participate in a dynamic fashion in membrane anchoring, and may thereby actively participate in cell signaling.

& Switch function of lipid anchors: — Signal-dependent exposure of lipid anchor. 2.9 Lipidation of Signaling Proteins 97

Figure 2.26 Model of the switch function of states may be controlled by specific cellular þ the myristoyl anchor in signal proteins. The signals (e.g., Ca2 , GDP/GTP exchange). In myristoyl anchor of a signal protein can exist the membrane-associated form, interactions either in a state accessible for membrane with membrane-bound effector proteins insertion or in a state buried in the interior of become possible and the signal can be the protein. The transition between the two transduced further.

Currently, two ways are known by which the myristoyl anchor can function as a switch in cell signaling:

Myristoyl-ligand switches: The orientation of the myristoyl moiety relative to the protein to which it is attached may be controlled by ligand binding. Depending on the presence of the ligand, the signaling protein can exist in a conformation where the lipid anchor is buried in the hydrophobic interior of the protein or in a conformation where it is exposed on the protein surface and accessible for membrane insertion. Membrane insertion of these proteins is reversible, and is regulated by a speciﬁc ligand in a signal path-controlled manner (Figure 2.26). þ Examples are the Ca2 -myristoyl switch of recoverin (Section 8.7.2) and the GTP- myristoyl switch of the Abl tyrosine kinase (Section 10.3.3). In both cases, ligand- induced conformational changes of the signaling protein are coupled to membrane binding. Myristoyl-electrostatic switches: Another type of myristoyl switch has been reported for the MARCKS proteins, which are substrates of protein kinase C (Section 9.5). The membrane binding of the MARCKS proteins is mediated by myristate plus basic motif. Protein kinase C phosphorylation within the basic motif introduces negative charges into the positively charged region. This reduces the electrostatic interactions with the acidic phospholipids and results in displacement of the MARCKS proteins from the membrane and into the cytosol. 98 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.9.7 The Glycosyl-Phosphatidyl-Inositol (GPI) Anchor

The glycosyl-phosphatidyl-inositol (GPI) anchor is a unique lipid anchor for protein attachment to the extracellular side of cells [39]. The core structure of this anchor consists of ethanolamine phosphate, trimannoside, glucosamine and inositol phospholipids, in this order. The anchor is linked to the C-terminus of the protein by the ethanolamine head (Figure 2.27).

& GPI anchors: — Extracellular anchoring of proteins — Examples: proteases, prion protein.

GPI-anchored proteins are found ubiquitously in eukaryotes, where they are involved in the uptake of nutrients, cell adhesion, and cell–cell interactions in the immune system. In T lymphocytes, GPI-anchored proteins participate in signal

Figure 2.27 Typical structure of a glycosyl phosphatidyl inositol (GPI) anchor. Ins, inositol; GlcN, 20-amino-20-deoxyglucose; Man, mannose; Etn, ethanolamine, P, phosphate. Questions 99 transduction processes which lead to the activation of T lymphocytes. Examples of GPI-anchored proteins include enzymes such as esterases and proteases, receptors, cell-surface antigens, and proteins with unknown functions, such as the prion protein. The GPI-anchored proteins can be released from the cell membrane by enzymatic cleavage, and are subsequently found in soluble form in the serum. Furthermore, GPI-anchored proteins can be released in the form of exosomes (extracellular lipid vesicles), and can be transferred to other cells in this form.

Questions 2.1. What are the major functional domains of signaling proteins? Give an example of a central signaling protein and explain the functional characteristics of such domains. 2.2. What is the advantage of preformed signaling complexes? Give an example of a signaling complex and describe the functional cooperation in such complexes. 2.3. Give examples of effectors that control the activity of signaling enzymes. By which mechanisms does effector binding regulate signaling enzymes? 2.4. What are the major posttranslational modifications on signaling proteins? Give the chemical structure of each group of these modifications. 2.5. By which binding properties can interaction domains be classified? Give examples of each group. 2.6. Give examples of the mechanisms used by interaction domains to interpret stored signaling information. 2.7. How is multisite protein modification used to produce distinct signals? 2.8. Phosphate groups in proteins can have many functional and structural consequences. What properties of the phosphate group are most important for its function in signaling? Explain why. 2.9. What are the major interaction domains that recognize Ser/Thr- and Tyr- phosphates? Give examples of the function of these domains in a signaling path. 2.10. What posttranslational modifications can be attached to Lys residues? Give examples of the biological functions of Lys modifications. 2.11. Which enzyme classes are responsible for Lys acetylation and deacetylation? Give examples of the biological function of Lys acetylation and deacetylation. 2.12. What is the mechanism of deacetylation by sirtuins? 2.13. What types of methylation are found on Arg, and which enzymes catalyze Arg methylation? 2.14. Describe the reactions involved in protein ubiquitination. 100 2 Structural Properties, Regulation and Posttranslational Modification of Signaling Proteins

2.15. What are the major subunits of Cullin-RING ligases? Which modifications may regulate substrate selection? 2.16. Describe the structure and function of Cbl ligase. 2.17. What types of Ub-linkage in poly-Ub chains are known? Give examples of the function of distinct types of Ub-linkages in signaling. 2.18. What are the non-proteolytic functions of Ub-conjugation? 2.19. Compare the features of Ub-conjugation with that of protein phosphorylation. Which aspects are common to the two types of modification? 2.20. Describe the functions of Ub-modification in the pathway of NFkB activation by the tumor necrosis factor receptor. 2.21. Give the structures of the major intracellular lipid anchors with examples of proteins modified by these anchors. 2.22. Which reactions are involved in prenylation? 2.23. How are proteins targeted to the cell membrane? Name the major tools and give examples.

References

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3 Organization of Signaling

3.1 Scaffold Proteins

Summary Signaling proteins with scaffolding function serve to organize and coordinate signaling events, especially at the cell membrane. Typically, scaffold proteins are devoid of enzyme activity and are of modular structure. The presence of multiple interaction domains and posttranslational modifications on scaffolds allows for the recruitment of partner proteins to form large signaling complexes in a coordinated fashion. Scaffolds may recruit different signaling enzymes at the same time, which serves to coordinate and regulate signal flow in a precise manner.

A major tool for the organization of cell signaling in time and space is the use of scaffold or adapter proteins that bring signal molecules together in a targeted fashion [1]. Typical scaffold proteins do not have enzymatic function themselves, but rather they function as a connecting link between different signal proteins, mediating a speciﬁc spatial neighborhood in signal conduction and assembling larger signaling complexes, especially at the inner side of the cell membrane.

3.1.1 General Aspects of Scaffold Proteins

From a structural view, scaffolds are extremely diverse proteins, many of which are likely to have evolved independently. Nevertheless, the diverse scaffolds are conceptually related in that they are usually composed of multiple modular interaction domains (Figure 3.1) that mediate interactions with complementary interactions motifs or posttranslational modiﬁcations (PTMs) on partner proteins. The occurrence of membrane-targeting domains, such as pleckstrin homology (PH) domains, and myristoyl modiﬁcations suggests that scaffold proteins are

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 104 3 Organization of Signaling

Figure 3.1 Domain structure of selected scaffolding proteins. PTB, phosphotyrosine-binding domain; PH, pleckstrin homology domain; P, phosphotyrosine-containing site; Pro, Pro-rich site; SH2, SH3, sarc homology domains 2 and 3. For details, see Section 10.2.1.

involved in particular in the coordination and assembly of signal complexes on the inner side of the cell membrane. Their exact domain composition and order, however, can vary widely depending on the pathway that they organize, and scaffolds are now recognized as ﬂexible platforms assembled through mixing and matching of interaction domains.

& Scaffolds: — Multivalent proteins — Multiple domains

Coordinate signaling by: — PKA — PKC — MAPKs — GPCRs — GTPases — Neuronal receptors.

Some scaffolds harbor homologous individual interaction motifs that mediate binding to particular signaling proteins. For example, the AKAP proteins (see 3.1 Scaffold Proteins 105

Figure 3.2 A model of the organization of the PLC is a b-type phospolipase C, which is the InaD complex. InaD is composed of five PDZ target of rhodopsin-activated Gqa. ePKC is an domains (PDZ1---5) which interact specifically eye-specific protein kinase C which inactivates with signaling proteins implicated in the vision the Trp channel by phosphorylation. þ process in Drosophila. TRP is a K -channel.

Section 9.3.3), which function as scaffolds in PKA signaling, all share a common short peptide motif that binds to the regulatory subunit of PKA. However, the other domains in the more than 20 different AKAP proteins known to date are highly variable, depending on which inputs and outputs the scaffold protein coordinates with PKA. Scaffold proteins can assemble and coordinate a variety of different signaling proteins and signaling paths. The first known have been the organization of protein kinase signaling such as PKA signaling and MAPK signaling (see Chapter 12). However, scaffolds also organize signaling by transmembrane receptors such as regulatory GTPases and signaling at neuronal synapses (see Chapters 7, 10 and 13). Synaptic scaffolds, such as the PDZ domain-containing protein, PSD-95 (see Section 10.2.1.2), contain a set of domains that bind to neuronal receptors [such as N-methyl-D-aspartate (NMDA) receptors], to other scaffold proteins, and to the actin cytoskeleton. These scaffolds build preformed assemblies that are precisely anchored at sites of cell–cell contact (e.g., the postsynaptic density), thus allowing cells to respond efficiently to stimuli (e.g., neurotransmitter release from the partner cell). Furthermore, scaffolds also target or anchor signaling complexes at the appropriate cellular location for receiving specific inputs. For example, the PDZ domain scaffold, InaD, organizes the visual response cascade in the fruit fly (Figure 3.2).

3.1.2 Scaffolds as Organizers of Signaling Circuits

Scaffold proteins can be thought of as multifunctional platforms that can organize a wide variety of regulatory interactions and signaling circuits between signaling 106 3 Organization of Signaling

Figure 3.3 Organization of signaling by the cAMP; (b) Scaffold proteins function to wire scaffolding proteins. (a) Different pathway input and output through alternative combinations of enzymes and substrates are possible routes; (c) Scaffold proteins can anchored by AKAP79/150 (Section 9.3.3). mediate branching of pathways to multiple Protein kinase A (PKA) stimulates calcium outputs; (d) Scaffold proteins are themselves channel (Cav1.2) currents while also negatively the targets of regulation; (e) Scaffold proteins regulating the action of b-adrenergic receptors can be the target of feedback phosphorylation (bAR) and adenylyl cyclase (AC), which are that tunes pathway responses. For example, responsible for PKA activation. PKA feedback phosphorylation of the KSR scaffold phosphorylation of b-adrenergic receptors by activated ERK blocks Raf (MAPKKK) binding leads to the desensitization and and attenuates MEK activation, thereby downregulation of these receptors, and shuts decreasing pathway output (see Chapter 12). off AC activity to cease the production of

proteins (Figure 3.3a). Some common core concepts of scaffold functions in signal organization have been recognized, as follows.

3.1.2.1 Organization of Sequential Signaling and Signaling Circuits The conceptually simplest scaffold proteins determine a specific linear input– output pathway among a set of partner proteins (Figure 3.3b) by a simple tethering 3.1 Scaffold Proteins 107 of partner molecules which have an enzyme–substrate relationship. Such enforced proximity is an important mechanism for scaffold proteins, because it increases the effective concentration of reactants and allows for efficient and specific signal transfer. For example, scaffolds of the MAPK cascade (see Chapter 12) have been shown to assemble three protein kinase modules. Furthermore, the exchange of modules within a scaffold complex can contribute to the diversity of input and outputs.

& Scaffolds: — Organize: Signaling complexes Signaling circuits Feedback loops — Are targets of regulation — May contain enzyme activity.

Scaffold proteins can also mediate pathway branching – the fanning out of signaling information to multiple outputs that are part of the assembled complex (Figure 3.3c).

3.1.2.2 Scaffolds Organize Signaling Complexes and are Targets of Regulation Most scaffolds carry multiple PTMs that serve to recruit signaling proteins into a signaling pathway and organize signaling complexes. Thereby, signaling can be turned on or off by inputs that modify the scaffold protein rather than the actual signaling enzymes (Figure 3.3d). One of the beneﬁts of scaffold-induced complex formation is that protein recruitment – and thus pathway function – can be easily regulated by external signals that modify association of other proteins with the scaffold. Numerous examples of such regulation are known; scaffolds – as for example the insulin receptor substrate (IRS; see Section 10.2.1.1) or LAT (Section 10.2.1.2) – become phosphorylated upon recruitment to transmembrane receptors, and the phosphorylation sites are then used as docking sites for the association of other signaling components.

3.1.2.3 Scaffolds Organize Feedback Loops One sophisticated role of scaffold proteins is to coordinate complex feedback loops in signaling pathways by, for example, coordinating mechanisms that can turn off the pathway. In these cases, the scaffold appears to play a central role in precisely shaping signaling response properties, such as dynamics or dose–response. For example, the KSR protein functions as a scaffold that assembles the three-tiered MAP kinase (MAPK) pathway in mammalian cells. Activation of the terminal MAPK creates a feedback loop that phosphorylates the KSR scaffold and the MAPKKK Raf. These modiﬁcations disrupt binding of the MAPKKK to the KSR scaffold and shut down pathway activation (see Section 12.2.1). Mutation of the KSR phosphorylation sites disrupts this feedback, and this results in a dissociation 108 3 Organization of Signaling

of the scaffold from the plasma membrane and abnormal pathway dynamics, including sustained pathway activation.

3.1.2.4 Scaffolds as Signaling Enzymes and Allosteric Regulators A scaffold can itself directly participate in signal transfer by delivering activating signals to components tethered to itself. Such a mechanism appears be involved in control of the MAPK pathway. The KSR scaffold (see above) has been shown to phosphorylate and activate the top component of the three-tiered MAPK cascade, namely the Raf kinase. When recruited to the KSR scaffold, Raf becomes phosphorylated by KSR and signaling to the downstream components of the cascade is further stimulated. KSR may be taken as a prime example of the sophisticated use of scaffolds in signaling, as KSR receives negative feedback inputs and this promotes Raf kinase activation.

3.2 Signal Processing in Signaling Paths and Signaling Networks

Summary The presence of multiple interaction domains and PTMs on signaling proteins allows for multiple regulatory inputs and for interactions with many protein partners, placing each signaling protein into complicated interaction and signaling networks. Branching, crosstalk, feedback circuits, control in terms of space and time are now each recognized as features that shape signal transfer in signaling networks. The challenge of how to provide for a coordinated and robust flow of information in such networks is solved by the use of PTM-induced assembly of signaling complexes, preformed signaling complexes, and by the signal-directed spatial organization of signaling at distinct subcellular compartments. Spatial control of signaling is achieved mainly by scaffolding proteins, lipid-modification, and the phosphorylation-dependent translocation of signaling proteins to membrane compartments. Another major control of signaling in networks uses a timely ordered sequence of reactions within the interaction networks.

Typically, a large number of signaling components participate in the transduction of an extracelluar signal into intracellular biochemical reactions that define the endpoint of signal transduction. Traditionally, in order to characterize and describe a signal transduction pathway, the number and type of signaling components involved, as well as their linkages, have been used. However, it is increasingly recognized that the multivalency and modular structure of signaling proteins, multisite modification, as well as the existence of subtypes of signaling proteins, allow different signals to enter and to be processed in the same type of signaling path, leading to variable outcomes. The description of signaling pathways in terms of their structural organization has therefore proven to become increasingly difficult. 3.2 Signal Processing in Signaling Paths and Signaling Networks 109

Although signaling pathways have been formerly described as being linearly organized, the presence of multiple PTMs and the modular structure of signaling proteins allows for a large number of different interactions with many means of how the signaling information can be distributed. Therefore, the features of branching and crosstalk make it necessary to describe cellular signaling in terms of networks, where several layers of signal transfer coexist that are extensively linked.

3.2.1 Specificity of Signaling

One major challenge is to uncover how the many signaling components function in concert, and how biological answers are produced in a distinct way with precise timing and at a particular cellular location. Therefore, the question regarding the specificity of signaling must be asked in terms of “which, when, and where” the interactions with signaling partners are formed and the signals are transmitted. It is now well established that cells employ a variety of sophisticated ways to ensure that distinct signals can trigger many biological answers in a cell type-specific manner. The tools for cellular signaling (as discussed in previous sections) are used in the following basic ways to channel signals through distinct routes and producing specific responses [2]:

Signal-dependent formation of protein complexes Processing of signals through preassembled multiprotein complexes Signaling in dependence of subcellular localization Temporal control of signaling processing.

3.2.2 PTM-Induced Formation of Signaling Complexes

The relaying of information from one cellular location to another often requires the dynamic formation of protein complexes. To achieve this goal, cells have evolved a variety of mechanisms, among which PTMs play a prominent role in being highly dynamic and largely reversible. As outlined previously, a multitude of PTMs can be speciﬁcally deposited and removed again from a signaling protein. so as to create a transient signal for the association of partner proteins for further signal transmission. The extensive array of PTMs found on typical signaling proteins creates modiﬁcation patterns that serve to store and relay information in a combinatorial and dynamic fashion.

& PTMs: — Transient signals for signaling complex assembly via binding to interaction domains. — Store information in the form of modifications patterns. 110 3 Organization of Signaling

One crucial aspect of signaling through PTMs is the presence of modular protein domains on partner proteins that recognize particular types of PTMs located on specific residues (see Section 2.4.2). The coupling of PTMs with PTM-interaction domains creates an attractive “decoding” mechanism for monitoring and responding to alterations in the cellular microenvironment. Currently, about 100 specialized protein-interaction domains have been identified that recognize a plethora of chemical signals. For most PTM-specific interaction domains, subtypes are known that have a core binding preference for the PTM but require specific neighboring sequences for efficient binding. The multiplicity of PTMs and cognate interaction domains allows for a high variability and for a fine-tuning of signaling processes. The recognition of PTMs by interaction domains typically serves to initiate the formation of signaling complexes that not only ensure efficient signal transmission but also allow for the downregulation and attenuation of signaling propagation. For example, signaling by RTKs involves the formation of p-Tyr on the receptor and downstream substrates or adaptors (Figure 10.4), and these modifications are recognized by SH2 domains or PTB domains to form multiprotein complexes at the inner side of the membrane. In this case, RTK phosphorylation is also used to recruit the E3 ligase Cbl for the purpose of downregulation and attenuation. Cbl recognizes the p-Tyr residues by its SH2 domain (Figure 2.17) which triggers ubiquitination, endocytosis, and subsequent proteasomal degradation of the receptor. Signaling by the tumor necrosis factor receptor (TNFR) is another example of the sequential use of PTMs to couple signaling components in a linear or branched manner. During signaling from TNFR to the transcription factor NFkB, the sequential engagement of distinct Ub-signals and a phosphorylation signal is used to release NFkB from the complex with inhibitory IkB components (see Figure 2.21).

3.2.3 Signaling through Preassembled Multiprotein Complexes

The passage of signals through preassembled multiprotein signaling complexes represents another means of managing intracellular communication. Prominent examples include signaling through receptors with associated tyrosine kinase activity (Chapter 13) and the MAPK/ERK modules (Chapter 12). The core principle of signaling through preassembled protein complexes is also widely used in transcription regulation, where preassembled multiprotein machines such as mediator, chromatin remodeling complexes are recruited to sites of transcription initiation and elongation (Chapter 4).

& Preassembled multiprotein complexes allow for: — Efficient signal transfer — Multiple regulation — Specific substrate access — Exchange of subunits. 3.2 Signal Processing in Signaling Paths and Signaling Networks 111

Conﬁguring enzymes in multiprotein complexes offers several advantages:

The precision of information ﬂow is improved. The organization in multiprotein complexes serves to segregate enzymes in a manner that prevents indiscriminate crosstalk. Enzymes in such pathways exhibit restricted substrate speciﬁcities and have limited spheres of action. In fact, their only true substrate may be the next enzyme in the cascade. Subunits of the complex may be exchanged, which may lead to different regulation and signaling properties. Scaffolding subunits may help to direct a multiprotein complex to distinct subcellular sites providing for differential signaling in space.

3.2.4 Signaling in Dependence of Subcellular Localization: Spatial Organization

The compartmentalization of enzymes in proximity to substrates is a widely used tool for spatially restricting cell-signaling events. Many proteins undergo characteristic changes of subcellular localization upon signal processing and these translocations are subjected to ﬁne-tuned regulations by a variety of mechanisms. The signal-directed translocation of signaling proteins includes shuttling between cytosol and nucleus, translocation into endosomal compartments and, most importantly, the shuttling of proteins between the cell membrane and the cytosol. Cells have available a palette of tools for the spatial organization of signaling:

& Spatial control of signaling: — PTMs, such as phosphorylation — Membrane-targeting domains — Lipid anchors — Clustering at membrane subdomains.

3.2.4.1 Spatial Control by Scaffolding As outlined in Section 3.1, typical scaffolds are hallmarked by their ability to simultaneously bind two or more signaling molecules that often have enzyme– substrate relationships. Furthermore, scaffolds often harbor domains that direct the scaffold and their associated “cargo” to the cell membrane. This can be achieved for example, by interaction with membrane-bound proteins carrying PTMs, as exempliﬁed by the recruitment of Grb2-mSos to activated RTKs via SH2- pTyr interactions (Figure 11.10). Another method of membrane recruitment of scaffolds employs PH domains (Section 8.6.1), the selectivity of which for different 112 3 Organization of Signaling

phospholipids causes PH domain-containing proteins, signaling enzymes or scaffolds sensitive to the activities of enzymes that either phosphorylate or dephosphorylate these sites on the inositol ring, such as PI3K or the lipid phosphatase, PTEN (Section 9.4). Accordingly, these enzymes can modulate the localization of downstream signaling proteins that sense distinct phospholipid products. This provides an effective means of assembling or disassembling signaling complexes in different subcellular compartments.

3.2.4.2 Spatial Control by Phosphorylation Many signaling proteins are known to shuttle between the cytosol and nucleus in dependence of their phosphorylation at distinct sites. This mechanism is used extensively to regulate the nucleocytoplasmic translocation of transcription factors. Examples include the shuttling of ERKs/MAPKs (Chapter 12) and the STAT proteins (Section 13.2.2).

3.2.4.3 Lipid Anchors The plasma membrane is a major platform for signal transduction, and many signaling proteins carry lipid anchors that are essential for stable membrane association and participation in signaling (Section 2.9).

3.2.4.4 Clustering of Signaling Proteins on the Nanoscale The architecture of the plasma membrane can dictate signaling properties by sequestering signaling proteins in both space and time. The plasma membrane is organized into ultraﬁne compartments by the engagement of transmembrane proteins with the submembrane actin skeleton. Within these compartments, distinct lipid assemblies – known as lipid rafts – can form that include short-lived complexes of glycosphingolipid and cholesterol [3]. As a result, the plasma membrane comprises a complex, nonrandom, dynamic array of lipids and protein–lipid complexes within which distinct membrane-anchored signaling complexes can form in dependence for example, on ligand binding to transmembrane receptors such as GPCRs. For example, transient, nanoscale protein clusters (nanoclusters) can form that operate as temporary signaling platforms. These clusters contain mixtures of different signaling proteins such as regulatory GTPases and protein kinases that are anchored either directly to the membrane, or by lipid-anchored regulatory proteins or subunits. This strategy is exempliﬁed by members of the Ras family of regulatory GTPases. Ras proteins are arranged on the plasma membrane as a combination of nanoclusters and freely diffusing monomers (see Section 11.5.5). The three Ras isoforms – H-RAS, K-RAS and N-RAS – use different C-terminal anchors for membrane binding: H-RAS and N-RAS undergo farnesylation and acylation, whereas K-RAS is only farnesylated and requires an adjacent polybasic domain for stable anchoring. As a result, H-RAS, K-RAS and N-RAS assemble into spatially distinct, non-mixed clusters. 3.3 Architecture of Signaling Pathways 113

3.2.5 Temporal Control of Signaling

Cellular signaling is not only organized in space but also exhibits distinct dynamic properties. The temporal aspects of signaling are governed to a large part by the transient nature of PTMs and by feedback mechanisms that limit the time window within which a signal can be received, stay active, and be transmitted further. The temporal control often involves phosphorylation, ubiquitination and subcellular translocation. A timely ordered sequence of modiﬁcation attachment, their removal, and subcellular translocation controls the time course of many signaling events. Again, signaling by the transcription factor NFkB is a prominent example of this common strategy.

& Temporal control of signaling by transient: — Phosphorylation — Ubiquitination — Subcelluar translocation.

As illustrated in Figure 2.21, the nuclear translocation of NFkBrequiresthe destruction of any inhibitory IkB subunits that sequester NFkBinthecytosol. A signal delivered by activated TNFRs leads to a timely ordered ubiquitination and phosphorylation of IkB subunits, which ﬁnally triggers a proteasomal degradation of IkB components and the release of NFkB for transport into the nucleus. In this system, the kinetics of overallsignaltransferareshapedby the opposing actions of ubiquitination versus de-ubiquitination, as well as by phosphorylation versus dephosphorylation. In order to model the dynamics of such a system quantitatively, it is necessary to know not only the reactant concentrations but also the rate constants of the individual reactions. Unfortunately, these parameters are not yet known, and quantitative modeling is not yet possible.

3.3 Architecture of Signaling Pathways

Due to the multivalency and modular nature of signaling proteins, the use of a broad array of PTMs and corresponding interaction domains, typical signaling proteins can receive and relay many different signals. Therefore, the current view of how signals are distributed and processed, and how the many signaling systems are interconnected, has changed dramatically and it is now a major challenge to describe the biological signaling networks in a feasible manner. 114 3 Organization of Signaling

3.3.1 Linearity, Branching, Crosstalk, and Networks

3.3.1.1 Linearity The classical view of signaling pathways has been that of a sequential transmission of signals in a linear signaling chain. Thereby, a signal is registered by an upstream component of a signaling chain and transmitted to the downstream component that then passes the signal on to the next protein in sequence, and so on. This description is useful to illustrate the main reaction steps in a signaling cascade, while focusing on the main steps serves to outline the logic of a signaling pathway. Consequently, many of the reaction pathways described in the following chapters of this book are still presented as reaction chains whereby signals are transmitted along linear tracts, resulting in the regulation of discrete biochemical reactions and cellular functions.

3.3.1.2 Branching Today, many signaling proteins are known which have multiple downstream reaction partners that they can activate for further signal transduction. This property leads to a branching of signaling pathways and provides for multiple outputs originating from the same type of signaling protein.

& Branching: — Signaling protein has multiple downstream partners.

Often, signal distribution to alternative routes depends on cell type and may be variable in both time and subcellular localization. In later chapters of the book, the dissipation of signals and the distribution to alternative reaction partners is only discussed for those cases where alternative reaction partners have been clearly identiﬁed.

3.3.1.3 Crosstalk Cells need to process a large number of signals at the same time, and these signals are mostly routed through different signaling pathways. The ﬂow of signals through the various pathways must be coordinated and properly balanced, which requires the presence of linkages between different pathways.

& Crosstalk: — Linkage of different pathways — Allows for the coordination of pathways.

This interdependence of signaling is also known as crosstalk. The multivalency of signaling proteins and multisite modiﬁcation allows the components of a signaling path to inﬂuence, regulate and modulate the signaling in other pathways. Many examples of crosstalk can be found in the following chapters, but of special note is the regulation of Raf kinase (which is a main component of the Ras-MAPK-pathway; see Section 11.6) by protein kinases that form part of other signaling pathways. 3.3 Architecture of Signaling Pathways 115

3.3.1.4 Networks Each signaling protein of a signaling path is subjected to regulatory inﬂuences from the same pathway or from different pathways, placing it into a network of interactions and regulatory inﬂuences which results in multifunctionality, variability, and interconnection of cellular signaling systems. Real cell signaling should therefore be described in terms of signaling networks that result from interconnections between pathways. In such a network, the same signaling protein is capable of receiving signals from many inputs; rather, it is the multivalency of the signaling proteins that forms the basis of branching and crosstalk, and it is now necessary to describe signaling in terms of signaling networks that include a large number of possible linkages within a signaling path, and between different signaling paths. The networking may occur within similar classes of signaling pathways, such as between the Rho and Ras pathway (Section

11.8), and between different pathways, such as between the Gs,a/cAMP and the MAP kinase pathway (Section 11.6). The following experimental observations serve to illustrate the complexity of intracellular signaling and signaling networks (Figure 3.4):

For a given hormone, different receptors can exist on the same or on different cells. These receptors can route the signal to different pathways, triggering very distinct reactions in different tissues or even within the same cell. An example of such a phenomenon is adrenaline, which can initiate on the one hand a cAMP- mediated signal transduction, and on the other hand an inositol triphosphate- mediated reaction. For a given receptor, signaling enzyme or adapter protein, subtypes are found which differ in their responsiveness to the incoming signal, in the nature and intensity of the reaction triggered in the cell, and in their capacity for regulation. The same secondary reaction can be triggered by different hormone-receptor þ systems and signaling pathways. This is exempliﬁed by the release of Ca2 , which can be regulated via different signaling pathways (see Chapters 7 to 9). Regulatory interaction modules or protein modiﬁcations can be engaged in dependence on time and subcellular location. The signal output in a given signaling system may depend on the amplitude of the incoming signal. Furthermore, the duration and frequency of an incoming signal can modulate the output of the system. An example of the latter is the þ regulation of CamK II by Ca2 (Section 9.6).

& Signaling networks are based on: — Modular structure and multivalency of signaling proteins and signaling complexes.

The major mechanisms of interactions within pathways and between pathways are described in the following subsections. 116 3 Organization of Signaling

Figure 3.4 (a) Linearity, branching, and to a high degree to the diversity and variability crosstalk in intracellular signaling. Crosstalk of hormonal signal transduction; (b) Variability refers to a situation where a signaling enzyme of receptor systems and signaling pathways. from one pathway activates (E4) or inhibits (i) For one receptor of a given binding (E00) signaling components involved in signal specificity (binding to hormone H) there can transduction of a different pathway; (b) be different subtypes in the same cell (R1, R2) Variability of receptor systems and signaling or in other cell types (R10). (ii) The hormone H pathways. For one receptor of a given binding can induce different reactions (X1, X2) upon specificity (binding to hormone H) there can binding the different receptor types (R1, R2). be different subtypes in the same cell (R1, R2) The receptor types R1 and R2 can be found or in other cell types (R10). The hormone H can simultaneous in one cell. (iii) The binding of induce different reactions (X, X0) upon binding two different hormones (H, H0) to different the different receptor types (R1, R2). The receptors (R10, R3) can induce the same binding of two different hormones (H, H0)to intracellular reaction. The characteristics (i) different receptors (R10, R3) can induce the and (ii) contribute to a high degree to the same intracellular reaction. These diversity and variability of hormonal signal characteristics illustrate the principle that transduction. Point (iii) illustrates the principle important cellular metabolites or reactions can that important cellular metabolites or reactions be controlled by different signal transduction can be controlled by different signal pathways. Branching and crosstalk contribute transduction pathways.

3.3.2 Regulatory Circuits and Responses in Biological Networks

Many ways exist to link the signaling molecules in a network, and each of these ways may lead to a distinct biological output. Based on a large number of experimental data relating to signaling pathways and their regulation, the modeling of distinct linkages in signaling pathways and their inﬂuence on network properties have revealed crucial regulatory principles in signaling pathways and networks [4,5]:

3.3.2.1 Circuits and Cascades A universal motif found in cellular networks is the cycle formed by two or more interconvertible forms of a signaling protein, which is modiﬁed by two opposing enzymes (Figure 3.5). This applies to nearly all PTMs, where the modifying and demodifying enzymes determine the lifetime of the modiﬁed state. Other examples 3.3 Architecture of Signaling Pathways 117

Figure 3.4 (Continued)

are the function of GTPase-activating protein (GAP) and guanine nucleotide exchange factors (GEFs) that control the switching properties of G proteins (see Chapter 7). Cascades of such cycles form the backbone of most signaling pathways that propagate external stimuli from the membrane to the nucleus or other distant targets. But, how these cycles and cascades can inﬂuence the dynamics of the reaction will depend on the rate constants of the individual steps, which can in turn be inﬂuenced dramatically by their subcellular location and recruitment to scaffolds.

3.3.2.2 Feedback Loops In general terms, feedback can be deﬁned as the ability of a system to adjust its output in response to self-monitoring. In biological systems, feedback is a general regulatory principle that is present both in intracellular and extracellular signaling, an example being during development. Typically, feedback is organized into loops that can have either a negative or a positive effect on signaling; whereas positive feedback will amplify the signal, negative feedback will attenuate it. However, as well as changing the steady-state responses, feedback loops may also favor the occurrence of instabilities, leading to an inhibition or enhancement of signaling. Furthermore, transient signals can be converted into permanent, irreversible responses.

3.3.2.3 Negative Feedback Negative feedback occurs when, for example, a signal induces its own inhibitor; it serves to dampen or limit signaling. The most obvious use of feedback signaling is 118 3 Organization of Signaling

Figure 3.5 Universal motifs of cellular transformation; (b) A cascade of cycles. signaling networks. (a) A cycle of the small Negative feedback provides robustness to GTPase Ras (for details, see Chapter 11). A noise, increasing resistance to disturbances guanine nucleotide exchange factor (GEF) inside the feedback loop, but brings about catalyzes the transformation of an inactive oscillations if it is too strong and the cascade is guanosine diphosphate (GDP)-bound form ultrasensitive. Positive feedback greatly (Ras-GDP) into an active guanosine increases the sensitivity of the target to the triphosphate (GTP)-bound form (Ras-GTP). A signal, and may also lead to bistability and GTPase-activating protein (GAP) is the relaxation oscillations. Adapted from Ref. [4]. opposing enzyme that catalyzes the reverse

to adopt the intensity and duration of a signal to the cellular requirements. Almost all signaling pathways are subject to negative feedback regulations in one way or another, with one of the best-modeled systems being feedback regulation of the MAPK/ERK pathway (see Figure 3.8). 3.3 Architecture of Signaling Pathways 119

Figure 3.6 Negative feedback loop in cytokine STATs activate the expression of SOCS receptor signaling. Ligand-bound cytokine proteins which are inhibitors of cytokine receptors (see Section 13.2.3) recruit protein receptors. As a consequence, JAK/STAT kinases, JAKs, which in turn phosphorylate the signaling is inhibited. transcriptional activator STAT proteins. The

& Negative feedback loops: — Dampen and limit signaling.

Another example of feedback regulation relates to the control of cytokine signaling through the JAK/STAT signaling pathway (Figure 3.6). JAKs are soluble tyrosine kinases that bind to cytokine receptors and transduce signals by the STAT proteins (see Section 13.2.1). Cytokine signaling is negatively regulated by proteins known as suppressors of cytokine signaling (SOCS; see Section 9.2.4) that participate in negative feedback loops. The physiological signiﬁcance of the negative feedback is exempliﬁed by SOCS1, which is induced by and inhibits interferon-c signaling. When the gene for SOCS1 is knocked out, mice die at the neonatal stage due to defects associated with excess interferon-c signaling.

3.3.2.4 Positive Feedback Positive feedback occurs when a signal induces more of itself, or of another molecule that ampliﬁes the initial signal, and this serves to stabilize, amplify, or prolong signaling. A generalized positive feedback loop is depicted in Figure 3.7; 120 3 Organization of Signaling

Figure 3.7 Circuit diagram of a simple positive targets, gene 2 activates gene 1, forming a feedback loop. Once turned on by an activator, positive feedback loop. When the initial gene 1 (shown in red) activates gene 2 (shown activator signal fades, these genes will remain in green). In addition to acting on downstream active.

this system has the interesting property that it remains activated even when the initial signal has disappeared.

& Positive feedback: — Amplifies, stabilizes, and prolongs signals.

Currently, many examples are known of positive feedback signaling, one such case being the positive feedback regulation of the small GTPase Ras by mSOS, a GEF for Ras (see Chapter 10). A positive feedback loop arises when Ras-GTP (produced when mSOS catalyzes the exchange of GDP for GTP in the nucleotide- binding pocket of Ras) binds to the mSOS allosteric pocket. This causes a signiﬁcant increase in the activity of this Ras-activating GEF, thereby stimulating further Ras activation.

3.3.2.5 Bistability Bistability describes the ability of a system to exist in two states, of ON and OFF. In this way, the system may behave as a switch that can toggle between the two states in response to concentration changes of the stimulus over a threshold value. Of the two alternative states, only one is populated; however, as the system cannot remain in an intermediate state, OFF and ON cannot exist simultaneously.

& Bistability: — Nonlinear, cooperative transition between “ON” and “OFF” — Switch-like behavior — No intermediate states.

Transition from the OFF-state to the ON-state by a transient stimulus occurs in a nonlinear cooperative manner, and the ON-state may persist even when the 3.3 Architecture of Signaling Pathways 121 stimulus has died away [6]. Such a system demonstrates ultrasensitivity to changes in the intensity of the stimulus, and has the characteristics of a cooperative transition. In such a bistable system the threshold intensity of the signal required for the system to be switched from the OFF to the ON state is different from that for transitioning in the reverse direction. Typical switches in biological systems are “robust,” which means that they behave as though they possess a memory of the signal. However, once an ON state has been achieved the system will continue to be at the ON state, even if the stimulus concentration is reduced to below the threshold level. In fact, in some special cases the system will stay ON even if the stimulus is completely removed. Such an irreversible behavior has been demonstrated in cell fate decision-making during the maturation of Xenopus oocyte [6].

3.3.2.6 Dynamic Behavior of Responses The outcome of cellular signaling is dictated to a large extent by the temporal dynamics of the underlying regulatory circuits, and thus by the rates of the reactions involved. This feature has been studied extensively using the example of the regulation of the MAPK/ERK pathways (Chapter 12) that are subject to several feedback controls. Computational modeling has shown that distinct modes of ERK spatiotemporal dynamics can emerge from different feedback conﬁgurations (Figure 3.8). Depending on the feedback topologies and kinetic parameters, a MAPK cascade can display markedly different temporal responses to an identical constant stimulus [5]: a monotone, sustained response (Figure 3.8a); a transient, adaptive response (Figure 3.8b); sustained oscillations (Figure 3.8c); and a switch-like, bistable response under conditions of a positive feedback in which two stable steady states, OFF and ON, coexist (Figure 3.8d). The modeling demonstrates that the details of the underlying reaction steps (i.e., the concentration of reactants, rate constants, positive versus negative feedback) determine the exact shape of the response. The same reaction scheme can show adaptive behavior, damped oscillations, persistent oscillations and, with positive feedback, even bistable properties. As the relevant parameters under in vivo conditions are unknown, it is not yet possible to explain the variations in the dynamics of pathways in different cell types.

3.3.3 Network Structures

Signal transduction through signaling networks depends on the types of interconnection of its components and the properties of the components itself. The large number of possible connections and the multiplicity of possible input and output signals at each component makes the description of signaling networks extremely complex. Several ways of depicting signaling networks have been employed. 122 3 Organization of Signaling

Figure 3.8 Versatile MAPK dynamics Each is half-maximal), and indicates the maximal panel shows schematically the RAF---MEK---ERK rate of a phosphatase reaction. (a) No ERK-- cascade (see Chapter 12); the feedback from -RAF feedback, F ¼ 1; (b) Negative ERK---RAF ERK to RAF, which is the initial mitogen- feedback, F ¼ 0.01, Kf ¼ 1 nM, V7 activated protein kinase (MAPK) activated by max ¼ 0.175 nM s 1; (c) Negative ERK---RAF Ras-GTP, is indicated (when present). The feedback, F ¼ 0.01, Kf ¼ 25 nM; (d) Positive different temporal responses of active, dually ERK---RAF feedback, F ¼ 5, Kf ¼ 100 nM. phosphorylated ERK (ppERK) to a constant Depending on the initial conditions (pre- Ras-GTP stimulus are obtained by changing existing activity level of the cascade), ppERK the parameters of ERK-mediated feedback. For either descends to the low activity state (blue details of evaluation, see Ref. [5]. Parameters F curves) or approaches the high-affinity state and Kf describe the feedback regulation (F ¼ 1, (red curves); the dashed line indicates a for no feedback, F < 1 for negative feedback, F threshold. Kholodenko 2010 [5], figure 1. > 1 for positive feedback; Kf equals ppERK Reproduced with permission of Nature concentration at which activation or inhibition Publishing Group.

3.3.3.1 Interaction Networks: Hubs One representation of networks uses all experimentally veriﬁed interactions radiating from a signaling protein in an apparently scale-free protein–protein interaction network, or “interactome” network. In such networks, most proteins interact with few partners, whereas a small but signiﬁcant proportion of proteins – the “hubs”–interact with many partners. Such scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. The complexity of such hub interactomes is shown in Figure 3.9, using an example of the transcriptional coactivator p300/CBP (see Section 4.5.2.1). 3.3 Architecture of Signaling Pathways 123

Figure 3.9 Interaction network of the histonesphere at the center. Where the network acetyl transferase p300, a gene regulatorytopology hub permits, interactors for which the protein (see Section 4.5.2.2). The figure strongest evidence is available are placed represents the interacting proteins for whichcloser to the hub. Larger spheres indicate experimental data have been annotated inproteins the for which some structural data are STRING resource (http://string.embl.de/). available. Gibson 2009 [7], figure 1. p300 is represented by the almost buried red Reproduced with permission of Elsevier.

& Hub proteins: — Multiple inputs and outputs — Many interaction partners — Multiple PTMs — Often disordered segments — Central importance. 124 3 Organization of Signaling

A general characteristic of “hub” proteins is their modular construction, the presence of natively disordered structures [7], and extensive PTM. The disordered regions of hub proteins frequently contain sequence elements that are recognized by interaction domains on partner proteins. Furthermore, these regions harbor sites of PTM (see e.g., p53; Figure 16.12) that serve to recruit partner proteins. The disordered segments of the “hub” proteins can be considered as platforms for multiple interactions, for the formation of regulatory complexes, and for molecular switching. Such hubs receive and transmit large number of different signals. As many of the interactions involved are mutually exclusive, the hubs often operate as molecular switches. A prominent example of this is the tumor suppressor p53, a central signaling protein which is intrinsically unstructured in its N- and C-terminus; moreover, theseregionsareextensivelymodified by diverse chemical modifications in a highly regulated fashion. The p53 modifications are recognized by a diverse set of upstream and downstream signaling components (Section 16.7.6) to generate a multiplicity of biological responses. Typically, hubs comprise a collection of related proteins that have a core activity in common, but differ in the details of domain order or PTM. The diversity within a hub protein family is generated to large part by alternative splicing or by genetically encoded isoforms. For example, p53 has two relatives – the p63 and p73 isoforms – that perform specialized functions (see Section 16.7). Most hubs have central functions in signaling networks which makes them sensitive to perturbations or mutations, with possible deleterious consequences for the organism. This point is illustrated by p53 which, when mutated, can lose its tumor-suppressing activity and thus promotes tumor formation.

3.3.3.2 Layered Networks A network architecture may be described as a layered structure that interfaces with diverse sources of input, and fans out into several outputs through conserved core processes. The core is conserved and consists of interconnected units that modify signals in a highly reproducible manner, whereas the output and input layers are less conserved and serve to diversify the biological answers. Such a layered network is ascribed a “bow-tie” or “hour-glass” structure. Figure 3.10 illustrates these features on the example of signaling through the ErbB network. The ErbB proteins belong to the RTK family of transmembrane receptors (see Chapter 10) that receive signals from growth factors and transduce these signals for example, via Ras GTPases and the MAPK pathway down to the transcriptional level (see Chapters 11 and 12). Depending on the exact combination of transcription factors and the cellular context, the output of the network regulates cell proliferation, differentiation and cell morphology, leading to distinct cellular phenotypes. 3.3 Architecture of Signaling Pathways 125

Figure 3.10 A systems perspective of the the system is conferred by structural ERBB network. A reductionist view of the bow- modularity and functional redundancy, along tie-architectured signaling network is with stringent system controls. An important represented. The heart of the system is a core positive regulator is ErbB2, a coreceptor. process, a collection of biochemical Heterodimerization between ErbB2 and any of interactions (Ras pathways, MAPK pathways, the three ErbB input modules enhances and þ PI3 kinase/Akt pathways, Ca2 -signaling prolongs the respective output. On the other pathways, non-RTKs), which are tightly hand, a ubiquitin ligase that is involved in coupled to each other and interface with two receptor degradation, Cbl, controls an sets of components: three input modules, important negative-feedback loop. Several each comprising an ERBB receptor tyrosine activation-dependent control loops fine-tune kinase; and a large group of partly redundant bistability. These include transcription of ErbB ligand growth factors. The output of the core ligands (positive regulation) and newly process is translated to gene expression synthesized negative regulators such as through multiple transcription factors. sprouty, an inhibitor of receptor tyrosine Depending on the exact combination of kinases (see Section 10.2.4). EGF, epidermal transcription factors and the cellular context, growth factor; EGR1, early growth response-1; the output of the network regulates cell HB-EGF, heparin-binding EGF-like growth behavior. The system maintains two steady factor; NRG1/3, neuregulin-1/3; TGFa, states (bistability), for which interconversions transforming growth factor-a. Adapted from depend on ligand binding. The robustness of Ref. [8]. 126 3 Organization of Signaling

& Layered networks: — Hour-glass structure — Conserved core — Redundant input modules — Variable output modules — Robustness due to multiple feedbacks.

Several ErbB subtypes are known that function as modules. The receptors can be activated by an array of growth factors, and transmit signals to the core machinery that uses conserved biochemical interactions to produce a panel of different downstream effects. The core process is composed of an array of strongly coupled subnetworks that employ a small set of molecular switches and cascades. In the case of ErbB signaling, such subnetworks comprise the reactions involved in signaling to the Ras GTPase, which functions as a node to distribute the signal further. The Ras node then distributes the signal further to other conserved core processes, such as the PI3 kinase pathway and the ERK/MAPK pathway. The output of the core process is translated to gene expression through multiple transcription factors, leading to the selected cell fate.

3.3.3.3 Modularity Complex biological networks consist of distinct semi-autonomous functional units that show strong internal connections, although they maintain weaker connections with the environment. A particular functional unit such as the ErbB network, may comprise different modules that can be used differentially. The modularity of the ERBB network is exempliﬁed by the use of different receptor heterodimers during development (for details, see Ref. [8]).

3.3.3.4 Redundancy and Robustness One common property of biological signaling networks is their robustness against small internal and external fluctuations and perturbations. Signaling in networks must be secured against such fluctuations in order to generate a stable output; for example, under conditions of small malfunctions of signaling components or variations in stimulatory inputs. Such protection confers stability and robustness to the system. An important contribution to robustness comes from redundancy. In the face of severe perturbations, cells can use functionally redundant signaling components or alternative modules to generate a robust output. Redundancy exists in the input layer as well as in the core of the network. Examples of redundant inputs to transmembrane receptor systems are abundant; an example is the ErbB network and signaling by cytokine receptors (see Chapter 12). Here, ligands with overlapping specificities, as well as variable receptor oligomers, essentially share downstream pathways, therefore displaying degeneracy. Redundancy is also seen at the richly connected core of networks. Not only enzymes, but also scaffold and adapter proteins, are present in multiple isoforms, References 127 and alternate connectivities coexist. The alternative wiring in redundant network systems may be illustrated once more by the example of the ErbB network, where signaling from ErbB to Ras GTPase can proceed via alternative ways for recruitment of mSos, the exchange factor required for Ras activation. ERBB1 can recruit mSos through either Grb2 or Shc (Figure 11.10), whereas Grb2 can associate with the receptor either directly or through Shc. Likewise, the E3 ligase Cbl is recruited to ErbB either directly, or indirectly, through Grb2. Other major mechanisms that contribute to the robustness of biological signaling are the use of positive and negative feedback circuits (see Figure 3.10). As outlined in Section 3.2.5.2, such feedbacks contribute to the robustness of the system (for a detailed discussion of feedback controls during ErbB signaling, see Ref. [8]).

Questions 3.1. What are the functions of scaffolding proteins, and on which properties are these functions based? 3.2. Name at least three different scaffolding proteins and their functions in signaling. 3.3. What are the major tools for organizing cellular signaling? 3.4. Give examples of negative and positive feedbacks in cellular signaling. 3.5. What are hub proteins, and how can these proteins manage to interact with the largest number of possible protein partners?

References

1 Good, M.C., Zalatan, J.G., and Lim, W.A. 5 Kholodenko, B.N., Hancock, J.F., and Kolch, (2011) Scaffold proteins: hubs for controlling W. (2010) Signalling ballet in space and time. the ﬂow of cellular information. Science, 332 Nat. Rev. Mol. Cell. Biol., 11 (6), 414–426. (6030), 680–686. PubMed PMID: 21551057. PubMed PMID: 20495582. Pubmed Central Pubmed Central PMCID: 3117218. PMCID: 2977972. 2 Scott, J.D. and Pawson, T. (2009) Cell 6 Chatterjee, A., Kaznessis, Y.N., and Hu, W.S. signaling in space and time: where proteins (2008) Tweaking biological switches through come together and when they’re apart. a better understanding of bistability behavior. Science, 326 (5957), 1220–1224. PubMed Curr. Opin. Biotechnol., 19 (5), 475–481. PMID: 19965465. Pubmed Central PMCID: PubMed PMID: 18804166. Pubmed Central 3041271. PMCID: 2766094. 3 Levental, I., Grzybek, M., and Simons, K. 7 Gibson, T.J. (2009) Cell regulation: (2010) Greasing their way: lipid determined to signal discrete cooperation. modiﬁcations determine protein association Trends Biochem. Sci., 34 (10), 471–482. with membrane rafts. Biochemistry, 49 (30), PubMed PMID: 19744855. 6305–6316. PubMed PMID: 20583817. 8 Citri, A. and Yarden, Y. (2006) EGF-ERBB 4 Kholodenko, B.N. (2006) Cell-signalling signalling: towards the systems level. Nat. dynamics in time and space. Nat. Rev. Mol. Rev. Mol. Cell Biol., 7 (7), 505–516. PubMed Cell. Biol., 7 (3), 165–176. PMID: 16829981.

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4 The Regulation of Gene Expression

4.1 The Basic Steps of Gene Expression

The transfer of genetic information from the level of the nucleic acid sequence of a gene to the level of the amino acid sequence of a protein or to the nucleotide sequence of RNA is termed gene expression. In eukaryotes, gene expression includes the following steps:

Transcription: the formation of a primary transcript, the pre-mRNA. Conversion of the pre-mRNA into the mature mRNA: this includes processing, splicing, and transport from the nucleus to the cytosol. Translation: synthesis of the protein on the ribosome.

The expression of genes follows a tissue- and cell-speciﬁc pattern, which determines the function and morphology of a cell. In addition, all development and differentiation events are characterized by a variable pattern of gene expression. The regulation of gene expression thus plays a central role in the development and function of an organism. Because of the multitude of individual processes which are involved in gene expression, there are many potential regulatory sites (Figure 4.1).

4.1.1 Regulation of Transcription

At the level of transcription, it can be determined whether a gene is transcribed at a given point in time. The chromatin structure plays a decisive role in this regulation, and chromatin structures exist that can effectively inhibit transcription and shut down a gene. This “silencing” of genes can be either transient or permanent, and is generally observed in development and differentiation processes. The regulated transcription of genes requires as an essential step the reorganization and modiﬁcation of the chromatin, which is a prerequisite for the initiation of transcription.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 130 4 The Regulation of Gene Expression

Figure 4.1 Levels of regulation of eukaryotic gene expression.

& Regulation of transcription includes: — Removal of repressive chromatin structures — Covalent modification of chromatin proteins and of DNA — Recruitment of RNA polymerase holoenzyme — Recruitment of specific transcription factors for gene selection.

Concomitant with chromatin reorganization and modification, the target genes must be selected and a transcription initiation complex must be formed at the starting point of transcription. A large number of proteins are involved in this step. The main components are the multisubunit RNA polymerase, general and gene- specific transcription factors, and cofactors that help to coordinate the chromatin structural changes and the process of RNA synthesis. The formation of a functional 4.2 The Components of the Eukaryotic Transcription Machinery 131 initiation complex is often the rate-limiting step in transcription, and is subject to a variety of regulation mechanisms. In addition to gene selection by gene-specific DNA-binding proteins, epigenetic changes in the form of DNA methylation, miRNA expression and chromosomal protein modifications shape the patterns of gene expression.

4.1.1.1 Conversion of the pre-mRNA into the Mature mRNA The transcription of genes in mammals often initially produces a pre-mRNA, whose information content can be modulated by subsequent polyadenylation or splicing. Various ﬁnal mRNAs coding for proteins with varying function and localization can be produced in this manner, starting from a single primary transcript.

4.1.1.2 Regulation at the Level of mRNA and Translation The use of a particular mature mRNA for protein biosynthesis is also highly regulated. The regulation can occur via the accessibility of the mRNA for the ribosome or via the initiation of protein biosynthesis on the ribosome. Further- more, the use of a mRNA can be modulated via speciﬁc RNAs with distinct regulatory functions, the micro RNAs (see Chapter 5). These mechanisms determine when and how much of a protein is synthesized on the ribosome.

4.1.1.3 Nature of the Regulatory Signals Regulation always implies that signals are received, processed, and translated into a resulting action. The nature of the signals which are employed during the course of the regulation of gene expression and which are ﬁnally translated into a change in protein concentration is highly variable. Regulatory molecules can be small molecular metabolites, hormones, proteins, or ions. The signals can be of external origin or can be produced within the cell. External signals originating from other tissues or from cells of the organism are transmitted across the cell membrane into the interior of the cell, where they are transduced by sequential reactions to the level of transcription or translation. Complex signaling networks are generally involved in the transduction.

4.2 The Components of the Eukaryotic Transcription Machinery

Summary Multiple regulatory signals impinge on eukaryotic promoters, and these signals are processed and transmitted to the transcription apparatus within the frame of chromosomal reorganization at the promoter. The proteins involved comprise RNA Pol I, II, and III, the general transcription factors (GTFs) TFII A, B, D, E, F, and H, specific transcription factors, Mediator, and chromatin-modifying and remodeling enzymes. 132 4 The Regulation of Gene Expression

Deposition of the transcription apparatus at genes to be transcribed is guided by sequence elements of the core promoter that are recognized by the transcription machinery and direct Pol II to the start site of transcription. The nature of the sequence signals is quite diverse and specifies the identity of a given promoter. Of the GTFs, TFIID may be characterized as a core promoter recognition complex that carries enzymatic activity and may exist in different cell type-specific forms. TFIIH is a multiprotein complex that harbors helicase activity and protein kinase activity in its CDK7 subunit. The latter is required for phosphorylation of the carboxyterminal domain (CTD) located on the largest subunit of RNA Pol II, Rpb1. By placing distinct phospho-marks on the CTD, CDK7 controls the transition from transcription initiation to elongation and to processive RNA synthesis. The mammalian Mediator is a multiprotein complex of 26 subunits that exists in multiple, biochemically distinct forms. Mediator is thought to form a bridge between specific transcription factors and the transcription apparatus. By interacting extensively with Pol II, the GTFs, and specific transcription factors, Mediator enhances Pol II recruitment and stabilizes transcription complexes at the promoter.

Both, prokaryotes and eukaryotes employ essentially the same basic steps for mRNA synthesis. RNA polymerase binds to the transcription start site, a pre- initiation complex forms, and initiation of RNA synthesis starts with the incorporation of the ﬁrst nucleotides. In this early stage of initiation, RNA synthesis may be eventually aborted and re-initiation may start. & Steps of transcription: — RNA Pol binding to promoter — Formation of pre-initiation complex — Initiation of RNA synthesis — Elongation — Termination.

Upon successful synthesis of the first five to eight nucleotides, the RNA polymerase escapes from the promoter and transits into the stable elongation phase, and finally into the termination phase. During all of these steps, transcription factors cooperate with the RNA-synthesizing machinery to allow for accurate and tightly controlled transcription. Accordingly, initiation factors, elongation factors and termination factors of transcription have been identified. Furthermore, the various phases of transcription are accompanied by on-going modifications and reorganizations of the chromatin.

4.2.1 The Basic Features of Eukaryotic Transcription

When comparing prokaryotic and eukaryotic transcription, the complexity of eukaryotic transcription is most impressive. The number of proteins required for 4.2 The Components of the Eukaryotic Transcription Machinery 133 regulated transcription in eukaryotes is much larger than in bacteria, and this is a reason why many aspects of eukaryotic transcription are still incompletely understood. While the basic mechanisms of eukaryotic transcription are quite well known, details of transcriptional control are only just beginning to be unraveled. Overall, the central components of the transcription machinery – the RNA polymerase and the transcription initiation factors – are similar in bacteria and eukaryotic cells. The RNA polymerases share a conserved core and a common transcription mechanism. The initiation factors – s in bacteria and a set of GTFs in eukaryotes – are more distantly related, but function in a similar manner in promoter recognition, promoter melting, abortive initiation, and promoter escape. In all organisms, gene transcription is tightly controlled. In response to internal and external signals, transcription may be activated or repressed and these signals are transmitted to the transcription machinery by the transcription factors in cooperation with accessory proteins. Major differences between bacteria and eukaryotic cells exist in transcriptional control, particularly in the way that regulatory signals are passed on to the transcription apparatus. In the case of bacteria, transcriptional activators or repressors bind to sequences adjacent to the promoters and exert effects directly on RNA polymerase by, for example, stabilizing RNA polymerase binding or – in the case of repressors – by preventing binding of the polymerase.

& Bacterial transcription: — Primarily controlled via direct interactions between regulators and RNA Pol. Eukaryotic transcription: — Primarily controlled via chromatin reorganization.

The situation is strikingly different in eukaryotes, where the multiple regulatory signals that impinge on promoters are processed and transmitted to the transcription apparatus within the frame of chromosomal organization of the DNA, and a structural reorganization must be induced at the promoter to allow the initiation of transcription. This requires the participation of a large number of accessory proteins, mostly organized in multiprotein complexes that result in features of the transcription machinery that are unique to eukaryotes.

& Major proteins involved in transcription: — RNA Pol I, II, III — GTFs — Specific transcription factors — Mediator — Chromatin-modifying and -remodeling enzymes. 134 4 The Regulation of Gene Expression

Figure 4.2 Function and cooperation of the main components of eukaryotic transcription. GTF, general transcription factor.

Based on biochemical and genetic studies, eukaryotic transcription in vivo – that is, on chromatin-embedded DNA – has been shown to be dependent on the following components (Figure 4.2): RNA polymerases I, II, and III carry the enzymatic activity for the synthesis of RNA on the DNA template, and are composed of 10–12 subunits. GTFs help to localize the RNA polymerase correctly on the promoter and to form a transcription-competent initiation complex. They help to impose a specific structure on the transcription start site, and some of them are required for elongation of the transcript. Specific transcription factors are sequence-specific DNA-binding proteins that mediate regulated transcription. They select the genes to be transcribed by binding to specific promoter or enhancer sequences, and they form activating or inhibiting contacts to the transcription machinery. The specific transcription factors receive signals for transcriptional regulation and transmit these signals to the mediator complex and to chromatin. Mediator is a multiprotein complex that forms a link between specific transcription factors and the basal transcription apparatus. This complex performs an essential function in transcriptional regulation, and is found as part of preformed transcription complexes that bind to eukaryotic promoters. 4.2 The Components of the Eukaryotic Transcription Machinery 135

Chromatin-modifying and chromatin-remodeling activities are required for establishing a transcription-competent status on chromatin-covered promoters. The proteins involved are found in multisubunit assemblies of varying composition. Speciﬁc transcription factors and the mediator complex communicate with the chromatin-modifying activities.

4.2.2 Elementary Steps of Eukaryotic Transcription

Transcription in eukaryotes can, as shown schematically in Figure 4.3, be subdivided into the following steps:

Restructuring of chromatin at the promoter: The promoter region must be transcriptionally activated, which requires the removal of repressive chromatin structures around the promoter in order to allow formation of the pre-initiation complex. The relief of repressive chromatin structures involves covalent modiﬁcation of the chromosomal proteins and a restructuring of chromatin at the promoter. A large number of different enzyme activities and accessory proteins participate in this process. Formation of a pre-initiation complex: This step includes promoter selection under the cooperation of general transcription factors and deposition of the RNA polymerase II holoenzyme on the promoter Activation of the pre-initiation complex and initiation of RNA synthesis: The DNA is unwound in the vicinity of the start site and RNA synthesis is initiated. Transition from initiation to elongation: Following a phase where abortive synthesis of short RNAs may occur, the transcription apparatus enters the phase of processive RNA synthesis. Termination: RNA synthesis ends at deﬁned sequence elements.

Transcription is regulated to a great extent at the start of transcription, that is, the steps preceding the transition into stable elongation. Hence, these steps form the focal point of the following discussion.

4.2.3 The Eukaryotic RNA Polymerases

Three types of RNA polymerase exist for the transcription of eukaryotic genes, each of which transcribes a certain class of genes. All three enzymes are characterized by a complex subunit structure.

& Eukaryotic RNA polymerases — Pol I: ribosomal RNA — Pol II: protein-coding mRNAs — Pol III: tRNAs, 5S RNA. 136 4 The Regulation of Gene Expression

Figure 4.3 Steps of eukaryotic transcription. 4.2 The Components of the Eukaryotic Transcription Machinery 137

RNA polymerase I (Pol I) is responsible for the transcription of the ribosomal RNA genes (class I genes), RNA polymerase II (Pol II) transcribes the genes encoding proteins (class II genes), and RNA polymerase III (Pol III) transcribes the genes for the tRNAs and the 5S ribosomal RNA (class III genes). The following discussion will be limited to Pol II and the genes transcribed by it, as this enzyme plays the most important role for regulatory processes and signal transduction. Aside from this, many characteristics of the transcription of the genes of class II are also valid for genes of classes I and III. Structural information is available for all three eukaryotic RNA polymerases (for details, see Ref. [1]). Most structural data have been accumulated for Pol II and its functional complexes, and these structures provide a comprehensive model of the key events of the RNA polymerase reaction, namely initiation at the promoter including promoter melting and nucleotide incorporation.

4.2.4 The Core Promoter and Structure of the Transcription Start Site

The Pol II core promoter represents the entry site for the basal transcription machinery, composed of Pol II and the GTFs. The core promoter comprises sequences that are recognized by the transcription machinery and direct Pol II to the start site of transcription. The nature of the sequence signals is quite diverse and speciﬁes the identity of a given promoter. In addition to the variable sequence signals, the location of the start site may be also variable. Two major types of core promoter are known, namely focused and dispersed (Figure 4.4). In focused core promoters there is either a single transcription

Figure 4.4 Focused versus dispersed about 50 to 100 nucleotides. Dispersed transcription initiation. In focused transcription is the most common mode of transcription, there is either a single major transcription in vertebrates. For instance, transcription start site or several start sites dispersed transcription is observed in about within a narrow region of several nucleotides. two-thirds of human genes. In vertebrates, Focused transcription is the predominant focused transcription tends to be associated mode of transcription in simpler organisms. In with regulated promoters, whereas dispersed dispersed transcription, there are several weak transcription is typically observed in constitutive transcription start sites over a broad region of promoters in CpG islands. After Ref. [2]. 138 4 The Regulation of Gene Expression

start site or a distinct cluster of start sites in a short region of several nucleotides. In contrast, dispersed core promoters show a number of transcription start sites distributed over a broad region that might range from 50 to 100 nucleotides. The dispersed type of promoter is less well characterized than the focused core promoter. In vertebrates, about 70% of genes have dispersed promoters, which typically are found in CpG islands. In general, focused promoters appear to be associated with regulated genes, whereas dispersed promoters are used in constitutive genes. The core promoters may contain many different sequence motifs that function as recognition signals for components of the core transcription machinery specifying different mechanisms of transcription initiation [2]. Analyses of focused core promoters have led to the discovery of sequence motifs such as the TATA box, BREu (upstream TFIIB Recognition Element), INR (Initiator), MTE (Motif Ten Element), DPE (Downstream Promoter Element), DCE (Downstream Core element), TCT (TCT motif), and XCPE1 (X Core Promoter Element 1) (Figure 4.5). In contrast, dispersed promoters generally lack BRE, TATA, DPE, and MTE motifs. The TATA box and BRE are the most ancient of the core promoter motifs, and are conserved from archaea to humans. Both elements have been identified as distinct binding sites for general transcription factors of the core transcription machinery. The TATA box, which was the first eukaryotic promoter element to be identified, is bound by the TATA box binding protein (TBP), a component of the multiprotein complex TFIID. BRE is recognized by TFIIB, while the initiation region (INR) is recognized by TAF subunits of TFIID. Based on functional assays and computational analyses of many transcription start sites, more or less stringent consensus sequences could be identified for these sequence motifs (for details, see Ref. [2]). None of the core promoter elements known to date is universal; rather, each is present in only a subset of core promoters of an organism. For instance, we know a large number of TATA-less promoters. Moreover, some core promoters appear to lack all of the known core promoter elements. Consequently, among higher eukaryotes the picture of core promoters is quite complex, and suggests that the

Figure 4.5 Some core promoter motifs for dictated by the presence or absence of specific transcription by RNA polymerase II (this core promoter elements. BREu, upstream diagram is roughly to scale). These motifs are TFIIB Recognition Element; INR, Initiator; typically found in focused core promoters. MTE, Motif Ten Element; DPE, Downstream There are no universal core promoter Promoter Element; DCE, Downstream Core elements, but it is likely that additional core element; TCT, TCT motif; XCPE1, X Core promoter motifs remain to be discovered. The Promoter Element 1. After Ref. [2]. properties of any particular core promoter are 4.2 The Components of the Eukaryotic Transcription Machinery 139 many different core promoters recruit distinct subtypes of the basal transcription machinery employing slightly distinct initiation mechanisms.

4.2.5 General Transcription Factors and the Basal Transcription Apparatus

Reconstitution experiments starting from naked DNA have helped to deﬁne a basal transcription apparatus assembled from three major components comprising more than 70 polypeptides:

RNA polymerase II The GTFs: TFII A, B, D, E, F, and H The mediator complex (Section 4.2.9).

Three major subcomplexes are found in the basal transcription apparatus, RNA Pol II, TFIID, and Mediator. The order of deposition of these subassemblies on the promoter is still open and their composition may vary depending on the gene structure. It is possible that various forms of the basal transcription apparatus exist in the cell, and these forms may be recruited to the promoter in different ways, depending on the transcriptional activators, the gene structure, and the local chromatin environment. On naked DNA, the GTFs may be associated with RNA Pol II in a defined order for the formation of a pre-initiation complex from which transcription is possible, albeit at a very low level. An increase in the basal transcriptional level requires the participation of the Mediator complex, which may also be considered as a GTF as it is essential for the transcription of all genes in eukaryotic cells. When nucleosome-covered DNA is used as a template for transcription, the restructuring, modification and mobilization of nucleosomes comes into play. One prerequisite for transcription initiation by the basal transcription apparatus on nucleosome-covered DNA is the induction of a favorable chromatin structure at the promoter and the start site. This structural reorganization is typically a complex process that requires the participation of gene-specific DNA-binding transcription factors, and also of chromatin modifiers and remodelers (Section 4.5). The processes involved also help to form a stable complex of the basal transcription apparatus at the transcription start site.

& GTFs for RNA Pol II transcription: — TFII A, B, D, E, F, and H.

The basic properties of the GTFs are summarized in Table 4.1. Only TFII D and TFII H will be characterized in more detail in the following subsections.

4.2.5.1 TFII D TFIID may be characterized as the core promoter recognition complex. It is a multiprotein complex typically consisting of the TATA box-binding protein (TBP) 140 4 The Regulation of Gene Expression

Table 4.1 General initiation factors of transcription by RNA polymerase II.

Protein No. of Subunit Function subunits size (kDa)

TFIID: TBP 1 38 Sequence-speciﬁc binding to TATA box, recruitment of TFIIB TAFs 12 15---250 Promoter recognition, regulation, chromatin modiﬁ- cation TFIIA 3 12,19,35 Stabilization of TBP-DNA-binding; antirepression TFIIB 1 35 Recruitment of RNA Pol II - TFIIF; selection of start site by RNA Pol II TFIIF 2 30,74 Assists in promoter binding by RNA Pol II RNA poly- 12 10---220 Enzymatic activity of RNA synthesis, binding of merase II TFIIF TFIIE 2 34,57 Binding of TFIIH, modulation of activities of TFIIH TFIIH 9 35---89 Helicase, protein kinase and ATPase- activity; promoter unwinding, promoter clearance

TAF: TATA box binding protein associated factor; TBP: TATA box binding protein; RNA Pol II: RNA polymerase II.

and TATA box-binding protein associated factors (TAFs). On TATA box-containing promoters, TBP specifically recognizes the TATA box, and its binding leads to a distinct bending of the DNA. In this manner, a particular topology of the DNA is created that serves as a prerequisite for the defined binding of RNA polymerase II and further basal transcription factors, such as TFIIA and TFIIB (for a review, see Ref. [1]). The TAFs fulfill numerous functions [3]. Typically, they are ascribed a structure-promoting function, with some of the TAFs displaying a high degree of homology to the histones H3 and H4. In fact, their structure matches the canonical histone-fold dimer, such that TAF-dimers are formed via the histone-fold. Thus, it is speculated that TAFs impose a distinct topology to the DNA and help to create a nucleosome-like structure at the promoter.

& TFIID subunits: — TBP: TATA box recognition Structural function. — TRFs: TBP-related factors. — TAFs: Structural functions Protein kinase activity HAT activity.

Other important tasks of the TAFs are promoter recognition and recruitment of the core transcription machinery. Those DNA elements of promoters that lack a 4.2 The Components of the Eukaryotic Transcription Machinery 141

TATA box may be recognized by TAF subunits of TFIID. As an example, the INR serves as a specific contact point for binding by the TAF components TAF1 and TAF2. Furthermore, the TAFs are targets for protein–protein interactions with transcriptional activators. TAFs also possess enzymatic activity; notably, TAF1 has both histone acetyltransferase and protein kinase activities. While the former enzyme presumably plays a role in reorganization of the nucleosome, the latter can lead to the phosphorylation of TFIIF. Importantly, the composition of TFIID may vary in a tissue- and development-specific manner, and it is now generally accepted that TFIID may exist in different cell type-specific forms. For instance, ovarian-specific paralogs of TAFs (e.g., TAF4b) have been identified that lead to distinct TFIID structures and promoter-selective activities. The extent of core promoter recognition complex diversity was expanded by the discovery of TBP-related factors (TRFs) that are widely expressed in varying levels in many mammalian cell types. Currently, at least three types of TRF (TRF1–3) are known that may replace TBP and perform cell type-specific functions. As an example, a genome-wide localization of Drosophila TRF2 showed that it occupies over a thousand promoters, most of which are not bound by TBP and lack a canonical TATA box [4]. Furthermore, the expression of TRF3 has been clearly associated with differentiation programs in vertebrates. A surprising example of the diversity of promoter recognition has been identified in myotubes, where a near- complete elimination of the whole TFIID complex is observed. Here, the role of TFIID is apparently taken over by the TBP cousin TRF3 and a single TAF, namely TAF3. It is assumed that TRF3/TAF3 can be directly contacted by myotube-specific transcription factors such as MyoD, to activate myotube- specific promoters.

4.2.5.2 TFIIH TFIIH is a multiprotein complex consisting of 10 different subunits, which can be separated into two subcomplexes: the core TFIIH complex comprising seven subunits; and the CDK-activating kinase(CAK) module comprising three subunits, the Ser/Thr-speciﬁc protein kinase CDK7, cyclin H, and the Mat1 protein. (Figure 4.6). The two subcomplexes can associate in a dynamic manner and are involved, either separately or together, in the regulation of major processes of the cell, namely transcription initiation and elongation, nucleotide excision repair (NER), and cell-cycle control.

& TFIIH participates in: — Transcription initiation — DNA repair — Cell-cycle control.

The various functions of TFIIH are mainly based on three enzymatic activities located on distinct subunits: 142 4 The Regulation of Gene Expression

Figure 4.6 Subunit structure of TFIIH. This ligase in yeast. The complex is involved both in 10-subunit complex is composed of a core (in RNA Pol I- and RNA Pol II-dependent yellow: XPB, p62, p52, p44, p34, and TTDA) transcription and in nucleotide excision repair associated with the CAK (in brown: Cdk7, (NER). The Cdk7 kinase can be found in three CycH, and MAT1) through MAT1 and the XPD different complexes: TFIIH, CAK-XPD, and subunit (in green). Four enzymatic activities CAK. When Cdk7 is in TFIIH, it functions in are found in TFIIH: XPB and XPD are 30 ! 50 transcription, whereas it functions in cell-cycle and 50 ! 30 helicases, Cdk7 is a kinase, and progression when it is in CAK alone or is p44 has been described as an E3 ubiquitin associated with XPD.

ATP-dependent DNA helicase (xeroderma pigmentosum subgroup D; XPD) ATP-dependent DNA helicase (xeroderma pigmentosum subgroup B; XPB) Ser/Thr-speciﬁc protein kinase CDK7.

Two elementary aspects of transcription are regulated by TFIIH, namely transcription initiation and transcription elongation. The kinase activity of the CAK module is required for an early step in transcription; the enzyme phosphorylates two serine residues (Ser5 and Ser7) in the CTD of RNA Pol II (Section 4.2.7). By placing distinct phospho-marks on the CTD, CDK7 controls the transition from transcription initiation to elongation, and to processive RNA synthesis. Furthermore, the CAK module has been shown to directly phosphorylate many transcription factors (e.g., nuclear receptors) to regulate the expression of speciﬁc genes. Another critical step in transcription initiation and elongation – opening of the DNA template – is assisted by the ATP-dependent helicase activity of TFIIH located on the XPB and XPD proteins.

& TFIIH: — 10 subunits — Helicase activity — ATPase activity — Protein kinase activity. 4.2 The Components of the Eukaryotic Transcription Machinery 143

The function of TFIIH in DNA repair is based on its participation in the NER of damaged DNA. It has long been known that transcription and the removal of bulky base adducts by NER are coupled. An increased repair of DNA damage by NER is observed while a gene is being transcribed. RNA polymerase II typically cannot transcribe past DNA lesions and will stall – with its associated general transcription factors – at sites of DNA damage. In order to activate repair of the damage, a dissociation of the CAK module from TFIIH is triggered, such that the TFIIH core can now associate with further NER repair proteins to form a productive NER complex, to remove the lesion, and to allow further transcription (for a review, see Ref. [5]). In addition, TFIIH is an essential component of repair processes not linked to transcription. It is also required for global genomic repair. The presence of the protein kinase CDK7 and of Cyclin H in the CAK subcomplex provides for the link to cell-cycle regulation. CDK7 is identical to CDK- activating kinase (CAK; see Section 15.3.2), which phosphorylates the activation loop of CDKs and thereby regulates cell-cycle transitions. Overall, the picture of the structure and function of TFIIH is complex, as TFIIH – or components of it – can assemble into different multiprotein complexes which perform central functions in the cell.

4.2.6 The Mediator Complex

The Mediator complex is a multisubunit complex required for transcriptional regulation from yeast to human [6]. Mediator was discovered on the basis of its coactivating activity in biochemical and genetic screens for coactivators of RNA polymerase. When puriﬁed, Mediator was shown to stimulate basal transcription and phosphorylation of the CTD of RNA polymerase II, but Mediator has now been ascribed a central role in transcription. There is a functional cooperativity between Mediator, Pol II and most of the other GTFs; in particular, Mediator interacts extensively with Pol II and the GTFs, enhancing Pol II recruitment and stabilizing transcription complexes at the promoter. In budding yeast, Mediator is as important as Pol II itself for expression of protein-coding genes, and Mediator subunits localize to promoters on a genome- wide scale. Therefore, Mediator may be considered a general transcription factor, in that it functions as a control panel which processes diverse signals in the form of sequence-speciﬁc transcription factors and coregulators, and then transmits this information to the core RNA polymerase complex.

& Mediator function: — Link between sequence-specific transcription factors and RNA Pol II

The Mediator from yeast consists of 21 polypeptides, and is found either in free form or in tight complex with RNA polymerase II. In contrast, mammalian Mediator is larger (26 subunits, 1.2 MDa) and exists in multiple, biochemically 144 4 The Regulation of Gene Expression

distinct forms. One problem with the characterization of Mediator is its low abundance and the inability to infer the functions of the subunits from bioinformatics. Mediator sequences contain almost no predicted functional motifs, but appear to contain an unusually high proportion of intrinsically disordered regions that are likely to contribute to Mediator’s structural plasticity and its vast potential for protein–protein interactions. These interactions include contacts to RNA Pol II and its CTD, to other GTFs, to sequence-specific transcription factors, and to chromatin-modifying enzymes. An important function of Mediator is to provide a link between sequence-specific transcription factors bound at regulatory cis-elements and the core transcription machinery at the promoter. Mediator may directly contact the transactivation domain of sequence-specific transcription factors, and thus convey regulatory information from these factors to RNA Pol II. Structural analyses have shown that the binding of sequence-specific transcription factors such as the vitamin D receptor (VDR), the tumor suppressor p53 or the VP16 activation domain to Mediator triggers major structural shifts within Mediator, suggesting a straightfor- ward means of spatially and temporally regulating Mediator activity (Figure 4.7). Interestingly, different activators induce different structural changes in Mediator, which allows for the association of distinct sets of coactivators (Section 4.3) and thus for a gene-specific control of transcription. The activating function of Mediator is thought to come into play at a state where RNA Pol II has already been recruited to the promoter and is ready to start productive elongation.

Figure 4.7 Structure of Mediator. Mediator is encircles and binds tightly to RNA Pol II; and a present in cells in at least three forms: a form in which the kinase module binds to the Mediator core, which is composed of more Mediator core in a manner that precludes than 20 subunits arranged in three modules Mediator binding to RNA Pol II. Conaway 2011 referred to as the head, middle, and tail; a [7], figure 1. Reproduced with permission of holoenzyme, in which the Mediator core Elsevier. adopts a more open conformation that 4.2 The Components of the Eukaryotic Transcription Machinery 145

& Mediator: — Multisubunit complex that interacts with: RNA Pol II GTFs Transcription factors Chromatin-modifying proteins.

An important control of Mediator activity appears to be exerted by the reversible association with a large protein complex, the CDK8 module, a 600 kDa assembly consisting of the protein kinase CDK8, cyclin C, Med12, and Med13. The association of the CDK8 module with Mediator may have either a repressive or stimulatory effect on transcription. The results of structural studies have suggested that the CDK8 module sterically hinders RNA Pol II from binding to Mediator, which would explain the repressive effect. The latter effect may also be explained by the observation that the CDK8 module can recruit a methyltransferase which catalyzes H3K9 methylation, a repressive chromatin mark.

& CDK8 module of Mediator: — Protein kinase activity — Recruits chromatin-modifying enzymes.

Activating functions of the CDK8 module may be linked to its intrinsic histone- modifying activity, and also to its interaction with and recruitment of histone acetyltransferase, GCN5L. The kinase activity speciﬁcally phosphorylates Ser10 of histone H3, while GCN5L acetylates Lys14 of H3. The double mark H3S10/H3K14 correlates with the activation of at least a subset of genes. These observations point to two important activities of the CDK8 submodule: (i) it can directly introduce activating marks on a histone; and (ii) it can recruit a chromatin-modifying enzyme for histone modiﬁcation. These observations indicate that Mediator and the CDK8 module represent a platform from which a variety of functionally divergent outcomes can be initiated. Overall, the Mediator complex can be ascribed a central role in transcription at nearly all protein-coding genes. These general regulatory roles include:

i) RNA Pol II recruitment and activation. ii) The coordination of pre-initiation complex assembly. iii) Control of TFIIH-dependent RNA Pol II CTD phosphorylation within this complex. iv) Sustained or transient repression of transcription initiation via Mediator– CDK8 submodule interactions.

The many subunits of Mediator provide a platform for dynamic interactions with a large number of target proteins, ranging from the transcriptional regulators to the general transcription factors, to core RNA polymerase (e.g., the CTD) and to 146 4 The Regulation of Gene Expression

chromatin-modifying enzymes. However, it remains largely unknown as to how these different interactions and functions are orchestrated during the various steps of transcription.

4.2.7 C-Terminal Domain (CTD) of RNA Polymerase II and the Onset of Transcription

Once a basal transcription complex has been deposited on the promoter, further signals are required to initiate RNA synthesis and to begin processive transcription elongation. Although all three eukaryotic RNA polymerases are very similar in structure and subunit conﬁguration, Pol II uniquely possesses an extra CTD on its largest subunit, Rpb1. This structural feature has been recognized as major control element for the onset of productive elongation. The CTD of Pol II is phosphorylated in a dynamic fashion, and this phosphorylation plays a key role in many aspects of the transcription process such as transcription initiation, transcription elongation, capping, splicing and polyadenylation of mRNA [8]. Transcription and mRNA processing are tightly coupled, and the phosphorylated CTD plays a key role in the linkage of the two processes. For this reason, the CTD dynamically associates with components of the splicing apparatus, including proteins with a high homology to splicing regulatory proteins such as SR-proteins (Section 5.1.3). Furthermore, proteins involved in the capping of mRNA and in polyadenylation associate with the CTD during transcription elongation, establishing a ﬁrm link between transcription and mRNA processing.

& CTD of catalytic subunit: — 52 copies of YSPTSPS — Multiply and dynamically phosphorylated — Involved in: Transcription initiation Capping Splicing Polyadenylation.

The CTD forms a tail-like, poorly structured extension from the catalytic core of

RNA polymerase II. It contains 52 copies of the heptamer sequence Tyr1-Ser2-Pro3- fi Thr4-Ser5-Pro6-Ser7 at which phosphorylation occurs. Ser2 and Ser5 were identi ed as the major phosphorylation sites, and multiple functions for these modifications have been elucidated. Furthermore, the phosphorylation of Ser7 and other covalent modifications have been described. Notably, different phosphorylation states predominate at each stage of transcription (Figure 4.8), and each preferentially binds a distinct set of proteins. These dynamic interactions provide a means for the coupling and coordinating of specific stages of transcription with other events necessary for correct gene expression. 4.2 The Components of the Eukaryotic Transcription Machinery 147

Figure 4.8 Stages of RNA Pol II CTD components. Early CTD Ser2 phosphorylation phosphorylation. Functional interactions are is triggered by an association of the Cdk9 shown as black arrows, and enzymatic kinase with Ser5P and/or capping enzymes. activities as red arrows. (a) Preinitiation: In the This kinase also phosphorylates the elongation preinitiation complex (PIC), transcription factor Spt4/5, which then contributes to activators (Act) recruit RNA Pol II by elongation and nucleosome methylation via interacting with Mediator. Once in the PIC, the RNA polymerase II-associated factor (PAF) Mediator stimulates the TFIIH kinase, which complex; (c) In late elongation, the phosphorylates CTD Ser5 and Ser7. CTD mammalian CDK9 kinases more extensively phosphorylation then triggers Mediator phosphorylate Ser2. This modification helps to dissociation; (b) Early elongation: In early recruit Spt6 and the H3K36 methyltransferase elongation, levels of CTD Ser5P and Ser7P are to cotranscriptionally modify chromatin. Ser2P high, but levels of Ser2P are low. CTD Ser5P also interacts with the polyadenylation and helps to recruit mRNA-capping enzymes, the termination machinery at mRNA 30 ends. After H3K4 methyltransferase, and other elongation Ref. [8]. 148 4 The Regulation of Gene Expression

CTD-phosphorylation is ascribed a major role in the transition from the initiation phase of transcription to the elongation phase. During preinitiation complex (PIC) assembly, the Mediator–coactivator complex bridges upstream activators and RNA Pol II. In addition to Mediator binding unphosphorylated polymerase, when incorporated into the PIC it strongly stimulates the CTD kinase activity of TFIIH located on CDK7. The subsequent phosphorylation of Ser5 and Ser7 disrupts Mediator binding, releasing it from the CTD (Figure 4.8), such that RNA Pol II can then enter the elongation phase. In addition to the release of Mediator, the bivalent phosphorylations introduced by CDK7 specify the cotranscriptional engagement of the RNA processing machinery and of histone-modifying enzymes. In early elongation, the phosphorylation of Ser5 helps in the recruitment of a H3K4 methyltransferase, mRNA capping enzymes, and further RNA processing enzymes. During transcription of the ﬁrst few hundred nucleotides, the levels of Ser5-P remain high but decline further downstream. The kinetics of Ser2 phosphorylation differ from Ser5 phosphorylation, and Ser2-P associates with a set of proteins distinct from Ser5-P. Levels of Ser2-P are initially low, but are increased in parallel to the decline of Ser5-P. CTDSer2-P mediates, for example, the association of H3K36 methyltransferase and of RNA processing factors involved in transcription termination. The phosphorylation of Ser2 is catalyzed by the protein kinase CDK9/ cyclin T, while removal of the phosphate is catalyzed by the phosphatase Fcp1.

& Protein kinases for CTD phosphorylation: — CDK7/cyclin H — CDK8/cyclin C — CDK9/cyclin T.

In summary, three different protein kinases, all of which are members of the cyclin-dependent protein kinase (CDK) family (see Section 15.2.1), have been identiﬁed that can phosphorylate the CTD:

CDK7/cyclin H: This protein kinase and its activating subunit, cyclin H, are localized on the general transcription factor TFIIH. CDK8/cyclin C: The Mediator complex may associate with the CDK8 module, which also participates in phosphorylating the CTD. This phosphorylation is implicated in transcription repression. CDK9/cyclin T: This protein kinase is the target of transcription activation by the retroviral TAT protein.

Furthermore, specific protein phosphatases are involved in shaping the phosphorylation pattern of the CTD and in recycling the polymerase for the next round of transcription. Altogether, CTD phosphorylation has proven to be a point where many regulatory signals may converge and influence the transition from initiation to elongation, the 4.3 The Principles of Transcription Regulation 149 efficiency of elongation, and the maturation of the mRNA. However, mechanistic details of how the various phosphorylated forms of CTD are recognized by the CTD-binding proteins, how the various kinases cooperate, and how they are regulated, remain to be established.

4.3 The Principles of Transcription Regulation

Summary Transcription is primarily controlled at the level of transcription initiation. The signals that mediate transcriptional control target chromatin, the transcription apparatus (including the GTFs), and gene-regulatory proteins with sequence- specific DNA-binding properties. The specific transcription factors are of modular structure. DNA-binding domains, transactivation domains and regulatory domains cooperate in conveying signals to the transcription start site and the neighboring chromatin structure. These processes include dimerization of the specific transcription factors on the cognate DNA elements, interaction with mediator and the transcription apparatus, as well as the attachment of multiple signal-directed PTMs. Furthermore, sequence-specific transcription factors interact with coregulators, including chromatin-modifying enzymes and chromatin-remodeling proteins, to promote transcription-permissive or -repressive chromatin structures at the transcription start site. In most cases, the regulatory DNA elements are found in clusters at a variable distance to the transcription start site, and multiple specific transcription factors cooperate in transcription activation.

4.3.1 Elements of Transcription Regulation

Transcription represents the most important point of attack for the regulatory processes, which control the flow of genetic information from DNA to mature protein. Primarily, it is the initiation of transcription that is regulated, as this represents the rate-limiting step. The regulatory signals that target gene transcription can originate from within the cell or from outside. In most cases, these signals are transmitted to the level of transcription via distinct signaling pathways that control the transcriptional activity in a positive (i.e., activating) or in a negative (i.e., repressing) manner. A large number of proteins are involved in transcription regulation. These proteins form part of a regulatory network where the concentration, activity, localization and modification of the protein components shape the flow of information down to the process of RNA synthesis. 150 4 The Regulation of Gene Expression

& Elements of transcription regulation: — DNA-recognition elements — Trans-acting transcription factors — Mediator — Chromatin structure.

The essential elements of regulation at the level of initiation in eukaryotes are (Figure 4.9):

DNA recognition elements: Recognition elements (REs) usually represent specific protein-binding sites on the DNA that lie near the transcription start site or are quite distanced from it. Protein binding to the RE by sequence-specific DNA-binding proteins interprets and transmits the information that is encoded in the primary DNA sequence to the factors and cofactors that mediate the synthesis of RNA from the DNA template. It is typical for higher eukaryotes that various REs are found in clusters forming composite control regions where several transcription factors act cooperatively in the initiation of transcription. If the activating REs are located far from the site of action, and their effect is also orientation-independent, then this region is termed an enhancer. Inhibitory REs of this type are typically found clustered in genomic regions called silencers. Sequence-specific transcription factors: Trans-acting, sequence-specific transcription factors function as the key interface between genetic regulatory information and the transcription system, allowing for the activation or repression of specific genes in response to external or internal cues (Section 4.4). They specifically bind the REs in order to select the gene to be transcribed or repressed. Mediator: The DNA-bound sequence-specific transcription factors must communicate with the transcription machinery and chromatin to allow for the formation of a pre-initiation transcription complex and to induce transcription-competent

Figure 4.9 The main steps of eukaryotic the deposition of trans-acting proteins at the transcription initiation. In a first step, cis-elements and of RNA polymerase and the chromatin is reorganized at the sites of general transcription factors at the promoter. transcription factor binding, the cis elements, The Mediator complex plays an essential role and at the promoter. As a consequence, by providing a link between the transcription nucleosome-free zones are created allowing activators and RNA polymerase. 4.3 The Principles of Transcription Regulation 151

chromatin structures. To this end, sequence-specific transcription factors use Mediator as a bridge to the transcription apparatus. Furthermore, coactivators or corepressors are recruited to the start site, inducing specific chromatin modifications. Chromatin modification and restructuring: Chromatin structure is a major attack point for transcription regulation in eukaryotes. Repressive and activating chromatin structures are known that depend on specific PTMs of histones and other components of chromatin. Efficient transcription initiation requires the removal of nucleosomes from the transcription start site and the recruitment of chromatin-remodeling complexes, allowing deposition of the core transcription machinery. Furthermore, binding sites for the activators must be available. To achieve these goals, the nucleosome structure and positioning must be changed, which requires the recruitment of histone-modifying and nucleosome-mobilizing complexes.

4.3.2 Regulation of Eukaryotic Transcription by Specific Transcription Factors

Primary control elements of the transcriptional activity in eukaryotes are sequence- specific DNA-binding proteins, often simply called transcription factors. The transcription factors bind the REs via a DNA-binding domain, and thus impart an activating or repressing influence on the initiation of transcription. The number of transcription factors with a DNA-binding domain increases with increasing genetic complexity. It is estimated that yeast contains about 300 transcription factors, whereas the human genome encodes about 2600 proteins that harbor DNA-binding domains of which a large proportion is thought to bind sequence-specifically to DNA elements. A typical transcription factor is of modular structure (Figure 4.10), and binds as a dimer or higher oligomer to the RE. A DNA- binding domain, one or more activation or repression domains, as well as dimerization domains and regulatory domains, are characteristic structural elements of sequence-specific transcription factors in eukaryotes.

& Sequence-specific transcription factors: — Bind to cis-elements — Register signals — Transmit signals to RNA Pol, Mediator, and chromatin.

Figure 4.10 Typical domains of transcriptional activators. The sequential order of the domains is variable. 152 4 The Regulation of Gene Expression

Aside from their DNA-binding property, transcription factors have the ability to register regulatory signals and to transmit these on to the transcription apparatus and chromatin.

& Modular structure of transcription factors: — DNA-binding domain — Dimerization domain — Transactivating domain — Regulatory domain.

The various domains of transcription factors do not function in isolation, as suggested from the linear representation in Figure 4.10. Rather, structural analyses of intact eukaryotic transcription factors have shown that the domains are tightly packed and are conformationally and functionally coupled. It is now well established that eukaryotic transcription factors are allosteric proteins that exist in different conformations and thereby may perform distinct functions. The various conformations are in equilibrium, and this equilibrium may be shifted by regulatory influences such as binding to the RE, PTM (e.g., phosphorylation), and the binding of cofactors and of chromatin-remodeling proteins. The nature of these influences specifies a distinct population of conformations and thereby a distinct function. The flexible nature of transcription factors is also illustrated by the observation that many of them harbor intrinsically disordered regions (see e.g., p53; Section 16.7) that are sites of PTMs and serve to recruit ligands as, for example, the RE or coactivator or corepressor proteins. The first crystal structure of the intact nuclear receptor PPARc-RXR heterodimer, a hormone-regulated transcription factor, shows many contacts between the various domains (see Figure 6.9), and this is thought to be the basis for the transcription factor’s ability to transmit hormonal signals in a DNA sequence-directed manner to the transcription apparatus.

4.3.2.1 Activation and Repression of Transcription The DNA of eukaryotes is ordinarily refractory to transcription because of its organization in nucleosomes, where the DNA is wrapped around a histone octamer. In a state that may be termed the “ground state” of chromatin, the nucleosomes are densely packed and may cover the control regions on the DNA required for the initiation of transcription. To activate transcription, the promoter region and the binding sites for the transcription factors must be accessible, but this requires the removal of nucleosomes from these regions. Nucleosome-covered DNA is transcriptionally inert, and histone modiﬁcation and nucleosome reorganization are required to overcome this repression. All proteins that are bound to DNA and serve to relieve the nucleosome-covered state favor transcription initiation and therefore function as transcription activators. On the other hand, all DNA-bound proteins that maintain the nucleosome-covered state at the promoter and prevent nucleosome mobilization may function as a transcription repressor. 4.3 The Principles of Transcription Regulation 153

4.3.3 Coregulators of Transcription

In addition to the transcription factors, the Mediator complex and the basal transcription machinery (Figure 6.9), other proteins are required for a regulated and highly efficient transcription of DNA in the context of chromatin. These proteins are loosely defined as coregulators, and may be classified by their function as either coactivators or corepressors.

& Coactivators: — Required for activation — Organized in large protein complexes. Examples: — DNA topoisomerases — HATs, HDACs — Poly-ADP-ribose polymerase — HMG proteins.

Typical coactivators or corepressors are found in large protein complexes that associate with the DNA-bound activator or repressor. Histone-modifying enzymes (see Section 4.5) such as protein lysine acetyltransferases (KATs, HATs) or protein lysine deacetylases (KDACs, HDACs) are found in these complexes, and exert either activating or repressing effects on transcription. Further coregulators have been described which participate in transcriptional regulation in a general sense. This loosely deﬁned class of general coactivators comprises proteins with enzymatic activity such as DNA-topoisomerase I and poly- ADP-ribose polymerase, as well as architectural or scaffolding proteins such as the HMG1 and HMG2 proteins (HMG ¼ high-mobility group). These proteins appear to be generally required for chromatin reorganization during transcription initiation and elongation.

4.3.4 DNA-Binding of Specific Transcription Factors

Regulatory DNA-binding proteins display speciﬁc and selective DNA-binding capacity by which they select particular genes for regulation.

& Sequence-specific transcription factors: — Select genes for regulation.

Only those genes which possess a copy of a particular DNA-binding element are subjected to control by the corresponding binding protein. 154 4 The Regulation of Gene Expression

4.3.4.1 DNA-Binding Domains Sequence-speciﬁc transcription factors contact their recognition sequences via deﬁned structural elements, termed DNA-binding motifs. These motifs are often found in the structural elements of a protein which can fold independently from the remainder of the protein, and therefore represent separate DNA-binding domains.

& DNA-binding motifs: — Helix-turn-helix — Zn-binding (Zn finger) — Basic: Helix-loop-helix Leucine zipper. — b-structures.

The region of the sequence-specific transcription factor which interacts with the recognition sequence often displays a characteristic small structural element which is stabilized through assistance from other structural elements, and is thereby brought into a defined position relative to the DNA. These DNA-binding motifs contain short a-helical or b-sheet structures that in most cases contact the DNA sequence within the major groove; moreover, the dimensions of the major groove make it well suited to accept an a-helix. Accordingly, a-helices are often utilized to contact the major groove, although many examples exist of interactions with the minor groove. Some transcription factors do not use well-defined structural elements to contact the DNA; rather, the unstructured part adopts a defined structure only when in complex with the DNA. An example of this is the transcription factor p53. Transcription factors are classified based on a common structural framework of the DNA-binding domain. Four superclasses have been identified by their DNA- binding motif: helix-turn-helix motifs, basic motifs, zinc-coordinating motifs, and b-scaffold motifs. Each superclass is further divided into a large number of further classes. The helix-turn-helix, zinc finger or leucine zipper motifs are found in >80% of the transcription factors of higher eukaryotes. The properties of these motifs may be briefly summarized, as follows:

Helix-turn-helix (HTH) motifs: This motif uses a recognition helix that is embedded in the large groove of the DNA. Additional a-helices stabilize the arrangement of the recognition helix. Transcription factors of the forkhead (FH) and homeodomain class belong to the HTH superclass. 2þ þ Zn -binding motifs: These motifs contain Zn2 complexed by four ligating Cys and/or His residues. Based on the stoichiometry of the complex, zinc ﬁngers of the

type Zinc-Cys2His2, Zinc-Cys4 and Zinc2-Cys6 can be distinguished. The zinc- binding motifs play, above all, a structuring role by ensuring that a recognition 4.3 The Principles of Transcription Regulation 155 helix is correctly oriented and stabilized and they may be involved in dimerization. The nuclear receptors are well-studied examples of transcription factors containing Zn-motifs (see Chapter 6). Basic motifs: This group of binding motifs displays as a characteristic structural element an extended bundle of two a-helices that are wound around each other in the form of a “coiled-coil.” At their end is a basic region which mediates the DNA binding. The basic region is often unstructured in the absence of DNA, and adopts a distinct structure only upon binding to the recognition element. This motif is used mainly for dimerization of transcription factor. Subclasses are distinguished by the presence of basic leucine zipper or helix-loop-helix motif. Important examples are the Myc/Max transcription factors (Section 16.2.3) b-scaffold: Proteins belonging to this class use b-sheets to contact the DNA. A well- characterized example is the transcription factor NFkB.

4.3.5 Structure of the Recognition Sequence and Quaternary Structure of DNA-Binding Proteins

Transcription factors are usually organized in a dimeric or tetrameric manner. Because the monomers bind to similar sequences, the REs typically consist of two half-sites each comprising three to eight base pairs. However, larger REs are also known with no immediately recognizable symmetry, as well as singular REs. The half sites can be arranged either palindromically (inverted repeat), or as direct repeats (Figure 4.11) separated by between zero and eight spacer nucleotides.

& DNA elements: — 3–8 base pairs — Inverted repeat or direct repeat — Binding of dimeric transcription factors.

4.3.5.1 Palindromic Arrangement Palindromic sequences with twofold symmetry are usually bound by dimeric proteins in which each subunit of the protein contacts one half-site of the DNA element.

4.3.5.2 Direct Repeats of the Recognition Sequence Direct repeat of the recognition sequence requires a nonsymmetrical spatial arrangement of the bound protein subunits. The protein–DNA complex has, in this case, a polar character and the two proteins bound on the two respective halves of the DNA element can register different signals and carry out different functions (see nuclear receptors; Chapter 6). 156 4 The Regulation of Gene Expression

Figure 4.11 Symmetry of DNA recognition major determinants of specific protein elements and the oligomeric structure of DNA binding; (b) Dimeric DNA binding proteins binding proteins. (a) The palindromic bind in a symmetric way to palindromic recognition sequence of the protein E2 from sequences; (c) Dimers formed at tandem papillomavirus contains an inverted repeat of repeats arrange in “tail-to-head” orientation. which four base-pairs (red arrows) are the

4.3.5.3 Identity of a RE The speciﬁc function of a given RE in transcription regulation is determined by the half-site arrangement, its sequence, and the number of spacer nucleotides. Furthermore, the neighboring sequences and the nature and number of neighboring REs is important for the regulatory role of a RE (Section 4.3.8). Many transcription factors tolerate the exchange of one or more nucleotides in the RE sequence, and can bind with no change in afﬁnity to related sequences. Therefore, REs are often represented in the form of sequence logos with weighted occurrences of nucleotides at a given position (Figure 4.12). However, examples are also known where every nucleotide counts and where the exchange of a single nucleotide may substantially alter the regulatory function of the transcription factor.

4.3.5.4 Homodimers and Heterodimers An important aspect of the occurrence of multimeric REs is the possibility of the formation of heterodimers. There exist related classes of DNA-binding proteins which recognize similar half-sites and which possess a common dimerization motif. Among these, both homodimers and heterodimers can be formed which bind to DNA, but with slightly different speciﬁcities. 4.3 The Principles of Transcription Regulation 157

Figure 4.12 Sequence logo of the p53 constructed from a set of aligned sequences. response element. The p53 protein is a tumor In this graphical method, the characters suppressor (see Section 16.7) that recognizes representing the sequence are stacked on top and binds to DNA containing p53 response of each other for each position in the aligned elements (REs) that comprise two decamer sequences. The height of each letter is made motifs RRRCWWGYYY in direct repeat. The proportional to its frequency, and the letters sequences of activating p53 REs (adapted from are sorted so that the most common one is on Ref. [9]) are shown as sequence logos top [10].

& Transcription factors form: — Heterodimers — Homodimers — Tetramers.

This property allows for the fine-tuning of transcription regulation, and is widely used to activate distinct transcriptional programs in a flexible manner to serve multiple biological functions. Notable examples are the nuclear receptors (Chapter 6). Transcription factors have also been identified that bind to the RE as tetramers; one such example is p53 (see Chapter 16)

4.3.6 Communication with the Transcription Apparatus: Transactivation Domains

The activation of transcription occurs in large assemblies where transcription factors communicate with other multiprotein complexes in a dynamic manner. Among these are chromatin-modifying and nucleosome-mobilizing complexes, Mediator, and the core transcription apparatus. Furthermore, the large assemblies involved in transcriptional activation contain enzymes that can modify the transcription factor posttranslationally and thereby transmit external regulatory signals in the form of chemical modiﬁcations to the level of transcription. Overall, transcriptional activation is a multistep process where the transcription factor is engaged in a dynamic fashion in a large number of interactions with other proteins. It is for this reason that the transcription factors of higher eukaryotes are of multidomain structure and can exist in multiple PTM states. The structural regions of transcription factors involved in activation are termed transactivation domains. These domains contain sequence motifs that are bound by complementary motifs on coactivators or corepressors. 158 4 The Regulation of Gene Expression

& Transactivation domains communicate with: — RNA Pol II — Coactivators — Corepressors — Mediator.

As outlined above, transactivation is a complex process where the transcriptional activator interacts with a variety of different components of the transcription apparatus in a dynamic manner. The presence of two activation domains in some transcription factors, such as in nuclear receptors (see Section 6.3.3), illustrates this aspect of eukaryotic transcriptional activation. In this case, each of the two transactivation domains is thought to contact distinct protein partners, and each contact may make differential contributions to transcriptional activation. In addition, the two transactivation domains may not act independently; rather, they can cooperate in transcriptional activation via allosteric mechanisms.

& Transcription factors: — May harbor one or two transactivation domains.

Unlike the well-defined DNA-binding modules, transactivation domains do not appear to be well-defined structural domains, and may not always require a defined structure. As an example, the transactivation domain of the tumor suppressor p53 is unstructured in isolation, but becomes structured upon interaction with the effector RPA (see Section 16.7) [11]. Most information on transactivation domains is available for the nuclear receptors (Section 6.3.3), and these data suggest that trans-activating domains can adapt to become complementary to a surface of the various binding partners via allosteric mechanisms. For example, the transactivation domain of steroid hormone receptors undergoes a ligand-dependent reorganization that allows receptor binding to the LDLL motif of the p160 coactivator (Section 6.4.2). The target surfaces of the binding partners appear to be less specific than might be intuitively assumed. Indeed, much structural evidence suggests that activation domains expose flexible hydrophobic elements to contact hydrophobic patches on the target. As these interactions per se are not highly specific, colocalization of the activation domain and the target protein on the chromatin-associated DNA template seems to be an important factor for activation. In addition, cooperative interactions of transcription factors bound to multiple sites at enhancers may help to increase the efficiency of chromatin remodeling and contact formation with the core transcription apparatus. Furthermore, it must be assumed that the transactivation domains contact different proteins in a sequential manner during the transcription initiation step. The Mediator is now considered as a main target of the transactivating domains, providing a link between transcriptional activators and the transcription machinery. Other targets belong to the group of basal transcription factors, such as TFIIB. Trans-activating domains also mediate contacts to the 4.3 The Principles of Transcription Regulation 159 chromatin-remodeling and chromatin-modifying complexes, such as lysine methyltransferases and lysine acetyltransferases, and recruit these to either the enhancer or the promoter region.

4.3.7 Clustering of REs and the Enhanceosome

On passing from unicellular eukaryotes to metazoans, an increasing complexity of the regulatory DNA regions and of the multiprotein transcription complexes that regulate gene expression is observed. One characteristic feature of metazoan genomes is the presence of distal control regions, comprising enhancers, silencers and regions termed insulators. A typical animal gene is likely to contain several enhancers that can be located in 50 and 30 regulatory regions, as well as within introns. Each enhancer is responsible for a subset of the total gene expression pattern; they usually mediate expression within a specific tissue or cell type. These regulatory DNAs are scattered over distances of approximately 10 kb in fruit flies and 100 kb in mammals. A typical enhancer will be about 500 bp in length and contain on the order of ten binding sites for at least three different sequence- specific transcription factors, most often two different activators and one repressor.

& Enhanceosome: — Operating at a large distance from the start site — Clustering of multiple REs — Cooperative binding of transcription factors — Multivalent interactions with coactivators.

The multiple REs within an enhancer may be separated by spacers and, together with the bound transcription factors and their cofactors, these constitute a unit. One of the best-characterized higher eukaryotic enhancers is that of the virus- inducible interferon (IFN)-b gene [12]. Expression of the INF-b gene requires the coordinate activation and DNA binding of four transcription factors, namely ATF- 2/c-Jun, IRF-3, IRF-7, and NFkB (p50/RelA). In vivo, these transcription factors bind cooperatively to a nucleosome-free region of the IFN-b promoter, spanning the interval from 102 to 47 bp relative to the transcription start site (Figure 4.13a). Cooperative binding of the activators requires a precise positioning of the DNA-binding sites and assembly into an enhanceosome, which is thought to provide stringent speciﬁcity and stability. The enhancer operates as a “coincidence detector,” as signals that induce individual transcription factors are not sufﬁcient to activate IFN-b transcription. Following enhanceosome assembly, a sequential recruitment of histone acetyltransferases GCN5 and CBP/p300 leads to nucleosome acetylation and chromatin remodeling by the SWI/SNF complex, so as to allow access to the core transcription machinery. The structure of the complexes formed at the IFN enhancer, as determined by crystallography and modeling, is shown in Figure 4.13b. The complete structure has revealed some important 160 4 The Regulation of Gene Expression

Figure 4.13 The enhanceosome of the virus- regulatory domains (PRDs); (b) Atomic model inducible interferon b (IFN-b) gene. (a) of the INF-b enhanceosome (PDB file). The Binding sites for transcription factors on the p50 is in light blue and RelA in dark blue; IRF- IFN-b enhancer. Expression of the IFN-b gene 7B and IRF-7D are in yellow, and IRF-3A and requires the coordinate activation and DNA- IRF-3C are in green; ATF-2 is in red and c-Jun binding of the transcription factors ATF-2/c- in blue. The DNA sequence is shown with Jun, IRF-3 and IRF-7, and NFkB (p50/RelA to a the core-binding sites colored accordingly. nucleosome-free region of the IFN-b promoter Panne 2008 [12], figure 5. Reproduced with that has been subdivided into four positive permission of Elsevier.

aspects of the multiprotein assembly on DNA that may also be valid for other higher-order complexes on promoters and enhancers: The cooperativity of activator binding is mediated by DNA-conformation, and thus by DNA sequence. The binding sites overlap, and the association of the eight transcription factors form a composite surface for recognition of the entire enhancer sequence. There are only a few direct protein–protein interactions between individual components. Each of the transcription factors can interact through their activation domains with the coactivator CBP (Section 4.5.2), and it is likely that multivalent interactions with these coactivators stabilize the enhanceosome assembly further. 4.3 The Principles of Transcription Regulation 161

4.3.8 DNA Recognition and Selectivity of Transcription Activation

Many transcription factors have been shown to control the transcription of a large number of target genes; for example, the tumor suppressor p53 directly regulates about 300 target genes. However, this raises the questions of how a transcription factor can recognize its cognate element in the presence of many similar elements, how many sequence variations are tolerated, and which factors determine this selectivity. The problem of binding site selectivity is illustrated by the fact that the human genome encodes about 2600 proteins with DNA-binding domains that may choose their binding site from many genetic regions. For a given transcription factor, the number of potential binding sites is very large: for example, a recognition element of 6 bp would occur 700 000 times in the human genome. Furthermore, transcription factors have been shown to tolerate changes from the consensus recognition sequence, which expands the large number of possible binding sites.

& Selection of REs by transcription factors is determined by: — Sequence of RE — Structural flexibility of transcription factor — Neighboring sequence — Chromatin surrounding.

The picture that has emerged from studies on transcription factor selectivity and their evolution is much more complicated than has been inferred from earlier studies with lower eukaryotes and bacteria. In fact, it is now increasingly accepted that the activation of speciﬁc gene loci depends on the following factors.

4.3.8.1 Sequence of the RE A complex landscape exists of a transcription factor binding to its RE. Typically, REs recognized by a particular transcription factor are often highly degenerate, and deviations from the consensus sequence by the exchange of one or two nucleotides and changes in spacer length may be readily tolerated. Binding to degenerate REs is highly variable; some degenerate REs are bound with a high affinity, and others with a low affinity. Interestingly, the binding profiles of a large number of mouse transcription factors could be best represented by more than one motif. Almost half of the 104 mouse transcription factors studied [13] showed such secondary DNA- binding preferences. Examples of secondary REs are shown in Figure 4.14. Binding to secondary binding sites occurs mostly with a lower affinity, and low-affinity sites may be shared between different transcription factors. Moreover, some transcription factors bind to sites that do not share any similarity with the consensus sequence. This is intriguing, as it suggests that some transcription factors recognize their DNA-binding sites through multiple, completely different interaction modes, either through alternate structural features or by switching between alternate conformations. 162 4 The Regulation of Gene Expression

A CA T G CG CGC G CC (a) Jundm2 TT A T T A

A G C C G A A A T G GT T CCTCA T

A G T G (b) Hnf4a AAGGTTCAA

A T ACAAGTCCACC

T T (c) Zfp187 C ATGTC ACCAAT

G C C G A ACC TC TAGTTTC

Figure 4.14 Transcription factor binding site in spacer length by one nucleotide; (b) secondary motifs. The sequence logos of Multiple effects: The transcription factor Hnf4a secondary DNA-binding motifs of mouse accepts multiple changes in the recognition transcription factors are shown [13]. Three element; (c) Alternate recognition interfaces: types of secondary motifs are shown: (a) The Zn-finger protein Zfp187 binds to a Variable spacer length. The transcription factor secondary motif that is completely different Jundm2 binds to secondary motifs that differ from the primary motif [13].

In humans, a surprisingly large variation exists in RE usage between individuals, and these differences are often correlated with differences in gene expression, which suggests a functional signiﬁcance of binding variation [14].

4.3.8.2 Allostery in Transcription Factor---RE Interactions Transcription factors and DNA may exist in distinct conformations, which allows for the allosteric regulation of recognition and modulation of subsequent events. Several cases are known where the precise nucleotide sequence of a RE, in addition to guiding the transcription factor to speciﬁc genomic loci, may also specify the mode of transcriptional regulation. DNA sequences may serve as such a signal, functioning as allosteric ligands that direct the activity of transcriptional regulators. For example, single nucleotide differences in NFkB binding site sequences can determine cofactor speciﬁcity for NFkB dimers [15]. Furthermore, the transduction of structural changes from DNA to domains of nuclear receptors has been described: DNA binding by glucocorticoid receptor induces a secondary structure in its activation domain AF1, while the activation domain AF2 of the estrogen receptor interacts with different cofactor peptides when the receptor is bound to different sequences [16]. The reverse scenario may be also important, as the nature of coregulator bound to the activation domain may be a determinant for the selection of a binding site.

4.3.8.3 Influence of Neighboring Sequences and Chromatin Surrounding Sequences adjacent to the consensus RE may modulate transcription factor binding. Furthermore, the tight packing of multiple transcription factors and coregulators on composite DNA regions such as enhancers may inﬂuence the sequence requirements for protein binding to the RE. Overall, transcription factors, their coregulators, and the bound DNA may be considered as conformationally ﬂexible ensembles where shifts in the 4.3 The Principles of Transcription Regulation 163 conformation of the components occur upon binding of the interaction partners. Especially in enhanceosomes, where multiple transcription factors and coregulators form a compact entity, a mutual adaptation of the components occurs so as to create a surface for the recruitment of further transcriptional regulators.

4.3.9 Repression of Transcription

The classical view of repressors, as based on observations in bacteria, can only be applied to a very limited extent to eukaryotes, due to the organization of eukaryotic DNA in chromatin. Bacterial repressors mostly function by competing with activators for binding to control regions near the promoter, or by preventing the binding of RNA polymerase to the transcription start site.

& Transcription repression: — Primarily by activation of enzymes that induce repressive chromatin structure — Includes recruitment of HDACs and histone-methyltransferases.

Although, originally, it was assumed that eukaryotic repressors use similar mechanisms, it is now generally accepted that chromatin is an integral part of transcription regulation, particularly of transcription repression. The function of nearly all eukaryotic repressors can be linked to the activation or recruitment of enzymes that covalently modify histones or other chromosomal proteins to maintain a repressive chromatin structure. One particular feature of eukaryotic gene repression is illustrated by the observation that the same regulatory protein can function as a repressor or an activator of transcription, depending on the nature of the regulatory signal that impinges on the regulator. When functioning as a repressor, the regulator recruits to the promoter a corepressor complex containing enzymes that modify histones to establish a repressive state of chromatin. An activating signal can then convert the repressor into an activator that recruits a coactivator complex. Enzymes within the coactivator complex then promote nucleosome displacement at the promoter and thus activate transcription. The switch between activator and repressor functions can be traced back in some cases to conformational changes in the transactivation domain that dictate the nature of the associating coregulator, either a corepressor or a coactivator. This point is illustrated in more detail in Chapter 6, using the example of hormone agonists and antagonists of nuclear receptors. The large number of distinct steps required for eukaryotic gene transcription suggests many possibilities for negatively controlling transcription (Figure 4.15). As stated above and presented in more detail in Section 4.5, the major part of the negative regulation of transcription is exerted via the chromatin structure. Transcription can be shut down very effectively by imposing a “closed,” repressive state on chromatin, which may occur in a signal-directed, regulated fashion. 164 4 The Regulation of Gene Expression

Figure 4.15 The various mechanisms for the repressors can block the activating function of repression of transcription. (a) Repressors can the latter; (e) Active repression is also affected induce a generally repressed state in chromatin by proteins that bind sequence-specifically to which is incompatible with transcription. To DNA elements and in their DNA-bound form allow transcription at all, the repressed state inhibit the transcription initiation. They do this must be relieved; (b) Repressors can target the mainly by recruiting enzyme activities (HDACs, transcription complex and thereby inhibit Lys methylases) to chromatin that help to transcription initiation; (c, d) By binding to free establish a transcription-incompetent state. or DNA-bound transcriptional activators, In addition, many gene-repression mechanisms that are not directly related to chromatin structure have been described, though in most cases the mechanistic basis is not clear. & Other repression mechanisms at the level of transcription factors: — Heterodimerization — Phosphorylation — Cytosolic retention — Binding of inhibitors. 4.4 The Control of Transcription Factors 165

Transcriptional repressors may function by binding to transcription activators, or by competing with the activator for overlapping binding sites. The extent of repression is then determined by the relative afﬁnity of both proteins to the DNA element, and their concentration ratios. Another repressive mechanism operates at the level of the subcellular distribution of transcriptional activators. As outlined in Section 2.8.5.3, using the example of NFkB, transcriptional activators may be kept in a repressed state by the binding of inhibitory proteins that prevent nuclear localization of the activator. Transcrip- tion repression can also result from phosphorylation of the basal transcription factors. By this token, the repression of transcription observed during mitosis (Chapter 15) is attributed – at least in part – to the hyperphosphorylation of TBP and TAFs.

4.4 The Control of Transcription Factors

Summary Cells use multiple mechanisms to control the more than 2000 transcription factors encoded in the human genome. The controls include binding of effector þ molecules such as hormones and Ca2 -ions and binding of inhibitor proteins. Furthermore, posttranslational modification of specific transcription factors is used to regulate the subcellular localization and interaction with chromatin- modifying proteins or protein complexes. The major PTMs found on specific transcription factors include Ser/Thr phosphorylation, Lys acetylation and ubiquitination. These modifications are recognized by cognate interaction domains of chromatin components with coregulator function.

4.4.1 Classification of Transcription Factors by their Function in Signal Transduction Networks

& Transcription factors may be: — Constitutively active — Under the control of external or internal signals.

In addition to classiﬁcation by structural features (Section 4.3.4), transcription factors with an activating function may be classiﬁed by their role within the signal transduction network of the cell [17]. On the basis of their biological and regulatory functions, transcription activators may be divided in distinct groups, as outlined below and illustrated in Figure 4.16. This grouping is based on whether, and how, the transcription activators are subject to internal or external regulation. 166 4 The Regulation of Gene Expression

Figure 4.16 Classification of transcription factors by their regulatory properties. For an explanation, see the text.

4.4.1.1 Constitutively Active Transcription Factors These transcription factors are present continually in the cell nucleus of all cells, but are not implicated in the speciﬁc control of individual genes. Rather, the constitutive activators seem to facilitate the transcription of many chromosomal genes, especially housekeeping genes that are always transcribed and whose products perform central structural or metabolic functions. This group includes the proteins Sp1, CCAAT, NF1, and others.

4.4.1.2 Regulatory Transcription Factors Most transcription activators perform regulatory functions in the cell, depending on the cell type and on internal or external signals. Two broad classes of regulatory transcription may be distinguished:

Developmental transcription factors: These accumulate in speciﬁc cells during development as a consequence of a regulated transcription of the genes encoding them. Members of this group enter the nucleus as soon as they are created, and often do not require any regulated PTM for their activation. Examples include early embryonic factors in Drosophila, and the HLH transcription factor Myo D, which is required for muscle differentiation. 4.4 The Control of Transcription Factors 167

Signal-dependent transcription factors: These proteins are inactive (or minimally active) until cells containing such proteins are exposed to the appropriate intra- or extracellular signal.

& Signal-regulated transcription factors: — Nuclear receptors — Sterol response element-binding proteins — Cell-surface receptor-activated transcription factors.

To date, three broad classes of signal-dependent transcription factors have been recognized:

i) The nuclear receptor superfamily: Activation of this group of transcription factors requires signals in the form of low-molecular-weight hormones, including steroid hormones (see Chapter 5). ii) Transcription factors activated by internal signals: Some pre-existing transcription factors become activated in response to signals formed intracellularly. Such signals may be internal sterol concentrations in the case of sterol response element-binding proteins (SREBPs), or damaged DNA that increases p53 concentrations (see Section 15.6). iii) Transcription factors activated by cell-surface receptor–ligand interaction. There are two major routes from cell-surface receptors to transcription activators. First, ligand binding to receptors activates signal transduction cascades that end at resident nuclear transcription factors. These become phosphorylated at Ser/Thr residues, leading to their activation. The best known example of this type of control is the receptor tyrosine kinase-Ras-MAPK pathway (see Chapters 9–11) that can activate a diversity of transcription factors. Second, a more limited number of resident cytoplasmic transcription factors are activated in the cytoplasm or at the cell membrane after receptor–ligand interaction, and then accumulate in the nucleus to drive transcription. Following receptor activation, these factors are activated by phosphorylation at Ser or Tyr residues and then translocated into the nucleus. Examples of this type of control include the Stat proteins (Section 13.2.2), Smad proteins (Section 14.1.3), and the transcription factor NFAT (Section 8.7.3). In some cases, proteolysis is required for the production of the mature, active transcription factor. This control is exempliﬁed by the NFkB (Section 2.8.5.3), Wnt (Chapter 16), and Notch (Section 14.2) signaling pathways.

4.4.2 Mechanisms for the Control of the Activity of DNA-Binding Proteins

Regulatory DNA-binding proteins are controlled by a multitude of mechanisms. These controls may operate at the level of the concentration of the binding protein, 168 4 The Regulation of Gene Expression

or they may act on pre-existing DNA-binding proteins by post-translational mechanisms. An overview of the most important mechanisms by which the transcription-regulating activity of speciﬁc DNA-binding processes in eukaryotes can be controlled is provided in Figure 4.17. These mechanisms include de novo

Figure 4.17 Major mechanisms for the control signal-directed translocation of transcription of the activity of transcription factors. factors into the nucleus is a major mechanism Regulatory DNA-binding proteins can occur in for transcriptional regulation. The amount of active and inactive forms. The transition available transcription factor can also be between the two forms is primarily controlled regulated via its degradation rate or rate of by the mechanisms indicated. The activation or expression. Furthermore, the interaction inactivation of transcription factors is between DNA-bound activators and the determined by signals that become effective transcription complex can be regulated by either in the cytoplasm or in the nucleus. The various signals. 4.4 The Control of Transcription Factors 169 synthesis and degradation, as well as the modiﬁcation and availability of pre- existing proteins.

& Transcription factor levels are controlled via: — Gene expression: Transcription Splicing Translation. — Targeted proteolysis — Subcellular localization.

4.4.2.1 Changes in the Concentration of Regulatory DNA-Binding Proteins The amount of sequence-speciﬁc transcription factors available for transcriptional control in the nucleus is, in many situations, a determinant of transcription activity. Three main approaches exist for controlling the level of transcription factors in the nucleus:

One main approach uses gene expression for the de novo synthesis of the transcription factor. Here, all of the mechanisms typical for eukaryotic transcriptional and translational control may be used. The speciﬁc expression of transcriptional activators is of great importance during the development and differentiation of organisms where long-term changes in gene expression are required. Concentration gradients of diffusible regulatory proteins may be also used for the control of gene expression. A second mechanism for controlling the amount of transcriptional factors uses targeted degradation. Speciﬁc signals can induce the degradation of a transcription factor via the ubiquitin-proteasome pathway (Section 2.8.5.3) and thus weaken the transcriptionally regulatory signal. A third important issue in the control of transcriptional regulators is that of subcellular localization. Currently, numerous examples are known of transcription factors that are controlled by a signal-dependent translocation from the cytoplasm to the nucleus, or vice versa (Section 4.4.3.1).

4.4.2.2 Regulation by Binding of Effector Molecules Low-molecular-weight effectors are commonly employed in bacteria to change the DNA-binding activity of repressors or transcriptional activators, and to control the amount of active DNA-binding proteins. Often, the effector molecules represent components arising from the metabolic pathway to be controlled. The goal of this regulation is to adjust the transcription rate to the current demand of the gene product. In higher eukaryotes, this type of regulation is used to a much lesser degree, and sequence-speciﬁc transcription factors are controlled by small effector molecules that are produced during the course of signaling pathways as second messengers or which function as circulating hormones: 170 4 The Regulation of Gene Expression

þ Calcium as an effector molecule:Ca2 is a key second messenger in eukaryotes þ since, by binding to intracellular signaling proteins, Ca2 is able to regulate a variety of cellular processes (see Chapter 8), including the transcription of specific genes. One example of transcription factors being regulated by metal ions is the transcriptional repressor DREAM, which binds to the cognate DNA þ þ þ element only in the absence of Ca2 . An increase in Ca2 in the form of a Ca2 signal leads to a reduced affinity to its DNA element and to an increased expression of the target gene. Hormones as effector molecules: As outlined in detail in Chapter 6, the nuclear receptors are sequence-specific transcription factors regulated by their hormone ligands, such as the steroid hormones. Here, binding of the hormone ligand activates the nuclear receptor for transcription regulation, either by relieving repression or by inducing the transport of the receptor from the cytosol into the nucleus. Inhibitory protein complexes: Transcription factors can be constrained in their ability to function as gene regulators by complex formation with inhibitor proteins. A notable example is the transcription factor NFkB, which is maintained in an inactive state by complexation with the inhibitor IkB (see Section 2.8.5.3). Here, incoming signals induce phosphorylation of the inhibitor IkB, leading to its proteolytic destruction and liberating NFkB for transport into the nucleus.

& Transcription factor activity is controlled via: þ — Ca2 ions — Hormones, for example, steroid hormones — Protein inhibitors — PTM.

4.4.3 PTM of Transcription Regulators

The post-translational covalent modification of transcription factors is a mechanism that is commonly employed among eukaryotes to control gene activation. In many cases, this occurs in response to external or internal signals, and provides for a rapid and effective adjustment of the activity of transcriptional regulators so that an immediate reaction within the framework of intra- and intercellular communication is possible. As outlined in Chapters 10, 12, 13, and 14, the final steps of signaling pathways targeting gene expression often comprise the covalent modification of sequence-specific transcription factors at distinct sites, leading to the activation or repression of transcription. The PTMs mainly serve two goals:

Control of subcellular localization: The covalent modiﬁcation of sequence- speciﬁc transcription factors by phosphorylation is widely used as a tool for regulating their nuclear localization. 4.4 The Control of Transcription Factors 171

Control of protein–protein interactions: Transcription factors are engaged in multiple protein–protein interactions during transcriptional activation, and these interactions often require distinct PTMs.

The spectrum of modifications observed on transcription factors is broad, and comprises all of the protein modifications used by the cell to control protein activity and function (Section 2.4). Importantly, most transcription factors contain several modification sites, and multiple distinct modifications have been detected in many transcription factors. The most frequent modifications found on transcription factors are phosphorylation, acetylation, and ubiquitination, while other modifications include Lys and Arg methylation and redox regulation. Each modification may serve a specific purpose and may be introduced in response to a specific stimulus. Most importantly, the modifications are reversible, which allows for dynamic modification patterns. As a result, transcription factors harbor variable modification codes that dictate specific functions in dependence of time and subcellular localization.

& Examples of transcription factor modification: — Phosphorylation — Methylation — Acetylation — Redox-modification — Proteolysis.

The complexities of the PTM of transcription factors are described in Section 16.6, based on the example of the tumor suppressor protein p53.

4.4.3.1 Regulation by Phosphorylation Most eukaryotic transcriptional activators are isolated as phosphorylated proteins. The phosphorylation occurs mainly on Ser and Thr residues, but can also be observed on Tyr residues. The extent of phosphorylation is regulated via specific protein kinases and protein phosphatases, each being components of signal transduction pathways (Chapter 8). In many cases, signal transduction chains use the phosphorylation of transcriptional activators as a final reaction in the control of gene expression. The influence of phosphorylation on the function of sequence- specific transcription factors will be illustrated in the following selected examples.

Regulation of Nuclear Localization by Phosphorylation The site of action of transcription factors is the nucleus, and newly synthesized transcription factors must translocate from the cytosol into the nucleus in order to participate in transcription regulation. Many signaling pathways use the nuclear translocation step as a switch to control the availability, and thus the activity, of transcription factors. Typically, phosphorylation is the regulatory signal, and many transcription factors are known that shuttle between the cytoplasm and nucleus in a 172 4 The Regulation of Gene Expression

phosphorylation-dependent fashion. The phosphorylations may occur in the nuclear localization sequences or in other structural parts of the transcription factor, and may have either a positive or negative inﬂuence on transport into the nucleus.

& Phosphorylation regulates: — DNA binding — Trans-activation — Recruitment of coregulators — Nuclear localization.

One well-characterized example of a negative inﬂuence of phosphorylation on translocation into the nucleus is the transcriptional activator SWI5 from yeast (Figure 4.18). The phosphorylated form of SWI5 is unable to translocate into the nucleus, and the action of a phosphatase is required to remove the phospho-signal

Figure 4.18 Regulation of the subcellular state, SWI5 is found in the cytoplasm, while in localization of the transcription factor SWI5 the under-phosphorylated state it accumulates from yeast by phosphorylation. The subcellular in the nucleus. Phosphorylation and localization of the SWI5 protein, that is, its dephosphorylation are catalyzed by protein transport into and out of the nucleus, is kinases (resp. protein phosphatases) that are regulated by phosphorylation/ part of signaling chains. dephosphorylation. In the phosphorylated 4.4 The Control of Transcription Factors 173 and allow SWI5 to enter the nucleus. The translocation-inhibitory phosphorylations of SWI5 are found in its nuclear localization signal, and appear to prevent the interaction of SWI5 with the nuclear import machinery. Another example of a negative inﬂuence of phosphorylation on nuclear translocation is the transcription factor NF-AT, which plays an important role in immune reactions (see Section 9.6.5). Phosphorylated NF-AT accumulates in the cytosol and requires dephosphorylation by the protein phosphatase calcineurin in order to be translocated to the nucleus. A positive inﬂuence of phosphorylation on the nuclear translocation of transcription factors has been documented by many examples in which phosphorylation is required for nuclear accumulation. Many signaling pathways originating from cell-surface receptors employ the activation of protein kinases and the phosphorylation of transcription factors to induce an accumulation of the transcription factor in the nucleus. Examples include the MAPK/ERK pathways (Chapter 12), which use the phosphorylation of various transcription factors to direct them into the nucleus for gene activation. Both, the STAT-proteins (Section 13.2.2) and the SMAD-proteins (Section 14.1.2) are further examples of phosphorylation-dependent nuclear accumulation. In order to reverse nuclear accumulation, the phosphorylated transcription factor must be dephosphorylated by nuclear phosphatases, and this leads eventually to a shuttling of the transcription factor between the cytosol and the nucleus.

Phosphorylation of the DNA-Binding Domain There are many examples of the specific phosphorylation of sequence-specific transcription factors within their DNA-binding domains. As an example, the DNA-binding activity of the serum response factor is inhibited by phosphorylation at a specific Ser residue within its DNA-binding domain, and this effect is used as a switch to direct target gene expression into proliferation or differentiation programs.

& Examples of phosphorylation-dependent nuclear localization: — SWI5 — STAT proteins — SMAD proteins — NF-AT.

Phosphorylation and Recruitment of Coregulators The phosphorylation of transcription factors may also serve to induce the binding of coactivators (or corepressors). The regulatory phosphorylations may occur within the transactivation domain or in other structural parts of the transcription factors. An example of this is the regulation of the CREB protein of higher eukaryotes, as displayed in Figure 4.19. The CREB protein is a transcriptional activator for genes with cis-regulatory, cAMP-sensitive DNA elements (cAMP-responsive elements; CREs). 174 4 The Regulation of Gene Expression

Figure 4.19 Regulation of the activity of a promotes a transcription-proficient state of the transcription factor by phosphorylation. The chromatin. CBP can only act as a coactivator if CREB protein is a transcription factor that the CREB protein is phosphorylated on Ser113. binds to cAMP responsive elements (CREs) The phosphorylation of CREB is controlled by a and thereby may activate the cognate genes. signaling pathway involving cAMP as an The CRE-binding (CREB) protein requires the intracellular messenger. The DNA element is assistance of the coactivator CREB-binding termed CRE because the cognate gene is protein (CBP) for efficient transcription regulated by a cAMP-dependent signaling activation. The coactivator function of CBP is pathway. based on its histone acetylase activity, which 4.5 Chromatin Structure and Transcription Regulation 175

& CREB: — Transcriptional activator — Binds to cAMP-responsive DNA elements (CREs). CBP: — Carries histone acetylase activity — CBP binding to CREB required for transcriptional activation; — Depends on phosphorylation of Ser133, catalyzed by cAMP pathways.

CREs are DNA sequences which mediate cAMP-regulated transcription. An increase in the cAMP concentration (see Chapters 6 and 7) activates protein kinases, which then leads – either directly or indirectly – to the phosphorylation and regulation of transcriptional activators. The transcription stimulation of the cognate genes requires the binding of CREB to the CREs, and the phosphorylation of CREB at Ser133. This phosphorylation event is mediated by a cAMP-dependent signal transduction pathway. Transcription activation by CREB depends on the action of a second protein, the CREB-binding protein (CBP), which binds speciﬁcally to CREB. CBP and a close relative, p300 (see Section 4.5.2), are widely used coactivators that have histone acetyltransferase activity. The interaction of CBP with CREB depends on whether it is phosphorylated at Ser133, since only if Ser133 of CREB is phosphorylated can CREB and CBP interact. It is assumed that CREB- bound CBP acetylates histones and other chromatin components, thereby relieving a repressed state of the chromatin.

4.4.3.2 Regulation by Acetylation Covalent modiﬁcation by acetylation at Lys residues is a widely used tool for the regulation of protein activity (see Section 2.6). Many transcription factors and their cognate coactivators or corepressors have been found to be acetylated, and this acetylation forms part of a network of regulatory modiﬁcations. Examples include the tumor suppressor p53 that has been found to be acetylated at multiple Lys residues (Section 16.7.4). Other examples include the transcription factor NFkB, the Foxo transcription factors, the STAT proteins, and the androgen, progesterone, estrogen, and liver X receptors (see Chapter 6).

4.5 Chromatin Structure and Transcription Regulation

Summary Chromatin structure plays a decisive role in transcription regulation. Activating and repressing chromatin structures are each well known, and both features are correlated with the pattern of PTMs found on histones and chromatin-modifying proteins. Furthermore, the placement and histone composition of nucleosomes 176 4 The Regulation of Gene Expression

regulate the access of the transcription complex to the start site, and thus transcriptional activity. Activating chromatin structures are correlated with histone lysine deacetylation, while repressing structures are correlated with histone lysine acetylation. Hence, histone methylation of Lys/Arg residues and phosphorylation of Ser/Thr represent further tools for regulating transcriptional activity. By interacting with cognate interaction domains, PTMs on histones can induce the recruitment of multiprotein complexes harboring enzyme activities that can mobilize and reshape the nucleosomes. There is extensive crosstalk among different histone PTMs, and a particular modification pattern on histones specifies a distinct transcriptional state. Both, chromatin structures and histone modification patterns form part of the epigenetic program of a cell, as distinct chromatin structures may be passed to daughter cells during cell division. the methylation of DNA at cytosine residues is another epigenetic mechanism that regulates transcriptional activity at distinct gene loci. Linkages between DNA methylation and chromatin modification have been discovered, explaining at least in part the correlation between DNA methylation and gene silencing.

The structure of chromatin imposes profound and ubiquitous effects on almost all DNA-related metabolic processes, including transcription, recombination, DNA repair, and replication. The DNA-encoded information is packaged into the chromatin polymer of chromosomes which adopts a complicated and dynamic organization in the three-dimensional space of the nucleus. Both, long-range and short-range interactions within a chromosome, as well as interactions between chromosomes, have been detected that are of great influence on the transcription of genes (for a review, see Ref. [18]). Chromatin loops and bridges bring distal parts of the genome into close physical proximity, with consequences for transcription activity. It is now well established, that long-range interactions may organize large units of transcription (also named “transcription factories”), and may establish states of the chromatin that are ready for coordinated and efficient transcription. The importance of the higher-order structure of chromatin is also exemplified by the properties of enhancers that can activate transcription of multiple promoters over a long distance: one single enhancer can stochastically communicate with multiple promoters, and a single promoter can be activated by multiple enhancers. The protein–DNA interactions underlying the long-range communication in chromosomes have not yet been characterized. Hence, at present the influence of chromosome structure on transcription must be described in terms of the basic structural unit of chromosomes, namely the nucleosome. Wrapping of the DNA around the nucleosome represents a basic means of compacting the genome, while further compaction is achieved by the packaging into higher-order structures, among which the so-called solenoid or 30 nm fiber is the best characterized. Interactions between adjacent nucleosomes and binding of the linker histones such as histone H1 are involved in the formation of the 30 nm fiber. This association hinders both nucleosome dynamics and active, ATP-dependent nucleosome remodeling, and is incompatible with transcription. 4.5 Chromatin Structure and Transcription Regulation 177

The wrapping of DNA in nucleosomes restricts the access of the core transcription machinery to the promoter, and the binding of transcription factors to their recognition elements. Consequently, the cell has had to develop ways of regulating transcription within the frame of nucleosome coverage of genes and their regulatory regions, and it is now generally accepted that chromatin structure is the most decisive factor for gene activity in eukaryotes. The following tools can help to regulate the transcription of nucleosome-covered DNA:

& Tools for modification of chromatin structure: — Covalent histone modification — ATP-dependent removal and sliding of nucleosomes — Incorporation of histone variants — DNA methylation.

Chromatin remodeling: ATP-dependent chromatin remodeling complexes reposition, reconfigure, or eject nucleosomes, thus exposing or occluding local DNA areas to interactions with the basal transcription machinery and with transcription factors. Chromatin modifiers: Post-translational covalent modification of the histones within a nucleosome, catalyzed by distinct enzymes, changes the structural and functional properties of a nucleosome. Most notably, the modifications serve to recruit accessory multiprotein complexes for further chromatin modification. Histone chaperones: Proteins with the function of chaperones assist in both the deposition and removal of promoter nucleosomes. Nucleosome replacement: Canonical histones in a nucleosome can be replaced by histone variants through a DNA replication-independent deposition mechanism. Histone variants harbor distinct information to respond to DNA damage conditions, or to override an established gene expression stage. DNA methylation: Methylation at the C-5 position of cytosine residues present in CpG dinucleotides by DNA methyltransferases facilitates static long-term gene silencing and confers genome stability through repression of transposons and repetitive DNA elements (Section 4.5.9).

4.5.1 Chromatin Architecture at Promoters

The basic structural unit of chromatin is the nucleosome, in which 146 bp of DNA are wrapped 1.65 turns around a histone octamer composed of two copies of each of the histones H2A, H2B, H3, and H4. Adjacent nucleosomes are separated by a linker DNA of 15 bp. The spacing of nucleosomes is such that interactions between the N-terminal tails of histones and adjacent nucleosomes are possible. This structural property represents a major obstacle for transcription initiation at promoters, and only DNA sequences in the linker DNA between the nucleosomes and DNA stretches at the edges of the nucleosomes have been shown to be 178 4 The Regulation of Gene Expression

accessible for protein binding. In order to be able to start activated transcription, the promoters and the binding sites for transcription factors must be freed of nucleosomes. The results of studies conducted in yeast have suggested that promoters can be classiﬁed as two contrasting architectural categories, namely “open” and “covered” [19], which in turn drive the two broad types of genes, constitutive and highly regulated, respectively. These two architectures are contrasting extremes, however, and many promoters will contain a “blend” of the attributes of the two, as well as more complex strategies, in order to achieve a correct regulation.

Open promoters: This type of promoter is often found at constitutive genes. A large (150 bp) nucleosome-depleted region directly upstream of the transcription start site can be detected at open promoters. Within this region, cis- regulatory sequences reside. Importantly, the exclusion of nucleosomes appears to be caused by specific sequences properties of the promoter DNA. Open promoters appear to favor the binding of transcription factors at the expense of nucleosomes (Figure 4.20a). Binding sites for transcription factors often reside within the nucleosome-depleted region itself, rather than being buried under nucleosomes, and their exposure in the nucleosome-depleted region promotes transcription factor binding and gene expression. Covered promoters: At regulated genes in their repressed state, nucleosomes often cover the transcription start site, the regions flanking the start site, and most of the binding sites for transcription factors. At such “covered” promoters, nucleosomes compete effectively with transcription factors for the occupancy of key cis- regulatory binding sites, rendering covered promoters more reliant than open promoters on chromatin-remodeling and -modifying enzymes to help “uncover” the regulatory sites and allow transcription activation. However, at least one binding site is typically exposed in the linker DNA between nucleosomes, or partly exposed at the nucleosome edge. This exposed site allows a “pioneer” transcription factor access to the promoter, but chromatin modification and remodeling are probably required to achieve full access to the regulators’ sites (Figure 4.20b).

The two types of chromatin architecture represent two extreme cases, and most promoters are assumed to reside somewhere between the two extremes. The extent of nucleosome coverage at the promoter will – among others – depend on the promoter sequence and the chromatin structure at the neighboring regions, and may therefore vary considerably between different promoters.

& Promoter types: — Open: Nucleosome-depleted start site Often at constitutive genes. — Closed: Start site covered with nucleosomes. 4.5 Chromatin Structure and Transcription Regulation 179

Figure 4.20 Properties of open and covered nucleosomes bear H2A.Z. Binding sites (BS) promoters. (a) Open promoters have a for transcriptional activators (ACT) are shown. depleted proximal nucleosome adjacent to the These are mainly exposed for open promoters transcription start site (TSS, black arrow), a and mainly occluded by nucleosomes (in the feature common at constitutive genes; repressed state) at covered promoters. (b) Covered promoters have a nucleosome Covered promoters typically have nucleosome- adjacent to the TSS in their repressed state, a positioning sequence elements of varying feature common at highly regulated genes. The strength and locations that help to define figure depicts features more common in each nucleosome positions (faint green) and contrasting promoter type, but most yeast promoter architecture. NDR, nucleosome- genes blend the features shown to provide depleted region. Cairns 2009 [19], figure 1. appropriate regulation. Green nucleosomes Reproduced with permission of Nature contain canonical H2A, whereas brown Publishing Group.

Once transcription factors are bound to their recognition elements and chromatin has been reorganized at the promoter, then transcription can be initiated. As the genes to be transcribed are typically covered by nucleosomes, a continuous removal of nucleosomes by nucleosome remodelers ahead of transcribing RNA Pol II is required. The presence of nucleosomes on the transcribed regions, and the need to recruit the remodeling complexes, is thought to be one of the reasons why a frequent pausing of RNA Pol II is observed during the elongation phase. The function of nucleosomes in transcriptional control is mainly shaped by three major processes, namely the incorporation of histone variants, nucleosome restructuring, and chemical modiﬁcation. 180 4 The Regulation of Gene Expression

4.5.1.1 Histone Variants The biological function of nucleosomes can be modulated by the incorporation of histone variants that differ from the major, canonical histones synthesized during S-phase [20]. There are four variants for H2A and ﬁve for H3, but no variants are known for H4 and only few for H2B and H1. Histone variants differ from their canonical counterparts at the level of the primary sequence; such differences can range from a few amino acid positions (e.g., H3.1 versus H3.3) to large protein domains (e.g., H2A versus macroH2A), and usually confer speciﬁc properties to the nucleosomes. In contrast to canonical histones, which are devoted to replication-coupled chromatin assembly, histone variants are expressed throughout the cell cycle and are available in nucleosome assembly pathways that occur in a replication-independent manner.

& Histone variants: — Four variants for H2A — Five variants for H3 — Specific functions in nucleosome reorganization.

The variants have been implicated in diverse biological functions, such as replication, transcription, heterochromatin formation, repair, chromosome condensation, or kinetochore formation. For example the H2A.Z variant, which is associated with an increased nucleosome turnover at transcription start sites, differs from canonical H2A not only in its amino-terminal tail sequence but also at key internal residues. These differences may cause changes in the variant’s interactions with itself and also with the H3/H4 tetramer in the nucleosome, thereby affecting nucleosome stability. H2A.Z is assembled into particular nucleosomes close to transcription start sites, replacing canonical H2A in a replication-independent manner in a reaction conducted by remodelers of the SWR1 family.

4.5.1.2 Chromatin Remodeling Chromatin structure at actively transcribing genes is highly dynamic. The nucleosomes can exist in distinct functional states due to chemical modifications and the incorporation of histone variants; furthermore, the nucleosomes can be moved, ejected, and newly synthesized. Large protein complexes that function as chromatin remodelers have an important role in these processes [19], as the modelers help to construct the initial chromatin states and catalyze transitions to alternative chromatin states (Figure 4.21), using the energy from ATP hydrolysis. Often, the subunits of the remodelers harbor modules which recognize distinct chemical modifications on nucleosome tails. In this way, remodelers can be targeted towards specific nucleosomes and may become active at distinct chromatin sites. Remodelers are specialized multiprotein machines that can be classified according to their main functions: 4.5 Chromatin Structure and Transcription Regulation 181

Figure 4.21 Types of chromatin remodelers. mainly through nucleosome movement or Remodelers use ATP hydrolysis to alter ejection. SWR1-family remodelers reconstruct nucleosomes and are specialized for certain nucleosomes by inserting the histone variant tasks. Most remodelers of the ISWI family H2A.Z into nucleosomes, specializing their (except for NURF and Isw1b) help to conduct composition. This can create an unstable chromatin assembly and organization and nucleosome in certain compositional and provide a consistent spacing of the temporal contexts, and might lead to ejection, nucleosomes. This organization can cover a sliding, or reconstruction at promoters. binding site (red) for a transcriptional activator Cairns 2009 [19], figure 2. Reproduced with (ACT). SWI/SNF-family remodelers provide permission of Nature Publishing Group. access to binding sites in nucleosomal DNA,

ISWI family: Remodelers in the ISWI family carry out nucleosome organization, which often promotes repression. ISWI complexes generally remodel nucleosomes that lack acetylation for example, at H4K16, conﬁning their activity to nucleosomes at transcriptionally inactive regions. They space nucleosomes by “measuring” the DNA linker between nucleosomes and sliding the nucleosome until the linker DNA reaches a ﬁxed distance, creating nucleosome arrays of uniform spacing. SWI/SNF family: Remodelers of this family can both slide and eject nucleosomes, and their functions are often correlated with nucleosome disorganization and promoter activation. Typically, gene activity is associated with histone acetylation, and SWI/SNF remodelers harbor domains that bind acetylated histone tails, promoting their targeting or activity in promoters undergoing activation. It is assumed that these remodelers help to generate the nucleosome-depleted region at the promoters and regulate the occupancy and position of the nucleosome nearby the transcription start site. SWR1 family: Members of the SWR1 family reconstruct nucleosomes by inserting the histone variant H2A.Z into nucleosomes, thereby specializing the composition of nucleosomes.

& Nucleosome remodeler: — ATP-dependent — Major families: ISWI SWI/SNF SWR1. — Removal of linker nucleosomes — Mobilization of histones at transcription start site and during ongoing transcription — Requires covalent histone modification. 182 4 The Regulation of Gene Expression

4.5.1.3 Chromatin Modification The crystal structure of a histone-octamer–DNA complex shows that the DNA wraps tightly around a cylinder-like core of the histones. Most important for the function of nucleosomes during transcription are the N-terminal tails of the histones, which do not adopt a specific structure in crystallographic studies. Between 15 and 38 amino acids from each histone N-terminus form the histone tails, providing a platform for PTMs that modulate the biological role played by the underlying DNA. The modifications are reversible, which allows for distinct, timely variable modification patterns characteristic of the activity state of chromosomal regions. As part of an epigenetic program, the chemical modification of nucleosomes labels the genetic information of nucleosome-bound DNA and indexes different regions of the genome, such as promoter regions, gene-encoding regions, enhancers, and regions that are stably or transiently repressed. Distinct patterns of modification (often interpreted as a “code”) are found on the nucleosomes, and these patterns are established by enzymatic activities that function as “writers,” whereas opposing enzymatic activities function as “erasers.” The information encoded by the modification patterns serves to change the structural properties of the nucleosome and, above all, to recruit other chromatin-modifying enzymes for the further modification and reorganization of chromatin. The chemical modifications are interpreted by “readers” (see Figure 2.3), which are proteins or protein complexes that bind via recognition modules to the chemical modifications, thereby recruiting activities for the further reorganization of chromatin in the neighborhood of the nucleosome. These activities include other chromatin modifiers, histone chaperones and chromatin-remodeling complexes. The histone tails are largely unstructured and carry most of the chemical modifications. Accessible regions of the core of the histones may, however, also be modified by for example, phosphorylation or ubiquitination. The modifications of histones comprise most of the PTMs summarized in Sections 2.5–2.8, such as:

Acetylation, mostly of lysine residues Methylation at lysine and arginine residues Phosphorylation at Ser/Thr residues, Ubiquitination at lysine residues Sumoylation at lysine residues ADP-ribosylation, at Arg residues Prolyl cis–trans isomerization.

4.5.2 Histone Acetylation

e ﬁ Acetylation of the -NH2 group of lysine residues was rst discovered on histones, and has subsequently been recognized as a general tool for regulating the activity and function of proteins (Section 2.6). The primary sites of acetylation are their 4.5 Chromatin Structure and Transcription Regulation 183

Figure 4.22 Patterns of histone modification. antibodies) are not shown. Note that Lys9, A, acetylation; M, methylation; P, Lys14, Lys23 and Lys27 in the H3 tail and Lys12 phosphorylation; U, ubiquitination. Post- and Lys20 in H4 can be either acetylated or translational modifications on the histone tails. methylated. Acetylation shown in purple; Modifications recently identified using mass methylation in blue; phosphorylation in spectrometry but unconfirmed (by mutational orange; ubiquitination in green. analysis and/or Western blot with specific

N-terminal tails. Speciﬁc lysine residues are modiﬁed (Figure 4.22) on all four histones, and the acetylation marks of the tails – together with other covalent PTMs – function either sequentially or in combination to bring about distinct changes in chromatin activity. There is a dynamic equilibrium of lysine acetylation in vivo which is governed by the opposing actions of the “writers” (the acetyltransferases) and the “erasers” (the deacetylases). The information stored in the acetylated lysine residues is interpreted by “reader” modules, the bromo domains, found on many proteins acting on chromatin. The following properties of the acetylated lysine residues have been recognized as being crucial to the biological function of histone acetylation:

The addition of an acetyl group to a lysine residue removes its positive charge and changes electrostatic interactions with the DNA, leading to a loosening of the nucleosome structure and increased histone mobility. 184 4 The Regulation of Gene Expression

Histone acetylation, especially of histone H4, leads to a decompaction of higher- order structures and decreased nucleosome-nucleosome interactions, thus facilitating transcription. Most importantly, acetylated lysine residues are used as docking sites for the association of chromatin modiﬁers carrying bromo domains, either as single proteins or, in most cases, in the form of multiprotein complexes. In terms of biological effects, histone acetylation is linked to transcriptional activation, and deacetylation is linked to repression. Whereas, increased histone acetylation promotes chromatin decompaction and increased DNA accessibility to transcription factors, histone deacetylation by histone deacetylases renders nucleosomal DNA less accessible to transcription.

& Histone acetylation: — At Lys — Removes positive charge — Catalyzed by histone acetyltransferases (HATs) — Activates transcription.

4.5.2.1 Histone Acetyltransferases Histone acetylation is catalyzed by lysine acetyltransferases (HATs or KATs; see Section 2.6), of which three major groups are known, the GNAT, MYST, and CBP/ p300 proteins. Members of the GNAT and MYST families have been identified as subunits of at least two types of multiprotein complexes with coactivator functions, one having a molecular weight of 2 MDa (SAGA; Spt-Ada-Gcn5 acetylase) and a second type with a size of about 700 kDa (ATAC, Ada Two-A Containing complex). These complexes possess global histone acetylation activity and function as locus-specific coactivators. In contrast, the p300/CBP proteins typically act as single proteins. Most of the HATs have a global regulatory function and target substrates other than the histones. Each HAT acetylates a defined spectrum of lysine residues that appears to be specified by the macromolecular complex of which it is a part. Examples of the variant substrates are histones, transcription factors such as p53 and GATA-1, structural proteins such as HMG1, and the basal transcription factors TFIIE and TFIIF. Many of the lysine residues targeted by HATs are also subject to other modifications, such as mono-, di-, or trimethylation, ubiquitination or sumoylation (see Section 2.4.1), indicating that competition exists between the different modifications. In these cases, acetylation precludes the other modifications and the presence of other modifications prevents acetylation.

& Histone acetyltransferases: — Organized in multiprotein complexes such as SAGA, ATAC — Often contain bromodomains — Examples: PCAF, p300/CBP. 4.5 Chromatin Structure and Transcription Regulation 185

Regulation of HATs As expected from their global regulatory role, HATs themselves are subject to a network of regulations [21]. The most interesting point here is the regulation of CBP/p300. These closely related HATs are regulated by autoacetylation, and P300 and CBP each contain an unstructured lysine-rich loop that, when unacetylated, decreases the enzymatic activity by acting as an inhibitory pseudosubstrate. Upon recruitment to a target promoter, multiple CBP/p300 molecules are arranged in close proximity and the loop becomes autoacetylated in trans between neighboring CBP/p300 molecules, leading to activation. Once the acetyltransferases are fully active, they can acetylate various targets that include histones, transcription factors and other components of the transcriptional machinery that are necessary for the robust transcriptional activation of the targeted gene. A similar activation in trans between neighboring enzymes is used by the receptor tyrosine kinases (see Chapter 10). Autoacetylation also allows for CBP/p300 release from the promoter, which might be a necessary step for the continued assembly of the basal transcriptional machinery. Another regulation of CBP/p300 uses phosphorylation. In this case, cell cycle-dependent CBP/p300 phosphorylation, mediated by the cyclin E–CDK2 complex (Section 15.4.2), activates the enzyme allowing for an increased transcription of target genes. Furthermore, transcriptional activation by CBP/p300 has been found to be regulated by competition between acetylation and sumoylation at the distinct lysine residues.

& HAT substrates: — Histones — Transcription factors — GTFs — Structural proteins.

Bromodomains Many members of the HAT families contain a conserved bromodomain, which has been shown to recognize and bind Ac-Lys residues. The bromodomain is widely distributed among enzymes that acetylate, methylate or remodel chromatin, and this highlights the importance of lysine acetylation in the self-maintenance of a transcriptional active state and the recruitment of other sources of chromatin-modifying enzymes [22].

4.5.2.2 Histone Deacetylation The removal of acetyl residues by histone deacetylases (HDACs) opposes the action of HATs. HDACs are grouped into two families, the Rpd3/Hda family and the Sirtuin family (Section 2.6). Generally, HDACs function as corepressors of transcription, and a high HDAC activity is associated with condensed, transcriptionally inactive chromatin. The deacetylation of histones leads to stronger protein– DNA interactions and a more compact nucleosome structure. The mobility of the nucleosome is decreased and the formation of higher-order structures is favored. As a result, accessibility for transcription factors is diminished and the chromatin is modiﬁed from an open gene structure, active euchromatin, to a closed gene structure, silenced heterochromatin. 186 4 The Regulation of Gene Expression

In transcription regulation, the Sirtuin family member SirT1 exerts a repressive effect by deacetylating H4K16Ac and H3K9Ac. When deacetylated, these residues may be marked with methyl groups inducing a repressive nucleosome structure. Interestingly, O-acetyl-ADP-ribose, the product of the deacetylation reaction, has been shown to be involved in the assembly of a repressive SIR–HDAC complex on hypoacetylated histone tails, thus coupling SIR–HDAC activity with transcriptional silencing (for a review, see Ref. [23]).

& Histone deacetylases: — Induce a repressive chromatin structure — Function as corepressors — Are organized in multiprotein complexes — Examples: NURD, SIN3, CoREST.

HDAC Multiprotein Complexes in Transcription Similar to HATs, the HDACs are often found in large multiprotein complexes. The deacetylase complexes are targeted to speciﬁc promoters by interactions with different types of transcriptional regulators, such as sequence-speciﬁc transcription factors, corepressors, methylated CpG-binding proteins and chromatin-remodelers. A number of repressive complexes containing HDACs have been described [24]. For example, HDAC1 and 2 are components of the repressive multiprotein complex (named “CoREST”) which inactivates the expression of neuronal genes in non-neuronal tissues. Other complexes containing HDAC1 and 2 are the NURD (nucleosome remodeling and histone deacetylation) and SIN3 repressor complexes. Furthermore, a complex of HDAC I and nuclear receptor-corepressors (see Chapter 6) binds to unliganded nuclear receptors and is believed to exercise a repressive effect. Another example is the tumor suppressor protein Rb (see Chapters 15 and 16), which functions as a transcriptional corepressor in the hypophosphorylated form and a transcriptional coactivator in the hyperphosphorylated form. The repressive form of Rb recruits the histone deacetylase HDAC I to the DNA and thereby initiates an active repression of the gene (Section 15.4.4).

4.5.3 Histone Methylation

Methylation by methyltransferases (Section 2.7) is another covalent PTM that occurs on the side-chain nitrogen atoms of lysine and arginine on histones. The most heavily methylated histone is H3, followed by H4.

& Histone methylation: — Catalyzed by methyltransferases — At Lys or Arg residues — Activates or represses transcription. 4.5 Chromatin Structure and Transcription Regulation 187

In contrast to acetylation, which correlates almost without exception with transcriptional activation, histone methylation can result in either transcription activation or repression (Figure 4.23), depending on the modified residue and the palette of other modifications decorating the histone simultaneously. Both, lysine methylation and arginine methylation are histone marks that can be removed or inactivated by the action of specific enzymes. The histone-demodifying enzymes oppose the action of the methyltransferases, and both types of enzymes represent major tools for establishing timely and regionally variable methylation patterns that dictate the activity state of chromatin.

Figure 4.23 Correlation of transcriptional categorized as transcriptional coactivators. activity with histone lysine methylation. H3K9me3 is a hallmark of constitutive Methylation of lysines H3K4 and H3K36 is heterochromatin; H3K27me3 is the readout of generally correlated with transcriptional PcG-mediated silencing. In the tail of histone activity, and demethylation of H3K4 is required H4, only K20 is targeted by KMTs. H4K20me1 for effective silencing. Methylation of H3K9 correlates with ongoing transcription, whereas and H3K27 are hallmarks of transcriptional H4K20me3 is an integral part of repression, and the antagonizing KDMs are heterochromatin-mediated silencing. 188 4 The Regulation of Gene Expression

4.5.3.1 Enzymes of Histone Lysine Methylation As compared to lysine acetylation, lysine methylation is much more diverse in its functions as it can promote transcription activation, mediate transcriptional repression, and trigger heterochromatin formation and even chromosome loss. As illustrated in Figure 4.23, distinct lysine residues on the core histones are subject to mono-, di-, or trimethylation, and these marks are associated with active and/or repressed chromatin. & Lysine methylation: — Multiple regulatory functions — Mono-, di-, or trimethylation — Catalyzed by KMTs — Removed by KDMs — Recognized for example, by chromodomains.

At least four different lysines within the N-terminal tail of histone H3 (K4, K9, K27, and K36), one located within the core of H3 (K79) as well as one within the H4 tail (K20), can be either mono- (me1), di- (me2), or tri- (me3) methylated. Some of these residues are also substrates for acetylation, and the modiﬁcation state will therefore depend on a competition of the activities of the “writers” and “erasers” for acetylation and methylation. The different methylation marks correlate with different activity states of transcription:

Activating marks: H3K4me2/me3, H3K27me1 and H3K36me2/me3 are enriched within euchromatic regions of less densely packed chromatin, where transcription takes place. Nucleosomes with di- or trimethylation at H3K4 are enriched at transcription start sites both of “active” and “inactive genes,” and it is thought that H3K4 methylation has an important role in maintaining a state of transcription that is “poised” for initiation. Repressive marks: Repressive lysine methylation marks are found predominantly at H3K9, H3K27 and H4K20, and there is a distinct influence of the number of methyl groups attached. Constitutive heterochromatin is distinguished by a high concentration of H3K9me3, while euchromatin/facultative heterochromatin is primarily marked with H3K9me1/2 and contains limited regions of H3K9me3. H3K27me2/me3 has been identified as another repressive modification on H3. Furthermore, transcriptionally inactive heterochromatic regions are enriched for H4K20me2/me3.

Histone Lysine Methylation Protein lysine methylation (Section 2.7.1) is catalyzed by protein lysine methyltransferases (KMTs, HMTs), while removal of the methyl group is catalyzed by protein lysine demethylases (KDMs, HDMs). Important KMT families in vertebrates are the SET and MLL families, the members of which are responsible for introducing the activating H3K4 marks. Whereas, there is only a single H3K4 KMT in yeast, about 10 different KMTs have been associated with H3K4 methylation in mammals. 4.5 Chromatin Structure and Transcription Regulation 189

4.5.3.2 Enzymes of Histone Lysine Demethylation Two classes of KDMs have been identiﬁed, namely the amine oxidase enzymes and the JmjC enzymes (see Section 2.7.1). One well-studied member of the amine oxidase class is the demethylase KDM1/LSD1, which is a component of the repressive CoREST complex. Of the 27 JmjC members found in humans, 15 have been shown to be active towards methylated histones. KDM enzymes regulate many of the cellular processes in which histone methylation has been implicated, including transcriptional regulation, the maintenance of genome integrity and the regulation of epigenetic memory. Depending on the location of the methyl mark, demethylases can function as either coactivators or corepressors of transcription. During the course of gene activation by the androgen receptor, LSD1 has been shown to directly associate with the androgen receptor (Chapter 6), and acts as a coactivator for transcriptional activation by removing the repressive H3K9 methyl marks. In contrast, KDMs that are active towards for example, H3K4me3 (an activating mark) can function as corepressors and are found as part of repressive complexes.

Recognition Modules for Methylated Lysine The methylated lysine residues are marks which are used to recruit coactivators and corepressors of transcription as well as chromatin-remodeling complexes, to specify a distinct activity state of chromatin. The binding partners contain recognition modules that bind selectively methylated lysine and are sensitive towards the number of methyl groups attached to a given lysine [25]. Currently, five different recognition domains are known that can bind selectively to methylated lysine residues (Section 2.7.1). Of these, the first to be identified was the chromodomain of the transcriptional repressor HP1, a component of transcriptionally inactive heterochromatin (Fig- ure 4.24). The methyltransferase SUV 39 catalyzes the trimethylation of H3K9, and the H3K9me3 formed is recognized by the chromodomain of HP1. As a consequence of HP1 recruitment, a stably repressed chromatin state is established.

& Lysine demethylases: — Remove methyl groups from Lys.

4.5.3.3 Histone Arginine Methylation

& Histone arginine methylation: — Is catalyzed by PRMTs — Yields mono- or dimethyl arginine — Regulates transcription.

Histone arginine methylation (see Figure 2.7) by protein arginine methyltransferases (PRMTs; see Section 2.7.2) has been implicated in both the activation and repression of transcription, depending on the site of methylation. The H3R2me2, H4R3me2a and H2AR3me2a marks are associated with actively transcribed 190 4 The Regulation of Gene Expression

Figure 4.24 Methylation of H3 during activity of SUV39 attaches a methyl group to silencing of pRB-E2F-controlled genes. The Lys9, providing a binding site for the repressor tumor suppressor pRb interacts with the HP1. Binding of HP1 then generates a highly transcription factor E2F and can repress compact chromatin structure by a still transcription by recruiting histone deacetylase unknown mechanism. HP1 and the SUV 39 activity to E2F-controlled genes. The methyltransferase are found in a tight complex deacetylase removes an acetyl group from Lys9 in the cell. of H3. In a subsequent reaction, the methylase

promoters, whereas the H3R8me2s and H4R3me2s marks are associated with repressed gene expression. The activating or repressing function of the marks appear to be based mainly on crosstalk with other modiﬁcations. For example, a functional synergy between arginine methylation and histone acetylation in transcription activation events has been shown. PRMTs interact physically with HATs and form coactivator complexes where the two enzyme activities cooperate in activating the transcription of speciﬁc genes, such as those for the transcription factor NFkB and the tumor suppressor p53. The recognition of methyl marks on arginine appears to be mediated by a single recognition domain, the Tudor domain, that is found on TDRD3, a coactivator of transcription.

4.5.4 Histone Phosphorylation

Phosphorylation, another covalent PTM of histones, occurs in all four core histones that constitute the histone octamer, as well as in some of the histone variants and histone H1.

& Histone phosphorylation: — At all histone types — Mostly at Ser/Thr — Protein kinases: for example, Msk1/2. 4.5 Chromatin Structure and Transcription Regulation 191

A major substrate for phosphorylation is histone H3 [26], that is phosphorylated on T6, S10 and S28, and these phosphorylations are correlated with transcription activation by crosstalk with other modiﬁcations. H3S10 phosphorylation is associated with transcriptional activation by promoting acetylation of K14 on the same histone tail. In mammalian cells, the protein kinases Msk1/2 catalyze the phosphorylation of H3S10. MSk1/2 enzymes belong to the AGC family of protein kinases (see Section 8.1), and can be activated via the ERK/MAPK pathway (Chapter 11) that in turn responds to mitogenic signals. In this way, a linkage between proliferation-promoting signals and transcription activation is provided (see Figure 4.25). Phosphorylation at H3T6 is another interesting example of crosstalk resulting in transcription activation; in this case, crosstalk occurs between H3T6 and H3K4. Upon androgen receptor-dependent gene activation, the protein kinase PKCb becomes activated and phosphorylates H3T6. When this site is phosphorylated, demethylation of H3K4me3 by the demethylase LSD1 is prevented. Trimethylation of H3K4 is a mark associated with transcription activation. H3 phosphorylation has been also recognized as part of a complex signaling mechanism that operates in the condensation/decondensation of chromatin

Figure 4.25 Signal-directed phosphorylation activated and phosphorylates H3 on Ser10. of H3S10 and activation of transcription. H3T10 phosphorylation then promotes H3S10 becomes phosphorylated by the AGC recruitment of the acetyltransferase GCN5 that protein kinase Msk1 in response to signals acetylates H3K14 on the same histone tail, originating from transmembrane receptors. leading to transcription activation. HAT, These signals are transmitted via the RAS/ histone acetyltransferase. MAPK pathway to Msk1 that becomes 192 4 The Regulation of Gene Expression

during the cell cycle. Furthermore, the phosphorylation of histone H1 has been linked to the relief of transcription repression.

4.5.5 Histone Ubiquitination

The ubiquitination (see Section 2.8) of histones also plays a specific role in regulating transcription [27]. As an example, the histone H2B from yeast is monoubiquitinated on Lys123, and this modification is associated with active transcription. A specific role of the 19S regulatory proteasome particle has been established in this process. H2B ubiquitination has been shown to recruit the 19S particle to the promoter, and this event facilitates the methylation of H3 Lys4 and the subsequent recruitment of the coactivator complex SAGA to the promoter. In mammals, histone H2A is the preferred substrate for ubiquitination, where the ubiquitination of H2AK119 is linked to a repression of transcription. The monoubiquitination of H2A is mediated by the E3 ubiquitin ligase Ring1b, which is a subunit of a repressor complex, the Polycomb group repressive complex 1 (PRC1), linking H2A ubiquitination with gene repression. As expected, the deubiquitination of H2A is associated with activation of transcription and the deubiquinating enzyme involved has been shown to function as a transcriptional coactivator.

4.5.6 Recognition of Histone Modifications by Protein Domains

In order to be translated into a biological function, the histone modiﬁcations must be interpreted by the proteins that shape the state of chromatin. The enzymes acting on nucleosomes must contain interaction domains or modules that recognize speciﬁc PTMs on histones. The characteristics of such interaction domains have been presented already in Chapter 2.

& Chromatin-binding domains: — Recognize specific histone modifications — Are located on chromatin-modifying enzymes and chromatin-remodeling complexes.

Domains that recognize PTMs on chromatin components have been identified among the chromatin-modifying enzymes (histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases) as well as among ATP-dependent chromatin-remodeling enzymes. The interaction domains are differentially distributed among the chromatin-modifying enzymes, which suggests that these domains confer specific chromatin-binding properties to the different enzyme families. The domains implicated in recognition of histone modifications are shown in Figure 4.26. 4.5 Chromatin Structure and Transcription Regulation 193

Figure 4.26 Major interaction domains recognizing PTMs on histones. The different sizes of the arrows indicate the preference for different methylated states of Lys. PHD, plant homeobox domain; UBD, ubiquitin-binding domain. For details, see Sections 2.4.2, 2.4.4, and 2.8.5.2.

Most importantly, the protein complexes that modify and shape chromatin often harbor several of the recognition domains of the same or of different selectivity for the chemical histone modiﬁcations. This feature allows for coupling and crosstalk between different modiﬁcations and the enzymatic activities that shape chromatin.

& Binding specificity of chromatin-binding domains: — Bromodomains: acetyl-Lys — Chromodomain: methyl-Lys — PHD domain: trimethylated Lys.

4.5.7 Histone Modification Crosstalk

It is now well established that histone modifications control the structure and/or function of the chromatin fiber, with different modifications yielding distinct functional consequences. These modifications may act either alone or in concert in 194 4 The Regulation of Gene Expression

a context-dependent manner to activate or repress chromatin-mediated processes. Currently, many examples are known of crosstalk among modifications (for a review, see Ref. [28]). Whilst one modification may promote the generation of another by recruiting or activating chromatin-modifying complexes to introduce a different histone modification, crosstalk can also direct the loss of a particular modification. Often, the presence of a combination of modifications is required to achieve a distinct effect on nucleosome function and on transcriptional activity. For instance, the combination of H4K8 acetylation, H3K14 acetylation, and H3 S10 phosphorylation is often associated with active transcription. Conversely, the trimethylation of H3K9 and a lack of H3 and H4 acetylation correlate with transcriptional repression in higher eukaryotes. Particular patterns of histone modifications also correlate with global chromatin dynamics, as the diacetylation of histone H4 at K4 and K12 is associated with histone deposition at S-phase, while the phosphorylation of histone H2A (at S1 and T19) and H3 (at T3, S10 and S28) appear to be hallmarks of condensed mitotic chromatin.

4.5.7.1 Crosstalk Mechanisms Several cross-regulatory mechanisms have been identiﬁed that allow a reinforce- ment of the actions of histone modiﬁcations in regulating gene expression (Figure 4.27):

i) One mechanism is mediated by an initial histone modification that recruits a histone-modifying enzyme and triggers increased activity of the enzyme. In this way, the deposition or removal of neighboring marks can be triggered. For example, the acetylation of histone H3 on Lys18 and Lys23 promotes the methylation of Arg17 by the CARM1 methyltransferase, resulting in an activation of estrogen-responsive genes. Modifications of nearby residues can also prevent the recognition of a substrate by a histone-modifying enzyme, and can also prevent the recruitment of proteins other than enzymes. As examples, methylation of histone H3R2 interferes with methylation of H3K4 (Figure 4.27b), and phosphorylation of H3S10 prevents the binding of heterochromatin protein 1 (HP1) to H3K9. Most interesting are trans-histone effects, where one histone and its modifications affect the modification of a different histone. This has been shown for the monoubiquitination of H2B from S. cerevisiae, a modification that modulates multiple methylation events on H3. Here, H2B ubiquitination is necessary to trigger H3K4 methylation and H3K79 methylation. Another example of a trans-tail crosstalk has been identified in enhancers where the H3S10 phosphorylation leads to the acetylation of H4 tails. ii) A second mechanism involves the coordinate action of different histone- modifying enzymes present in the protein complex. Chromatin-modifying complexes often contain more than one different histone-modifying enzyme. These enzymes may act independently, but in some cases they work together to coordinate histone modification. 4.5 Chromatin Structure and Transcription Regulation 195

Figure 4.27 Crosstalk between histone modifications can span more than one modifications. (a) Interplay among different histone. The monoubiquitination of histone PTMs. “Compatible” modifications (those H2B on Lys120 of the C-terminal helix can lead which facilitate other modifications to occur to the trimethylation of Lys4 in the histone 3 and/or can coexist) are represented by green tail (H3K4) by the KMT enzymes Set1/ arrows. “Incompatible” modifications (those COMPASS. However, H3K4 methylation by which negatively affect other modification and/ COMPASS and COMPASS-like complexes can or cannot coexist) are shown in red; (b) be blocked if the nearby arginine of H3 is Crosstalk among PTMs located on different already methylated. histones. Crosstalk among histone 196 4 The Regulation of Gene Expression

iii) A third, totally different mechanism uses proteolytic cleavage of histone H3 tails to irreversibly erase modiﬁcation marks. This approach may serve to generate a distinct distribution of histones in heterochromatic and eukaryotic regions during processes of differentiation.

& Histone modification patterns: — Variable patterns at multiple sites — Modulate nucleosome structure — Recruit histone-modifying enzymes — Recruit chromatin-remodeling complexes — Positive and negative crosstalk of different modifications.

4.5.7.2 Is There a Histone Modification “Code”? The patterns of histone modifications have been often interpreted as a histone modification “code” which might be read by various cellular machineries. It has been hypothesized that specific tail modifications and/or their combinations constitute a code that determines the transcriptional state of the genes. According to the histone code hypothesis, multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple tails, specify unique downstream functions. As modifications on one histone can affect modifications on another histone, the histone code was expanded to a “nucleosome code,” suggesting that the presence of all post-translational histone modifications in one nucleosome regulate the underlying DNA sequence. Particular importance has been attached to so-called “binary switch modules” in which modifications on two neighboring residues can influence each other (see above). In order to create a code, “writers,”“readers,” and “erasers” of the code components are necessary. In the case of the histone code, modifications are “written” by histone modifiers that are specific for a residue and encode information. For example, the methylation of H3K27 is “written” by the methyltransferase E(z) and “read” by proteins carrying specific binding domains, such as bromodomains for lysine acetylations and Chromo, Tudor and PHD domains for lysine methylations. The “readers” can fulfill distinct tasks, such as recruiting other proteins or an enzymatic activity, and thus can act as translation machinery for the information encoded within the histone modifications. Since histone modification patterns are highly dynamic, “erasers” such as deacetylases and demethylases are required to remove the modifications in a regulated manner. The interplay between writers, readers and erasers provides for the plasticity of histone modifications and chromatin structure, as well as for the timely variable biological information stored in distinct chromatin regions. The existence of a histone code has, however, been questioned following genome-wide studies of histone modification patterns [29]. It has been postulated, based on the results of these studies, that histone modifications should be interpreted as a language rather than a code. Moreover, within the modification 4.5 Chromatin Structure and Transcription Regulation 197 language the biological meaning of a modification depends both on the timing and the context, that is, the chromatin surroundings. 4.5.8 Histone Modification and Epigenetics

The term “epigenetic” describes inheritable changes in gene activity that are not encoded by the DNA sequence. Epigenetic phenomena include DNA methylation, histone variants, nucleosome remodeling, RNA binding and the PTMs of histones, as discussed above (for a review, see Ref. [30]). The epigenetic mechanisms are most important for the maintenance of stable expression patterns observed in multicellular organisms during cellular differentiation. Once established, these patterns are frequently maintained over several cell divisions, despite the fact that the initiating signal that triggers differentiation is no longer present. The fixation of transcriptional activity over cell divisions implies that the structural and functional characteristics of histones and nucleosomes can be largely retained during cell division. Differential packaging into chromatin and the inheritance of these structures during cell division is one such mechanism that allows fixation. However, it remains largely unclear as to how distinct modification patterns of histone or nucleosomes are transferred during DNA replication to the newly incorporated nucleosomes. One major contribution to the maintenance of distinct chromatin structures during cell divisions appears to derive from the lysine methylation of histone tails and from DNA methylation, both of which processes are interconnected (see Section 4.5.9.3). Genome-wide studies of the dynamics of histone modifications [30] have shown that acetylation and phosphorylation marks are rather short-lived and have a quite high turnover rate (Figure 4.28). Both marks are associated primarily with actively transcribing regions and affect mostly histones located near the promoter region. Histone lysine methylation, by contrast, is mostly a long-lived mark with a low turnover rate. Most importantly, there is a linkage between histone lysine methylation and DNA methylation, which is also a quite stable epigenetic mark (Section 4.5.9). The stable lysine methylation marks are bound by structural proteins that show high specificity for a given methylation mark and can impose a condensed, repressive structure on chromatin. For example, the Chromo domain of HP1 binds only methylated K9 within histone H3, whereas the similar domain in the repressive complex PcG recognizes only methylated K27 within the same molecule. This distinct binding affinity is reflected also by the in vivo localization of the two proteins: HP1 binds to constitutive heterochromatin, while PcG binds to facultatively repressed genes.

4.5.9 DNA Methylation

The methylation of DNA is the most abundant epigenetic modiﬁcation in vertebrate genomes [31]. Overall, the methylation of cytosine bases is associated with a repressed chromatin state and an inhibition of gene expression. 198 4 The Regulation of Gene Expression

Figure 4.28 Dynamics of histone modifications such as H3K79me are present in modifications on active and silenced genes. the body of these genes. During transcription Modification patterns differ on actively elongation, further distinct patterns of histone transcribed and silenced genes, which is modifications have been detected; (b) Inactive displayed in this figure as a schematic view of genes have a fairly even distribution of modification distribution over the gene. (a) silencing modifications, such as H3K9 Active genes: At the transcriptional start site methylation and H4K20 methylation, whereas there is a nucleosome-depleted region (NDR) H3K27 methylation is enriched in the within the promoter. Nucleosomes 50 to the promoter. These modifications can be bound NDR carry inactivating modifications such as by heterochromatic proteins such as HP1; H3K9me2/3, H3K27me2/3, and H4K20me3. hence, this chromatin area can condense, as Nucleosomes within the promoters of actively seen in heterochromatin. For details see transcribed genes carry high levels of active Ref. [30]. Barth 2010 [30], figure 1. Reproduced modifications, such as acetylations and with permission of Elsevier. methylation of H3K4. Other active

& DNA methylation: — 5-methylcytidine at CpG — Associated with a repressed state — Catalyzed by DNA methyltransferases — Inhibitor: 5-azacytidine. In the mammalian genome, methylation takes place only at cytosine bases that are located 50 to a guanosine in a CpG dinucleotide (Figure 4.29); the product of methylation is 5-methyl C. A high CpG content is found in regions known as CpG islands that are often located in the vicinity of promoters. In normal cells, CpG islands are generally hypomethylated, which allows for an open chromatin structure and favors transcription. When CpG islands located within promoters are methylated, the corresponding genes are persistently silenced. 4.5 Chromatin Structure and Transcription Regulation 199

Figure 4.29 The methylation of DNA: 5- pattern of DNA remains intact upon DNA methyl-cytidine and maintenance methylation. replication and is passed on to the daughter (a) The methylation of cytidine residues on cells. The newly synthesized strands are DNA is catalyzed by a methyltransferase that unmethylated immediately after DNA employs S-adenosine methionine as a methyl replication. The methyltransferase uses the group donor. The preferable substrate for the previously methylated parent strand as a methyltransferase are hemimethylated CpG matrix to methylate the CpG sequences of the sequences. 5-Azacytidine is a specific inhibitor newly synthesized strand. of methyltransferases; (b) The methylation 200 4 The Regulation of Gene Expression

Furthermore, the CpG-poor regions found in repetitive elements within the intergenic and intronic regions of the genome are typically methylated and thereby maintain a closed chromatin structure. In cancer cells and on the inactive X chromosome, many CpG islands become methylated, forcing these regions into a closed chromatin structure with concomitant gene repression (see Section 16.2.2.1).

4.5.9.1 DNA Methyltransferases The enzymes responsible for DNA methylation at CpG sequences are the cytosine DNA methyltransferases (DNMTs), whereby the methyl group is derived from S- adenosyl methionine. An important inhibitor of DNA methyltransferases is 5- azacytidine, which blocks DNA methylation and leads to a change in the DNA methylation patterns of cells.

& Two types of DNA methylation: — Maintenance methylation — De-novo methylation.

Mammalian cytosine DNMTs ﬁt into two general classes based on their preferred DNA substrate:

De novo methyltransferases DNMT3a and DNMT3b: These are mainly responsible for introducing cytosine methylation at previously unmethylated CpG sites. Such a de novo methylation at CpG sequences has been clearly shown. The mechanisms by which the de novo methyltransferases select their substrate sites appear to be varied. Targeting to speciﬁc CpG sequences can be accomplished by speciﬁc domains within the DNMT, by recruitment through protein–protein interactions with transcriptional corepressors, and by the RNA-mediated interference system (Section 5.3). Maintenance methyltransferase DNMT1: This enzyme copies pre-existing methylation patterns onto the new DNA strand during DNA replication. A characteristic distribution pattern of 5-methylcytidine (m5C) is found within each cell, which remains intact upon cell division. DNMT1, which is responsible for this maintenance methylation, has a preference for DNA sequences in which the complementary strand is already methylated at CpG sequences (Figure 4.29). Such a hemi-methylation of DNA is found, for example, in regions that are just being replicated.

4.5.9.2 Coupling DNA Methylation to Gene Repression DNA methylation is an epigenetic modiﬁcation that is generally linked with the transcriptional silencing of associated genes.

& DNA methylation and transcription repression: — MeCpG recognized by specific binding proteins: MBPs — MBPs recruit HDACs and methyltransferases. 4.5 Chromatin Structure and Transcription Regulation 201

Several models have evolved to explain the relationship between CpG methylation and gene repression:

Direct interference with transcriptional activators: DNA methylation can directly repress transcription by blocking transcriptional activators from binding to cognate DNA sequences. Methyl CpG-binding proteins: Methylated DNA is recognized by methyl CpG- binding proteins (MBPs) that recruit corepressors to silence gene expression directly. An important link between DNA methylation and chromatin modiﬁcation is provided by the ability of MBPs to engage in protein–protein interactions with histone-modifying enzymes. Members of the MBP family are known to associate with corepressor complexes at the sites of its occupancy. For example, the MBP member MeCP2 recruits the corepressor complex Sin3a which contains the histone deacetylases HDAC1 or HDAC2 as subunits. In this way, the deacetylases are directed to sites of CpG methylation and can deacetylate histone tails so as to maintain a repressed state and link DNA methylation and histone modiﬁcation.

4.5.9.3 Linking DNA Methylation and Histone Methylation It is now well established for various organisms that histone modification and DNA methylation are intimately associated. Genome-scale DNA methylation profiles suggest that DNA methylation is strongly correlated to histone lysine methylation patterns. Specifically, DNA methylation is positively associated with the presence of H3K9 methylation, a mark associated with gene repression. By contrast, a strong negative correlation exists between DNA methylation and H3K4 trimethylation, a mark associated with active transcription. The mechanisms underlying the linkage between DNA methylation, unmethylated H3K4 and H3K9 methylation are only beginning to be unraveled [32]. A subtype of the de novo DNA methyltransferase DNMT3, namely DNMT3L, has been shown to bind histone tails only when H3K4 is unmethylated. It has been suggested that DNMT3L acts as a sensor for H3K4 methylation: when methylation is absent, DNMT3L induces de novo DNA methylation by docking another DNMT subtype, namely DNMT3A, to the nucleosome. In this way, DNMT3L provides a linkage between unmethylated H3K4 and de novo DNA methylation (Figure 4.30), coupling histone modification and chromatin structure to de novo DNA methylation. A linkage between hemimethylated CpG5me and trimethylated H3K9, a repressive mark, appears to be provided by an accessory protein named UHFR1 that harbors multiple binding modules. First, UHFR1 can bind to hemimethylated DNA as well as to the maintenance methyltransferase DNMT1 and, in addition, UHFR1 can bind via a Tudor domain to trimethylated H3K9. The cooperation between the different binding modules of UHFR1 appears to couple maintenance methylation to a repressive chromatin structure, a coupling that is thought to be required during DNA replication. 202 4 The Regulation of Gene Expression

Figure 4.30 Model of the coupling of DNA nucleosome. Maintenance methylation by methylation to histone H3 methylation. During Dnmt1 appears to couple to the repressive DNA replication, a coupling between DNA histone H3K9me mark through the UHFR1 methylation and histone modification patterns protein that functions as an accessory protein exists. This is necessary to preserve for for Dnmt1. The methylation status at K4 and example, a repressive chromatin structure at K9 of H3 is determined by the activity of the methylated CpG sequences. The maintenance Set1 methylase and LSD1 demethylase, among methylase dimer Dnmt3a/Dnmt3L is thought others. The actions of proteins and arrows are to act as a sensor for H3K4 methylation, a indicated by color: green for activities histone tail mark associated with transcription associated with increased gene expression, activation. When H3K4 methylation is absent, and red for those tending to decrease the level Dnmt3a/Dnmt3L induces de novo methylation of gene expression. After Ref. [32]. by promoting association with the

4.5.9.4 Biological Functions of DNA Methylation DNA methylation is an essential part of the epigenetic program of multicellular organisms. In speciﬁc situations of cells, for example, during cell-cycle progression and differentiation, the methylation pattern can be quite dynamic. Importantly, somatic DNA methylation contributes to differentiation by repressing key genes in the germline and irreversibly forcing the cell on a path to differentiation. Enzymes have been identiﬁed that function as demethylases and can alter the methylation pattern. Due to its general repressive functions, DNA methylation is involved in the following biological processes:

Transcription initiation and transcription elongation. Heterochromatin formation. 4.5 Chromatin Structure and Transcription Regulation 203

Methylation of foreign DNA, such as viral DNA, provides a defense mechanism against the expression of exogenous DNA. DNA methylation participates in genetic imprinting and in X-chromosome inactivation. The term “genetic imprinting” describes a situation where genes are expressed unequally depending upon whether they were maternally or paternally inherited. Normally, both copies of the parental genes are equally transcribed in a diploid chromosome; however, with imprinting a gene inherited from either the mother or father is selectively inactivated. Methylation is obviously involved in such an inactivation. The inactive copy is more strongly methylated than the active copy. DNA methylation has been recognized as an important aspect of tumorigenesis. Changes in the methylation pattern have been linked in many tumors to a decreased expression of tumor suppressor genes (see Chapter 16).

4.5.10 Summary of the Regulatory Steps in Transcription

Changes in the state of chromatin, the various histone modifications, and the start of transcription may be ordered in a sequence of events, as summarized below. Many aspects of the coordination of transcription with chromatin remodeling remain to be clarified, and the proposed scheme must therefore remain speculative. In particular, the order of the events summarized below will depend on the gene context, the nature of the activating signals, and the specific state of chromatin near the promoter.

Relief of the repressed state: In a first step, chromatin must be prepared for transcription by relieving a closed, repressed state. This process involves the removal of marks that conceal the repressed state and the deposition of marks that promote the disruption and mobilization of nucleosomes in advance of initiation. Examples of repressive marks to be removed are H3K9me3, H3K27me2,3 and H4K20me2,3. Deposition of activating marks: In addition, activating marks such as H3K4me3 and H3K36me2,3 must be introduced in order to prepare chromatin sites for transcription. Lysine acetylation of histones at regulatory sites is another step required for destabilization of the chromatin structure. The activating marks are recognized by binding modules present in chromatin-modifying enzymes and chromatin remodelers. Thereby, these proteins are recruited to distinct chromatin sites, allowing for further modification and reorganization of nucleosomes. Creation of a nucleosome-depleted region: The region near the transcription start site must be freed of nucleosomes. In a step preceding or following chromatin modification at the promoter, chromatin-remodeling complexes are targeted to the transcription start site to mobilize or eject nucleosomes from the promoter region. A common characteristic of the remodeling complexes is the presence of distinct ATPase subunits. The ATP-dependent remodeling complexes use ATP hydrolysis 204 4 The Regulation of Gene Expression

to disrupt nucleosome–DNA interactions and thereby participate in nucleosome mobilization and the removal of nucleosomes from the transcription start site. Histone variants such as H2A.Z may be used to mark the border of the start site. Binding of sequence-specific transcription factors: Once the structure of chromatin has been “loosened” and the region surrounding the transcription start site has been depleted of nucleosomes, sequence-specific transcription factors gain access to their cognate DNA-binding elements and can engage in protein–protein interactions with coactivators to promote further modification and reorganization of chromatin. Furthermore, transcriptional activators interact with Mediator and the core transcription machinery, allowing the formation of a pre-initiation complex. Multiple mechanisms are available to control the transcriptional regulators, including the binding of activators, chemical modification (e.g., phosphorylation, acetylation), and nuclear translocation. Formation of a pre-initiation complex: Once the region around the transcription start site has been depleted of nucleosomes, binding of the basal transcription factors (e.g., TFIID) to the core promoter sequences is possible and the core transcription apparatus is deposited at the transcription start site. Start of RNA synthesis: The onset of transcription depends on the cooperation of Mediator and transcription factors bound to the regulatory sequences. The interaction with transcription factors and Mediator stabilizes the RNA polymerase complex at the promoter and facilitates the transition from the pre-initiation phase to the elongation phase. Transition into elongation: An early step in the transition from transcription initiation to transcription elongation includes phosphorylation of the CTD of RNA polymerase II that serves as a trigger for the start of the elongation phase. Various protein kinases are involved in this phosphorylation, and a dynamic pattern of phosphorylation is then established on the CTD, providing a platform for the association of the mRNA-processing machinery. Furthermore, chromatin-modifying enzymes travel along with the RNA polymerase during mRNA elongation. This serves to modify nucleosome structure and to remove nucleosomes in front of the elongating transcription machinery.

The 50 and 30 ends of genes are marked differentially during transcription. Chromatin at the 50-end control regions is destabilized by acetylation to allow binding of the transcription factors and deposition of the transcription machinery. The chromatin at the 30-ends of genes, however, must be stable enough to prevent inappropriate initiation at degenerate start sites internally. To this end, the transcription process itself marks the gene as having been transcribed with methylation at histone H3 Lys36. This occurs through cotranscriptional methylation by the methyltransferase Set2, which is bound to the CTD of RNA polymerase II during the elongation process. It has been shown in yeast, that H3 Lys36 methylation is used to direct a histone deacetylase to the transcribed regions that deacetylates and stabilizes nucleosomes within a transcribed gene. Questions 205

Overall, a complex cooperation of chromatin modifications and transcription has emerged where the epigenetic modifications of chromatin and DNA play a crucial role in determining the transcriptional state of a gene. Transcription is a dynamic process where the main players – sequence-specific transcription factors, Mediator, RNA polymerase II, the nucleosome and the chromatin-modifying enzymes – are engaged in a multitude of protein–protein interactions that are variable in time during rounds of transcription. Many of the main players are organized in multiprotein machines of variable composition, and it is this variability and the dynamics of the process that until now has precluded a detailed picture of the regulation of transcription. However, the sequence of events proposed above is speculative, and is dependent on chromatin context and the specific gene structures.

Questions 4.1. What are the distinguishing structural features of eukaryotic promoters? 4.2. What are the main protein components required for eukaryotic transcription, and what is their main function? 4.3. Which enzymatic functions are found on TFIIH, and what is their function? 4.4. Describe the characteristics of the CTD of RNA Pol II and its function in transcription. 4.5. Give examples of structural motifs found on the DNA-binding domain of specific transcription factors. Give examples of such transcription factors. 4.6. Typical specific transcription factors are found as dimers or higher oligomers. Give some examples of these. How is this feature reflected in the structure of the cognate recognition element, and how are these organized? Which types of interactions are involved in the recognition of DNA elements by transcription factors? 4.7. Give an example of a eukaryotic enhancer. Describe its properties and structural organization. Can you explain why it is advantageous for a cell to organize DNA elements as enhancers? 4.8. Which features determine the selectivity of transcription factors for the cognate recognition element? 4.9. Which mechanisms contribute to gene repression? Give examples. 4.10. Describe the main mechanisms for the control of transcription factors and give examples. 4.11. Give at least two examples of transcription factors that are regulated by phosphorylation. Describe the underlying mechanisms. 206 4 The Regulation of Gene Expression

4.12. Which processes are required for deposition of the transcription apparatus at the start site? Describe these processes. 4.13. Which PTMs are found on the core histones? Name the enzymes catalyzing these modifications, and give examples. 4.14. Describe the reaction catalyzed by sirtuins. 4.15. Which PTMs can be found on Lys residues of histones, and which interaction domains are used for reading these modifications? 4.16. Give some examples of crosstalk among histone modifications. 4.17. Explain the two major types of DNA methylation at C-residues. Which enzymes are involved? 4.18. How might DNA methylation be linked to the establishment of repressive chromatin structures?

References

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5 RNA Processing, Translational Regulation, and RNA Interference

5.1 Pre-mRNA Processing

Summary The information encoded in the primary product of transcription, the pre-mRNA can, by alternative splicing, be used to generate multiple mRNAs from a single pre-mRNA. Alternative splicing vastly expands the repertoire of proteins in a cell, and the subtypes generated by the alternative splicing of a pre-mRNA may have quite different functional and regulatory properties. Splice site selection and splicing itself are intimately linked to transcription by RNA polymerase II and to processes upstream and downstream of transcription, including translation and mRNA decay. The features of alternative splicing important for splice-site selection have been only partially characterized. The structure of the primary transcript, the components of the spliceosome, chromatin structure and proteins involved in transcription elongation, have been all found to cooperate in splice site selection.

Transcription and translation are spatially separated events in eukaryotes. The primary product of nuclear transcription is pre-mRNA, that is characterized by three structural features: (i) capping at the 50-end; (ii) polyadenylation at the 30-end; and (iii) the presence of introns interspersed between the coding regions of the pre- mRNA, the exons. The introns are removed in the process of splicing to yield the mature RNA that is transported out of the nucleus for translation. How much protein, and which protein is formed by translation, depend heavily on the identity and quantity of processed, mature RNA. Capping, polyadenylation and splicing are highly regulated and coordinated events that have been shown to be intimately linked to the transcribing RNA polymerase complex.

& Pre-mRNA processing includes: — Capping — Polyadenylation — Splicing.

5.1.1 Capping and Polyadenylation

5.1.1.1 Capping This process involves the addition of a 7-methyl-guanosine residue at the 50 end to protect this end from nuclease-mediated degradation. The cap structure in eukaryotes can be of three types, namely m7GpppNp, m7GpppNmp, and m7GpppNmpNmp (where m indicates a methyl group attached to the respective nucleotide), and is used as a docking point for the cap-binding protein complex that mediates recruitment of the small ribosomal subunit to the 50 end of the mRNA. Capping at the 50 end of the pre-mRNA occurs immediately after the incorporation of about 30 nucleotides in the primary transcript. The enzymes involved in capping associate with the C-terminal domain (CTD) of RNA polymerase II once the CTD has become activated by Ser5-P formation, through the action of the TFIIH- associated cyclin-dependent kinase, CDK7.

5.1.1.2 Polyadenylation Polyadenylation occurs after cleavage of the pre-mRNA at the 30 end, and involves the addition of up to 500 adenine (A) residues by the enzyme poly(A) polymerase (PAP). Overall, polyadenylation is a highly complex process where more than 85 proteins collaborate in forming and tailoring the poly(A) tail in coordination with transcription (for details, see Ref. [1]). The regulation of poly(A) tail length of translationally controlled mRNAs is a recurring theme in the oogenesis and early development of many animal species. In most cases, long poly(A) tails (80–500 A residues) correlate with the activation of translation, and short tails (20–50 A residues) with the repression of translation.

5.1.2 Alternative Splicing

The genetic information encoding a protein in higher eukaryotes is usually found in pieces of coding sequence, termed exons, interrupted by noncoding sequences, termed introns.

& Alternative splicing: — Large source of proteome diversity — Differential combination of introns.

In order to form mature mRNA, the introns must first be excised and the exons rejoined in the correct order; this process is termed splicing. Almost all protein- 5.1 Pre-mRNA Processing 211 coding genes are spliced, and this process removes a large part of the sequence of the pre-mRNA. In an average human gene, more than 90% of the pre-mRNA is removed as introns, while only about 10% is joined as exonic sequences by pre- mRNA splicing. The number of introns in eukaryotic genes can be very large; for example, there are 50 in the human dystrophin gene. Splicing occurs in a large protein–nucleic acid complex, termed the spliceosome, the components of which, apart from the pre-mRNA, include a number of proteins and small RNAs, termed U1, U2, U4, U5, and U6. The RNAs found in the spliceosome are bound to specific proteins, and the complexes thus formed are termed small nuclear ribonucleoproteins (snRNPs). Depending on the type of RNA that is bound, U1, U2, U5, and U4/U6 snRNPs are produced. The major pathway for extracting different information from pre-mRNAs employs alternative splicing. Exon usage is often an alternative; in this case the decision is taken by the cell whether to remove a part of the pre-mRNA as an intron or to include this part in the mature mRNA as an alternative exon. Starting with a single pre-mRNA, multiple alternative mRNAs can be formed via the rejoining of various exons, each coding for proteins with different activities and functions (Figure 5.1a). Some alternative splicing events appear to be constitutive, whereas others are regulated in a tissue-specific manner in response to developmental or physiological signals. In this way, a flexible adjustment of specific mRNA levels in response to a transduced external signal can be achieved. The use of alternative splice sites provides a powerful mechanism for expanding the number of proteins in a cell. In the extreme example of Drosophila melanogaster Dscam, over 38 016 isoforms are generated for the neural wiring and immune defense (Figure 5.1b). In the human genome, there are about nine exons per gene on average, which allows for the generation of a large number of gene products; indeed, it is estimated that alternative splicing affects more than 88% of human protein-coding genes. The coding capacity of the human genome is largely increased by alternative splicing, and protein isoforms are generated that differ in biological properties as, for example, protein–protein interaction, subcellular localization, or catalytic ability. The misdirection of splicing has been recognized as a frequent cause of inheritable genetic diseases, and numerous changes in splicing patterns have been identified in cancer cells. In fact, based on an inspection of about 10 000 disease- causing mutations, it has been estimated that about 15% of such mutations and about 8% of all human deaths can be linked to splicing mutations in splice sites [3].

5.1.3 Regulation of Alternative Splicing

The splicing of pre-mRNAs allows a large number of combinations of exons. However, only a few of the theoretically possible combinations are realized and only a few of the splice sites are used. A major question in alternative splicing relates to the sequence and structure determinants in pre-mRNA that specify the selection of splice sites. Furthermore, auxiliary protein factors must be involved in 212 5 RNA Processing, Translational Regulation, and RNA Interference

Figure 5.1 Alternative splicing can generate a immunoglobulin cell-surface proteins from multitude of different proteins from a pre- Drosophila melanogaster that mediates mRNA. (a) A pre-mRNA with four exons recognition events between neurons. Dscam (colored bars) and three introns (black bars) has 24 exons; exon 4 has 12 variants, exon 6 can produce three different mRNAs via an has 48 variants, exon 9 has 33 variants, and exon skipping mechanism (one of the major exon 17 has two variants. The combination of forms of alternative splicing), which in turn exons 4, 6, and 9 leads to 19 008 possible correspond to three different protein isoforms with different extracellular domains. structures; (b) Alternative splicing generates a With two different transmembrane domains large number of isomers with different protein-- from exon 17, the total possible protein -protein interaction possibilities. An extreme products could reach 38 016 isoforms. Tsai example of mutually exclusive alternative 2009 [2], figure 4. Reproduced with permission splicing is observed in the Dscam family of of Elsevier.

this selection, as splicing exhibits distinct tissue-specific patterns and it must be assumed that these factors are available in a tissue-specific manner. Another major issue in the regulation of splicing is the intimate coupling between splicing and transcription by RNA polymerase which provides for a linkage of chromatin structure, ongoing transcription, and concomitant splice site selection. The following features of alternative splicing have been recognized as being important for splice-site selection and the generation of tissue-specific splicing patterns:

Transcript structure: A detailed analysis of splicing characteristics in different tissues has led to the proposal of a splicing code [4]. This code uses a combination of a large number of RNA structural features such as cis-acting RNA sequence motifs, intron sequence, exon length and transcript secondary structure to 5.1 Pre-mRNA Processing 213 predict tissue-speciﬁc splicing patterns. Some of the cis-acting, regulatory sequences are known as exonic or intronic splicing enhancers and exonic or intronic splicing silencers. These conserved RNA sequences are typically 10 nucleotides in length, act either in isolation or in clusters, and stimulate (enhancers) or inhibit (silencers) the use of splice sites through the binding of trans-acting regulatory proteins. Regulatory binding proteins: Splice site selection involves the binding of proteins and/or protein–RNA complexes to sequence and/or structural motifs that make up the splicing code. These proteins are part of splicing complexes and/or are associated with RNA polymerase during ongoing transcription. Examples of such regulatory splicing proteins are the SR proteins and proteins classiﬁed as hnRNPs (heterogeneous nuclear ribonucleoproteins) which bind to sequence motifs of splice sites and target the splicing complex to distinct splice sites.

& Alternative splicing is influenced by: — Structure of primary transcript — Protein binding: SR proteins, hnRNPs.

It is well established that the selection of a splice site can be altered by numerous extracellular stimuli such as hormones, immune response, neuronal depolarization, and cellular stress. The signaling pathways that direct regulatory signals to the splicing machinery are not well characterized. Most functional alterations of splicing factors appear to be mediated by phosphorylation on Ser/Thr residues of SR proteins and hnRNPs.

5.1.3.1 SR Proteins Members of this family of phylogenetically conserved and structurally related splicing factors are characterized by a domain rich in serine-arginine dipeptides, termed the SR-domain. The SR proteins are required for constitutive splicing and for alternative splicing, and are considered as multifunctional adapter proteins with multiple roles in RNA metabolism [5]. SR proteins have a modular structure consisting of one or two copies of an N-terminal RNA-recognition motif (RRM) followed by a C-terminal RS domain. The RRMs determine RNA-binding specificity, whereas the RS domain functions as a protein–protein interaction module by recruiting components of the core splicing apparatus to promote splice site pairing. Splicing enhancers located within exons have been identified as specific binding sites for SR proteins, and binding of the latter proteins to these sites serves to recruit the splicing machinery to the adjacent intron. SR proteins are phosphorylated within the SR domain in response to a variety of extracellular stimuli. The phosphorylations affect many functions of SR proteins, including formation of the splicing machinery and alternative splicing. For example, phosphorylation of SR proteins is required for efficient splice-site recognition, and dephosphorylation for splicing catalysis. 214 5 RNA Processing, Translational Regulation, and RNA Interference

5.1.3.2 hnRNPs The hnRNPs are a set of primarily nuclear proteins that bind to nascent transcripts produced by RNA polymerase II, and thereby perform multiple functions. These proteins are of a modular structure and typically carry RNA recognition motifs. One hnRNP member, hnRNP A1, has been identiﬁed as an antagonistic partner of SR proteins in splicing.

& Regulation of alternative splicing: — Complex mechanism — Controlled by external and internal signals.

& SR proteins: — Essential for alternative splicing — Ser-, Arg-rich — Multiple roles in RNA metabolism — Regulated by phosphorylation.

5.1.4 Chromatin Structure and Splicing

In higher eukaryotes, splice site selection and splicing itself are intimately linked to transcription by RNA polymerase II. Such cotranscriptionality of splicing means that splicing takes place, or is committed to occur, before the nascent RNA is released from RNA polymerase II. The coupling of splicing and transcription is based on the recruitment of splicing factors to the transcription machinery by the CTD of the catalytic subunit of RNA Pol II (see Section 4.2.7). Efficient splicing, at least of long transcripts, has been shown to require a phosphorylated CTD and it is coupled to ongoing transcription. Transcription in vivo occurs on nucleosome- covered DNA, while transcription elongation is accompanied by the removal of nucleosomes ahead of, and the reformation of nucleosomes behind, RNA Pol II when it travels along the DNA template. The restructuring of chromatin during transcription elongation is a major determinant of the rate of transcription, and it thereby also influences the recruitment of splicing factors to the CTD. Currently, there is much experimental evidence that chromatin structure – specifically histone modifications – plays a major role in regulating alternative splicing [6]. A chromatin-adapter model of alternative splicing postulates that histone modifications along a gene determine the binding of adapter proteins that read the histone modification, and in turn recruits splicing factors. As the recruitment of splicing factors is a major driving factor in determining splicing outcome, the chromatin architecture and histone modifications can greatly influence – via the binding of adapter proteins – the selection of splice sites. Examples of such adapter proteins are the GCN5 acetyltransferase (Section 4.5.2), the heterochromatin-binding protein HP1, and the chromatin remodeler CHd1 (Section 4.5.1.2). These adaptors 5.1 Pre-mRNA Processing 215 contain binding modules for distinct histone modifications, and have been also shown to interact with distinct splicing factors.

& Alternative splicing: — Coupled to transcription — Influenced by CTD phosphorylation — Influenced by chromatin structure — Coupled to transport and translation.

5.1.5 Coupling of pre-mRNA Processing, Transcription, and Translation

It is now well established that pre-mRNA processing is intimately linked to upstream and downstream events in transcription, including translation and mRNA decay. The pathway from pre-mRNA synthesis to the mature RNA ready for translation includes a series of events that are highly interdependent and interconnected. The following steps can be discerned in this pathway.

5.1.5.1 RNA Pol II Poised for Transcription The formation of a pre-initiation complex at a promoter does not necessarily mean that the transcription complex also engages in productive elongation. Many gene promoters are permanently engaged with RNA Pol II initiation complexes, without starting productive elongation. Rather, 50-proximal abortive transcripts are formed. By having Pol II already plugged into the gene promoter, the complex process of uncovering the gene from repressive chromatin structure and recruiting the Pol II initiation complex from its component parts is bypassed. The molecular trigger that switches Pol II from abortive initiation into functional elongation mode remains to be identiﬁed. However, pre-mRNA processing has been implicated in this process.

5.1.5.2 Formation of 50 Cap The ﬁrst RNA processing event to occur on the nascent transcript is 50 end capping, and this may well be a key component of the switch that pushes RNA Pol II from an abortive early elongation into a fully processive elongation across the body of the gene. Other components of this switch involve both negative and positive elongation factors that may themselves be regulated by phosphorylation dictated by cell signaling cascades connected to the extracellular environment through membrane receptors. One well-characterized positive factor in higher eukaryotes is the heterodimeric protein PTEFb, comprising the CDK9 kinase and associated cyclin T, which generates CTD Ser2-P patterns on RNA Pol II elongating into the body of the gene (see Figure 4.8).

5.1.5.3 Chromatin Modification and Start of Productive Elongation The transition into the stable elongation phase is accompanied by an ongoing modiﬁcation of chromatin, with the CTD of RNA Pol II playing a critical role. CTD 216 5 RNA Processing, Translational Regulation, and RNA Interference

Ser5-P acts to recruit the histone methyltransferase Set1, which trimethylates H3K4 over the promoter-proximal regions. H3K4me3 marks also correlate with the enhancement of splicing and the recruitment of splicing factors that act on the emergent nascent RNA from the elongating RNA Pol II complex. The switch in CTD phosphorylation from Ser5-P to Ser2-P during productive elongation also serves to recruit a set of splicing factors and chromatin modiﬁers to transcription machinery.

5.1.5.4 Transcription and Nuclear Pores Gene activation is a dynamic process often accompanied by a translocation of the transcription target to nuclear pores where interaction with the nuclear pore complex (NPC) can be shown. In doing so, the gene to be transcribed adopts a looped conformation that brings the promoter and terminator region in close proximity. Such a structure effectively allows a direct injection of the gene transcript into the cytoplasm during the transcription process.

5.1.5.5 Splicing During Transcription Elongation A direct linkage between transcription and splicing has been demonstrated for nearly all eukaryotes. There is an ordered recruitment of spliceosome components to the transcript, initiated by the direct interaction of splicing components with the CTD of RNA Pol II. The intron sequences appear to be cleaved cotranscriptionally and are removed by the action of exonucleases, although as the adjacent exons are retained on the polymerase elongation complex, splicing can still occur. The growing RNA chain is then packaged into an expanding complex with RNA- binding proteins. It is of note that the splice junctions are marked by binding a protein complex named the exon junction complex. This complex is stably deposited at a late stage of splicing and accompanies the mRNA to the cytoplasm.

5.1.5.6 RNA Pol II Termination The ﬁnal stages in transcription of a gene occur when the polymerase reads through functional poly(A) signals, generating pre-mRNA sequences recognized by the poly(A)-binding complex. This complex is recruited to elongating RNA Pol II in part through direct interaction of its components, with the CTD Ser-2P elongation mark. The poly(A) tail forms, facilitating mRNA release from the transcription site and its ultimate export through the nuclear pore complex to cytoplasmic translation.

5.1.5.7 Linkage to Cytoplasmic Events The growing RNA is continuously covered with RNA-binding proteins, forming a complex termed messenger ribonucleoprotein (mRNP). Part of the protein components of mRNP remain associated with the mRNA until it reaches the cytoplasm. These proteins inﬂuence a series of cytoplasmic events such as the localization of the mRNA to subcellular locations, uptake by the translation machinery, and nonsense-mediated mRNA decay. 5.2 Regulation at the Level of Translation 217

5.2 Regulation at the Level of Translation

Summary The translation of mature mRNAs is controlled mainly at the level of translation initiation, with such control either being targeted at specific mRNAs or affecting the entire population of mRNAs. When controlling specific mRNAs, protein binding to the regulatory sequence elements at the 50 or 30 end of the mRNA leads to a halt in translation. In the case of global mRNA control, the translation initiation factors eIF-4E (together with its binding partner 4E-BP) and eIF-2 are the primary targets of regulation. In this case, signal-directed phosphorylations are used as tools for translation regulation.

5.2.1 Overview of Translation Initiation

Eukaryotic translation is controlled – analogously to transcription – primarily via initiation which, in most cases, is the rate-limiting step in the translation process. The initiation of translation of nearly all mRNAs requires a mature transcript that is correctly capped and polyadenylated. Three substeps can be discerned in translation initiation: (i) recruitment of the small (40S) ribosomal subunit to the 50 end of the mRNA; (ii) scanning along the 50-untranslated region (UTR) and initiator AUG recognition; and (iii) large (60S) ribosomal subunit joining. Translation initiation in eukaryotes requires the ordered assembly of a complex of evolutionarily conserved proteins, which starts with binding of the translation initiation factor 4E (eIF-4E) to the 7-methylguanosine (m7GpppN) cap structure at the 50 end of the mRNA. In parallel, a free 40S subunit binds – with the help of further initiation factors – a ternary complex consisting of eIF-2, GTP and met- Met tRNAi to form a 43S preinitiation complex. Next, the eIF-4G factor is recruited, allowing additional factors (PABP, eIF-4A, eIF-4B, eIF-1, eIF-1A, eIF-2, eIF-3, and the 60S subunit) to form a complex that, after mRNA circularization, initiates translation (Figure 5.2). In this assembly, eIF-4G functions as an adapter that helps to bring together the 50 and 30 ends of the mRNA. This contains specific binding sites for both eIF-4E and poly(A)-binding protein (PABP), thus forming a complex that circularizes the mRNA. In this context, the 50-cap and the 30-poly(A) tail are modifications necessary for efficient translation. In the next step, the 40S ribosomal subunit and the Met initiator tRNA i join the cap complex to form the 43S initiation complex.

& mRNA circularization: — Required for translation — Involves eIF-4E, eIF-4G and poly(A)-binding protein. 218 5 RNA Processing, Translational Regulation, and RNA Interference

eIF-2 eIF-4B eIF-1 eIF-4A eIF4-E eIF4-G PABP m7GpppN AAAAA

mRNA

Figure 5.2 Circularization of mRNA. The translation factors eIF-4E, eIF-4G and poly(A)-binding protein (PABP) cooperate in the circularization of mRNAs. Other translation factors join this complex.

ThemRNAofeukaryotesdoesnotpossessspecific initiation sequences, as is the case for prokaryotes. Rather, the AUG start codon is identified by scanning the eukaryotic mRNA: the 40S subunit of the ribosome threads the 50-untranslated end of the mRNA and uses the first AUG codon encountered to initiate translation. Whether an AUG codon is used as an initiator depends, additionally, on the sequence context. If the sequence environment is unfavorable for initiation, then the scanning is continued and initiation will occur at one of the next AUGs. With the help of this “leaky scanning” strategy, it is possible to produce proteins with different N-termini from the same mRNA. As signal sequences are often found at the N-terminus, this mechanism may lead to the alternative compartmentalization of a protein. Unfortunately, the details of these processes cannot be provided here, and the reader is referred to textbooks on cell biology for this information.

5.2.2 The General Mechanisms of Translational Control

Translational control is especially relevant in situations where transcription is silent, or when local control over protein accumulation is required. Translational regulation can be either mRNA-speciﬁc or global (Figure 5.3), and most translational regulation mechanisms target the rate-limiting initiation step [7].

& Control of translation: — mRNA specific: via protein binding to mRNA elements — Global: via eIF-4E availability. 5.2 Regulation at the Level of Translation 219

mRNA-specific control Global control

Positive negative positive negative signals signals

Sequence-specific RNA binding proteins miRNAs eIF4G eIF4E PABP

Cap PolyA Cap PolyA

Control of specific All mRNAs pos. or neg. mRNAs controlled!

Figure 5.3 Global and specific mRNA control. employs two major approaches. First, the Global mRNA control is mainly based on the binding of RNA binding proteins to specific signal-directed modification of translation structural elements of the RNA can inhibit or initiation factor eIF-4E that promotes activate specific mRNAs. A second mechanism circularization of mRNAs in cooperation with uses RNA interference by miRNAs that bind eIF-4G and the poly(A)-binding protein specifically to sequences in the 30-control (PABP). The control of specific mRNAs regions of the mRNAs.

5.2.2.1 mRNA-Specific Control of Translation This control is driven by RNA sequences and/or structures that are commonly located in the 50-or30-UTRs of the transcript. These features are usually recognized by regulatory proteins or microRNAs (Section 5.3). Embedded within the UTRs of eukaryotic mRNAs is information specifying the way in which the RNA is to be utilized, and diverse proteins bind specifically to these sequences thus interpreting this information. As a result, the translation of specific mRNAs can be either activated or repressed. The binding of regulatory proteins to mRNA-specific sequence elements often interferes with formation of the cap-binding complex, thus repressing translation in a mRNA-specific manner.

5.2.2.2 Global Control of Translation In response to external or internal signals, the translation of all mRNAs may be either activated or repressed. The treatment of cells with hormones, mitogens or growth factors generally leads to an increase in protein biosynthesis. Conversely a 220 5 RNA Processing, Translational Regulation, and RNA Interference

lack of nutrients, or environmental stresses such as heat, UV irradiation or viral infections, generally repress translation. The regulatory mechanisms underlying these controls target, above all, the translation factors eIF-2 and eIF-4E. The translational control can be global, with almost all mRNAs being affected. Another translational control is mRNA-speciﬁc and regulates the translation of only some mRNAs (Figure 5.3). Selected examples of the mRNA-speciﬁc and global control of translation are presented in the following subsections.

5.2.3 mRNA-Specific Regulation by 50-Sequences: Control of Ferritin mRNA Translation by Iron

There is one well-characterized example illustrating the control of mRNA translation by a specific ion, namely the control of ferritin mRNA by iron. In this system, it is the concentration of iron that regulates protein binding to regulatory sequences at the 50 end of the ferritin mRNA. This control of ferritin mRNA by iron is an example of translational regulation by protein binding to the 50 end of the mRNA that interferes with the stable association of the small ribosomal subunit with the mRNA, leading to translational repression and subsequent degradation of the mRNA. Ferritin, a protein employed for the storage of iron, plays an essential role in iron metabolism. When increased amounts of iron are available, the production and level of ferritin must be increased to provide sufficient iron storage capacity, and this control is based on the coupling between iron concentration and the translation of ferritin mRNA. The regulation of ferritin concentrations occurs at the level of translation initiation, and is mediated by hairpin structures at the 50 end of ferritin mRNA. The hairpin structures, termed iron-responsive elements (IREs), offer binding sites for specific RNA-binding proteins, the iron regulatory proteins (IRPs). Two types of IRPs are known, IRP1 and IRP2, that have 60% sequence homology. The binding of IRPs to the hairpin structure prevents association of the 40S ribosomal subunit with the ferritin mRNA, leading finally to ferritin mRNA degradation. The binding of IRPs to the hairpin structures is controlled by the amount of iron in two ways (Figure 5.4). The first pathway involves IRP2 activity being regulated via an iron-induced protein oxidation, followed by ubiquitination and proteasomal degradation. In a second pathway, IRP1 is regulated via iron-dependent binding to the IREs. In response to the iron concentration, IRP1 can switch between two distinct conformations that have different cellular functions and which differ one from another in terms of their 4Fe-4S cluster content [8]. When iron levels are low, IRP1 will function as a repressor of ferritin mRNA translation; however, as the 4Fe- 4S clusters cannot form at low iron levels, IRP1 will exist in an IRE-binding- competent form that will bind to the IRE and prevent access of the ribosome to the coding sequences. Under these conditions, the translation of ferritin mRNA is halted and the amount of ferritin stored is decreased. 5.2 Regulation at the Level of Translation 221

Figure 5.4 Regulation of the stability of the with binding of the 40S ribosomal subunit, þ mRNA of ferritin by Fe3 . Translation of the prevents translation initiation, and induces mRNA of ferritin is subject to regulation by Fe degradation of the ferritin mRNA. If high levels concentration. Fe exerts its regulatory effect via of Fe are present, then the IRP1 is in its the iron regulatory protein type1 (IRP1). The binding inactive form, the IRE is free, the 40S latter binds to a control segment at the 50- ribosome can bind, and translation can start. terminal region of the ferritin mRNA, known as Binding-active and -inactive forms of the IRP1 the iron responsive element (IRE). Binding of differ in the content of Fe-S clusters. IRP1 to a hairpin structure of the IRE interferes

þ & Regulation of Fe2 uptake by ferritin: þ — Low Fe2 : IRP2 binds to IRE at the 50 end of ferritin mRNA Translation of ferritin mRNA is blocked þ — High Fe2 : IRP2 does not bind to IRE Translation of ferritin mRNA is possible þ Ferritin is available for Fe2 storage and uptake

High iron concentrations, however, favor the formation of a IRE-binding- incompetent form by inducing the insertion of the 4Fe-4S cluster into the protein. As a consequence, IRE1 cannot bind to the hairpin structures, the 40S subunit can associate with the ferritin mRNA, translation is initiated, and the de novo synthesis of ferritin for the storage of iron is possible. Formation of the 4Fe-4S cluster converts IRP1 into an active aconitase, an enzyme that is responsible for the conversion of citrate into isocitrate in the citric acid cycle. 222 5 RNA Processing, Translational Regulation, and RNA Interference

Another protein that is important for iron metabolism – the transferrin receptor – is also subject to translational control by iron. In such regulation, the IRPs become involved by controlling the degradation of the transferrin mRNA in an iron- dependent manner.

5.2.4 mRNA-Specific Translational Regulation by Protein Binding to 30-UTRs

One essential step in translation initiation is the circularization of the 50 and 30 ends of the mRNA into a closed-loop complex that contains eIF-4E, eIF-4G and PABP as the key protein components (Figure 5.2). This complex is thought both to stabilize the association of the cap-binding initiation factors and to facilitate the recycling of ribosomes that have terminated their translation of the mRNA. The binding of eIF-4G to eIF-4E is mediated by a speciﬁc sequence motif in eIF-4G, and interference with this association greatly reduces normal, cap-dependent translation. A large number of mRNAs has been found to contain regulatory sequences at the 30 end that mediate the binding of regulatory proteins to eIF-4E [9].

& Control by repressor binding to 30 RNA elements: — Translational repressors compete with eIF-4G for binding to eIF-4E.

These proteins contain eIF-4E binding motifs and therefore may function as translational repressors by competing with eIF-4E for binding to eIF-4G. Several such repressors have been shown to play important roles in developmental processes by preventing the untimely translation of stored mRNAs. Examples include the Bicoid protein and the Pumilio protein (both from Drosophila), the Maskin protein, and the RNA helicase Xp54. The modes by which these repressors regulate translation differ in detail, and may involve cytoplasmic polyadenylation signals. A model of the regulatory function of Bicoid is shown in Figure 5.5. The Bicoid protein is both a DNA sequence-speciﬁc transcriptional activator and a translational repressor. Apart from its DNA-binding capability, the Bicoid protein binds to 30-untranslated sequences of the mRNA of another homeodomain protein (caudal protein) to inhibit its translation. Bicoid forms a morphogen gradient in Drosophila embryos that represses translation of the uniformly distributed caudal mRNA, which in turn encodes a transcription factor necessary for posterior segmentation. An eIF-4E motif is found in Bicoid that competes with eIF-4E for binding to eIF-4G, preventing the recruitment of eIF-4G to the cap complex and repressing the translation of caudal mRNA.

5.2.5 Global Translational Regulation of mRNAs by Targeting eIF-4E

A global control of translation is exerted by regulating the availability of the initiation factor eIF-4E for cap complex formation in response to hormonal or other 5.2 Regulation at the Level of Translation 223

eIF4-E

m7GpppN eIF4-G

Caudal mRNA

Bicoid AAAA

Figure 5.5 Model of translational repression the translation initiation factor eIF-4E. Bicoid by Bicoid. The bicoid protein forms a gradient binds to RNA elements at the 30 end of caudal in Drosophila embryos and acts both as a mRNA and to eIF-4E. This excludes eIF-4G transcriptional and a translational regulator. from binding to eIF-4E and prevents the Translation of the caudal mRNA from formation of a functional initiation complex. Drosophila is repressed by binding of Bicoid to external signals, such as insulin or stress signals. The signal transduction pathways linking external signals such as insulin to the translation apparatus are mainly directed towards translation initiation, and speciﬁcally to the initiation factor eIF-4E [10]. The mechanism for global control of translation involves the signal pathway- regulated complex formation between eIF-4E and a family of inhibitory proteins named 4E-binding proteins (4E-BPs), of which 4E-BP1 is the best characterized.

& Global mRNA control: — Binding of 4E-BP1 to eIF-4E represses translation — Signal-directed phosphorylation of 4E-BP1 releases repression.

4E-Bp1 contains an eIF-4E-binding motif and is therefore able to compete with eIF-4G for binding to eIF-4E, sequestering eIF-4E away from eIF-4G. By excluding eIF-4G from the cap-binding complex, 4E-BP1 exerts a general repression of a cap- dependent translation. The binding of 4E-BP1 to eIF-4E is regulated by the phosphorylation of 4E-BP1. In the hypophosphorylated form, 4E-BP1 binds strongly to eIF-4E, preventing cap-binding complex formation. Upon signal- mediated phosphorylation of a critical set of Ser/Thr residues on 4E-BP1, its interaction with eIF-4E is abrogated, eIF-4E is free for cap-complex formation, and the translation block is relieved (Figure 5.2).

& Phosphorylation of 4E-BP1 is triggered by: — Insulin signaling pathways — MAPK/ERK pathway.

The phosphorylation of 4E-BP is under the control of at least two signaling pathways, namely the insulin-Akt-pathway (see Section 10.4) and the MAPK/ERK 224 5 RNA Processing, Translational Regulation, and RNA Interference

pathway (see Chapter 12). Under the influence of for example, insulin (and other hormones or growth factors), a signaling chain is activated that results in an activation of the serine/threonine-specific protein kinase Akt/PKB (Section 8.6.3), and – via further steps – of the further downstream protein kinase mTor (mammalian target of rapamycin) that is the core of a larger complex, the mTOR complex 1 (mTORC1). The serine/threonine-specific protein kinase mTOR belongs to the class of PI3-like protein kinases, and was named according to its specific binding to the complex between the immunosuppressant rapamycin and the FK504-binding protein (see Section 8.6.5). Activated mTORC1 then phosphorylates multiple substrates [11], among which 4E-BP1 and another protein kinase, the S6 kinase, are predominant. The phosphorylation of both proteins generates a signal that results in a stimulation of translation (Figure 5.6a).

5.2.5.1 4E-BP1 Phosphorylation When 4E-BP1 becomes phosphorylated, it can no longer bind to eIF-4E and the free eIF-4E is now available for cap-complex formation to initiate translation (Figure 5.6b). Nutrients such as glucose and amino acids influence translation via another pathway, which also includes TORC1 and has eIF-4E as a target. When sufficient nutrients are available, mTORC1 is activated and phosphorylates 4E-BP1, allowing the initiation of translation. A lack of amino acids such as leucine leads to a reduction in mTOR activity and of 4E-BP1 phosphorylation, and hence to an inhibition of translation. The regulation of translation is accomplished in these systems via a specific inhibitory protein and an initiation factor of translation. The binding activity of the inhibitor protein is regulated by protein phosphorylation, and thus by protein kinases. Signals from diverse signaling pathways may use different protein kinases to achieve phosphorylation of the same target protein, namely 4E-BP1.

I Figure 5.6 Regulation of translation initiation protein of translation initiation. The multiply by insulin. (a) Pathways leading from insulin phosphorylated 4E-BP1 protein strongly binds (or insulin-like growth factor; IGF) to the level to the initiation factor eIF-4E making it of translation initiation. For PI3 kinase, PDK1, unavailable for translation initiation. eIF-4E is PKB and mTor, see Section 8.5. The figure required, together with the proteins eIF-4A and does not show all steps of the insulin signaling eIF-4G, for binding of the 40S subunit of the cascade. IRS, insulin receptor substrate; IGFR, ribosome to the cap structure of the mRNA insulin-like growth factor receptor; PtdInsP3, and for mRNA circularization. If the 4E-BP1 phosphatidylinositol 3,4,5-triphosphate, protein becomes phosphorylated as a result of membrane-bound second messenger, see insulin-mediated activation of the PI3-kinase/ Chapter 7; mTOR, mammalian target of Akt kinase cascade, then eIF-4E is liberated rapamycin; (b) Regulation of eIF-4E by insulin from the eIF-4E4E-BP1 complex, the ternary and nutrients. Insulin and nutrients activate complex between eIF-4E, eIF-4A and eIF-4G signaling cascades that finally trigger the forms, mRNA can circularize and protein phosphorylation of 4E-BP1, a regulatory biosynthesis can begin. 5.2 Regulation at the Level of Translation 225 226 5 RNA Processing, Translational Regulation, and RNA Interference

5.2.5.2 S6 Kinase Phosphorylation A further susceptible target of the mTOR pathway is the ribosomal protein S6 of the 40S ribosomal subunit. Under the inﬂuence of insulin, S6 is phosphorylated by a speciﬁc protein kinase, the S6 kinase1 (p70S6K1, also called RSK or p70RSK) which results in increased levels of translation of certain mRNAs. Like 4E-BP1, the S6 kinase becomes phosphorylated and activated by mTorC1 [12]. Several pathways triggered by growth factor receptors, including the MAPK/ERK pathway (see Chapter 12) and the Insulin/Akt kinase pathway, can contribute to the activation of p70S6K1.

5.2.6 Regulation of Translation via eIF-2

The regulation of protein synthesis in response to hormonal signals and stress conditions uses another attack point that is centered around the binding of the methionyl-initiator tRNA to the 40S ribosome, leading to formation of the 43S initiation complex.

& Regulation of translation by hormones and stress targets eIF-2–eIF-2B interaction: — Phosphorylation of eIF-2B by protein kinases releases its inhibitory effect: HRI: regulated by heme PKR: regulated by dsRNA PERK, GCN2: regulated by stress.

Binding of the methionyl-initiator tRNA to the 40S ribosomal subunit is mediated by the eukaryotic initiation factor-2 (eIF-2), which belongs to the superfamily of regulatory GTPases (Chapter 7), and fulfills the task of bringing the methionyl-initiator-tRNA to the 40S subunit of the ribosome. The function of eIF-2 is illustrated schematically in Figure 5.7. The active eIF-2GTP form binds the methionyl-initiator-tRNA, associates with the cap structure of the mRNA, and then commences to scan along the mRNA. Once an AUG codon is encountered, the bound GTP is hydrolyzed to GDP, resulting in dissociation of the eIF-2GDP from the 40S ribosome. Transition from the inactive eIF- 2GDP form into the active eIF-2GTP form requires a G-nucleotide exchange factor, termed eIF-2B, which is composed of five polypeptides that can be divided into a regulatory subcomplex (subunits a, b,andd) and a catalytic subcomplex (subunits e, c). The regulatory subcomplex interacts with eIF-2a in a phosphorylation-dependent manner. In response to external signals, the a subunit of eIF-2 is phosphorylated on Ser51, and this phosphorylation converts eIF-2 from a substrate to an inhibitor of eIF-2B. The affinity of eIF-2 for the nucleotide exchange factor eIF-2B is increased, without inducing nucleotide exchange. Translation factor eIF-2 is found in excess of eIF-2B in the cell, so that phosphorylated eIF-2 binds the entire reservoir of 5.2 Regulation at the Level of Translation 227

Figure 5.7 The function of eIF-2 in eukaryotic structure of mRNA to initiate the scanning translation. The initiator protein for process. eIF-2 undergoes an activation cycle translation, eIF-2, is a regulatory GTPase that typical for regulatory GTPases: the inactive eIF- occurs in an active GTP-form and in an inactive 2GDP form is activated with the assistance of GDP form (see Chapter 7). The active eIF- the eIF-2B protein into the active eIF-2GTP 2GTP forms a complex with the initiator- form. eIF-2B acts as a G-nucleotide exchange tRNA, fMet-tRNAfmet and the 40S subunit of factor in the cycle. the ribosome. This complex binds to the cap eIF-2B. As a consequence, no further eIF-2B is available for nucleotide exchange, and protein biosynthesis is halted (Figure 5.8). Four different protein kinases have been identiﬁed that speciﬁcally phosphorylate eIF-2 on Ser51 [13] and thereby mediate a stress-induced repression of translation. The eIF-2a kinases comprise the families of HRI, PKR, PERK, and GCN2 kinases:

HRI: The protein kinase HRI (heme-regulated eIF-2 kinase) was first identified in studies on the regulation of protein biosynthesis in erythroid cells. A decrease in the heme concentration in reticulocytes leads to an inhibition of globin synthesis at the level of translation. This regulation mechanism ensures that only so much globin is produced as is heme available. If the level of heme falls, then HRI becomes activated; the activated HRI then phosphorylates the eIF-2a subunit, which in turn shuts off protein biosynthesis (Figure 5.8). The mechanism of regulation of HRI kinase by heme is not well understood, though heme binding sites have been identified on the N-terminus and the kinase domain of HRI. 228 5 RNA Processing, Translational Regulation, and RNA Interference

eIF-2 x GDP α

dsRNA PKR HRI + heme

PERK stress GCN2 active- heme inactive

eIF-2 x GDP α

P eIF-2B

eIF-2 x GDP

α eIF-2B P

GDP/GTP exchange blocked

translation arrest

Figure 5.8 Control of eIF-2 by kinase (HRI) which is regulated via the heme phosphorylation. Phosphorylated eIF-2GDP concentration. Another protein kinase that can binds strongly to the eIF-2B complex without phosphorylate and regulate eIF-2 is the RNA- nucleotide exchange occurring. Initiation of dependent eIF-2a-kinase (PKR). The latter is protein biosynthesis is not possible in this induced by interferons and activated by case. At least four different protein kinases double-stranded RNA. Stress influences control the phosphorylation state of eIF- activate the protein kinases PERK and GCN2, 2GDP. In reticulocytes, eIF-2 is subject to allowing also for the inhibition of protein phosphorylation by the heme-regulated eIF-2- synthesis via eIF-2 phosphorylation.

PKR: The protein kinase PKR (RNA-speciﬁc eIF-2 kinase) is regulated by the binding of double-stranded (ds)RNA and by interferon on the level of expression. PKR contains two dsRNA binding sites, and it is thought that dsRNA binding disrupts inhibitory interactions in PKR, leading to its activation. The infection of 5.3 Regulation by RNA Silencing 229

cells by viruses containing dsRNA as the genetic material can therefore trigger activation of PKR, leading to a stop in protein biosynthesis. The activation of PKR by dsRNA, and its induction by interferon, identify PKR as a component of cellular antiviral defense. Consistent with this notion, a large number of viruses express inhibitors of PKR. PERK and GCN2: The third eIF-2a kinase, PERK, participates in the endoplasmic reticulum stress response, while the fourth eIF-2a kinase, GCN2, is activated under conditions of amino acid starvation.

Importantly, the inhibition of translation via phosphorylation by the eIF-2a kinases can have both a general and a gene-specific effect, and can even lead to an enhanced translation of specificmRNAs.Whereas,thegeneral level of translation may be reduced under these conditions, specificmRNAs are preferentially translated. This upregulation of specificmRNAsis explained by a leaky scanning of AUG codons and the use of alternative initiation sites.

5.3 Regulation by RNA Silencing

Summary A major pathway for posttranscriptional gene regulation uses small noncoding RNAs of 20–30 nucleotides to negatively control the expression of specific genes via processes collectively subsumed under the heading of RNA silencing. Two major classes of small RNAs that participate in RNA silencing are dominant: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Both classes of small RNAs guide translational repression and the degradation of target mRNAs by base-pairing with the targets. The miRNAs are transcribed from a noncoding miRNA gene and processed further to produce a mature miRNA that can then associate with a member of the Ago family of proteins to form the core of the miRNA-induced silencing complex (miRISC). The assembly of this complex on the mRNA is thought to interfere with mRNA circularization, and to trigger deadenylation and degradation of the target mRNA. miRNAs appear to regulate gene expression mostly through repression, and it is this property which provides the miRNAs with a specific role in the regulatory programs of the cell by allowing a dampening of the action of transcription factors and setting up specific gene expression profiles. Regulation by miRNAs has also been shown to play a critical role in all major cellular processes in eukaryotes. Multiple mRNAs may be the target of a particular miRNA, which allows for a repression of many mRNAs acting in a common biological process. Importantly, aberrant functions of miRNAs have been linked to tumorigenesis. 230 5 RNA Processing, Translational Regulation, and RNA Interference

& The regulatory small RNAs function by binding to complementary sequences of target mRNA: — miRNAs — siRNAs — piRNAs Other regulatory RNAs: — sncRNAs — lncRNAs

Regulation by RNA silencing has attracted much interest as a novel tool for regulating gene expression in eukaryotic systems, and RNA silencing is now recognized as a major gene-regulatory mechanism in eukaryotes by which the expression of mRNA encoded information can be inhibited. However, small RNAs that regulate gene expression posttranscriptionally are also well known and have been studied in prokaryotes [14]. Cells of higher eukaryotes employ RNA silencing to regulate the expression of a large number of genes during developmental processes, as well as many other vital cellular functions. The effects of RNA silencing target major levels of genome organization, including translation, transcription, RNA stability, RNA processing, and chromatin structure. Furthermore, protection against the proliferation of transposable elements and viruses can be provided by RNA silencing. Several RNA silencing pathways are known that differ in details, within an organism and between organisms. While it appears that a basic pathway has been conserved, specialization has adapted the common RNA silencing machinery for different purposes.

5.3.1 Basics of RNA Silencing

Three major classes of small RNAs participating in RNA silencing can be distinguished (Figure 5.9):

MicroRNAs (miRNAs): This class of small RNAs is derived from the RNA polymerase II-directed transcription of miRNA genes. Small interfering RNAs (siRNAs): These are produced from long dsRNAs that can be formed endogenously or originate from exogenous sources (viruses, transfected siRNA, transposons) piwi-interacting RNAs (piRNAs) [15]: This class of small RNAs exerts its function mostly in the germline. piRNAs are 24–30 nt in length, are Dicer-independent, and bind the PIWI-subfamily of Ago proteins. They are transcribed from genome-integrated transposons and repetitive elements. The function of piRNAs has been linked to transposon binding and degradation and heterochromatin silencing. 5.3 Regulation by RNA Silencing 231

Figure 5.9 Classes of small regulatory RNAs. processing, mature miRNAs function through Small interfering RNAs (siRNAs) originate degradation of protein-coding transcripts or from exogenous or endogenous sources and translational repression. PIWI-interacting function through translational repression or RNAs (piRNAs) are mainly expressed as RNA degradation. MicroRNAs (miRNAs) are ssRNAs from mono- or bidirectional clusters. transcribed as individual units (primary piRNAs are involved in transposon miRNA, pri-miRNA) or together with host degradation and heterochromatin silencing. genes (mirtrons). Following multistep-

Further classes of regulatory noncoding RNAs have been identiﬁed [15], including small noncoding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). The sncRNAs are intermediate-sized RNAs of 60–300 nt that perform regulatory functions in the nucleolus. lncRNAs are a heterogeneous group of noncoding transcripts more than 200 nt long that are involved mostly in transcription regulation. The precise mechanisms of biogenesis and function have not been well established for sncRNAs and lncRNAs, and only the best studied pathways – namely the miRNA and siRNA silencing pathways – will be discussed at this point. Overall, the small RNAs can be described as speciﬁcity factors that direct bound effector proteins to target nucleic acid molecules – most often mRNAs – by base-pairing interactions. The effector proteins, when bound to the small RNA, then interfere with the function of the target nucleic acid. The siRNAs and miRNAs invariably have members of the Ago family of proteins as effectors, and use further dsRNA-binding proteins as helper proteins. The piRNAs bind to different effectors, the Piwi family of proteins, which are related to Ago proteins. 232 5 RNA Processing, Translational Regulation, and RNA Interference

& Major components of gene silencing: — miRNAS, siRNAs — Ago proteins — Adapter proteins — RISCs.

Together, the small RNAs and their associated proteins act in distinct but related RNA silencing pathways that have a major impact on shaping the transcriptomes and proteomes of eukaryotic cells (Figure 5.10). One class alone – the miRNAs – is predicted to regulate at least one-third of all human genes. Although the RNAs are small, their production, maturation and regulatory function require the action of a large number of proteins. RNA silencing by miRNA and siRNA can be roughly divided into the following steps:

Figure 5.10 Core features of miRNA and target RNA recognition through Watson---Crick siRNA silencing. Double-stranded RNA base-pairing. Once the target is recognized, its precursors of various types are processed by a expression is modulated by one of several Dicer protein into short (ca. 20---30 nt) distinct mechanisms, depending on the fragments. One strand of the processed duplex biological context. Carthew 2009 [16], figure 1. is loaded into an Argonaute protein, enabling Reproduced with permission of Elsevier. 5.3 Regulation by RNA Silencing 233

Double-stranded RNA precursors of various types are processed by an endoribonuclease named Dicer into short (ca. 20–30 nt) fragments. Association of the small RNAs into protein complexes with Ago proteins and the formation of the “RNA-induced silencer complex” (RISC). Binding of the RISC-incorporated small single-stranded RNAs to target sites on mRNA via complementary base-pairing Silencing of the mRNA by repression of translation and mRNA degradation.

The target mRNAs are selected for silencing by varying degrees of base complementarity between the small RNAs and sequences in the target RNA. The base-pairing of small RNAs often occurs in the 30-UTR of the target mRNA, although the small RNAs can also bind to other regions of mRNA, including exon- coding regions. The production, maturation and silencing functions of the small RNAs occurs in RNA–protein complexes that form in a sequential order and involve a large number of different proteins.

& RISC: — Multisubunit RNA–protein complex — Mediate binding of the guide strand of miRNA or siRNA to complementary sites on target RNA — Interfere with mRNA circularization — Induce repression of translation — Induce degradation of target mRNA.

Two pathways have been characterized in more detail that use a similar core machinery for production, namely the RNA interference (RNAi) pathway involving siRNAs, and the miRNA pathway involving miRNAs. Whilst these two pathways differ in the origin of the small RNA, the major steps required for their biological effects appear to be similar.

5.3.2 The miRNAs

The miRNAs are endogenous RNAs of 20–23 nt length that are the ﬁnal product of a noncoding miRNA gene. RNA silencing by miRNAs is used to modulate gene expression by triggering a stop in translation and subsequent degradation of mRNA (for a review, see Ref. [16]).

5.3.2.1 miRNA Biogenesis The miRNA genes resemble protein-coding genes in that they may contain introns and they are transcribed by RNA polymerase II. Like other RNA Pol II transcripts, the transcripts from miRNA genes are capped, spliced, and polyadenylated [17]. Approximately one-third of the about 1000 miRNAs identiﬁed in humans are located in clusters of 51 kb. These miRNA gene clusters are coexpressed, based on evidence from miRNA proﬁling data from a variety of tissues and cell lines. A 234 5 RNA Processing, Translational Regulation, and RNA Interference

transcript may encode several distinct miRNAs, or it may encode a miRNA and protein. In the latter case, the miRNA encoding sequence is transcribed together with host genes that are collectively called mirtrons.

Transcription The primary transcript of a miRNA gene, the pri-miRNA, contains the mature miRNA within a stem–loop structure about 70 nt long (Figure 5.11). Several processing steps are required for production of the mature miRNA. In a ﬁrst step, a nuclear RNase III endonuclease, named Drosha, liberates the stem– loop from the pri-miRNA to yield a pre-miRNA. For precise excision of the pre- miRNA, Drosha relies on the help of an anchoring protein.

Figure 5.11 Action of nucleases in miRNA comprising the miRNA (guide strand) and production. (a) miRNAs are produced by the miR strands (passenger strand) of the pre- successive actions of two RNase III miRNA. The miRNA must then be unwound ribonucleases, named Drosha and Dicer. After and selectively incorporated into RISC by the their transcription by RNA polymerase II, miRNA-specific RISC assembly machinery; primary miRNAs (pri-miRNA) are cleaved in (b) The successive action of Drosha and Dicer the nucleus by the nuclease Drosha to generates the mature miRNA. The guide generate the precursor miRNA, pre-miRNA. strand of the miRNA duplex is incorporated Following export into the cytoplasm, Dicer into RISC, whereas the passenger strand is cleaves the pre-miRNA stem defined in the discarded. nucleus by Drosha. This liberates a duplex 5.3 Regulation by RNA Silencing 235

& miRNAs: — Are processed from primary transcript by Drosha and Dicer — Are incorporated in miRISC — Guide miRISC to complementary sites on 30 end of target mRNA.

The transcription of mirtrons yields a pre-mRNA that is subsequently spliced, while the miRNA encoding part is excised in the form of a “lariat,”, the processing of which then generates a pre-miRNA that is subsequently exported from the nucleus into the cytosol by the protein exportin 5.

Processing by Dicer The pre-mRNA is then processed in the nucleus by Dicer, a double-stranded RNA-speciﬁc enzyme of the RNase III family, to produce the mature miRNA (Figure 5.12). This step involves excision of the terminal loop in pri-mRNA.

5.3.2.2 Formation of miRISC The silencing function of the miRNAs is performed by the incorporation of miRNA as single-stranded RNA into the miRNA-dedicated miRISC that guides the translational repression and degradation of its target mRNAs by base-pairing with the targets [18]. The mature miRNA is a short-lived imperfect duplex with exact ends (see Figure 5.11b) that must pass through an ordered pathway of protein- associations in order to base-pair correctly with the target sequence on the mRNA. In a first step, the mature miRNA associates with a member of the Ago family of proteins that form the core of the RISC. To date, four Ago proteins have been identified in mammals, and the regulatory function of all small RNAs is thought to depend on one or more of these Ago proteins. Because the miRNAs in the RISC must anneal to their target mRNAs, the two strands of the miRNA duplex must be separated and one strand discarded to form the functional RISC. The discarded strand is called the miRNA strand (“passenger strand”), whilst the strand retained in the RISC is called the miRNA strand (“guide strand”). Transfer of the miRNA strand to one of the Ago proteins also requires the participation of the RNase Dicer and TAR RNA binding protein 2 (TRBP), a helper protein. Once Ago has been loaded with the miRNA strand, further RNA-binding proteins associate to form a mature miRISC. The composition of the mature RISC appears to be variable; in mammals, the protein G182 has been identified as an essential component of the miRISC, while miRNA-bound Ago in association with G182 is thought to form the functional miRISC.

5.3.2.3 Posttranscriptional Repression and mRNA Decay The miRNA acts as an adapter for the miRISC to guide the latter towards mRNA binding. With few exceptions, miRNA-binding sites in animal mRNAs lie in the 30- UTR and are usually present in multiple copies. Most animal miRNAs bind with 236 5 RNA Processing, Translational Regulation, and RNA Interference

Figure 5.12 Biogenesis of miRNAs. The protein 2 (TARBP2) generates mature miRNAs are transcribed as individual units miRNAs, which are loaded into the RNA- (pri-miRNA) or together with host genes induced silencing complex (RISC). miRNAs (mirtrons). Following processing by the function through degradation of protein- Drosha complex or the lariat-debranching coding transcripts or translational repression. enzyme, respectively, pre-miRNAs are exported Esteller 2011 [15], figure 1. Reproduced with from the nucleus by exportin 5 (XPO5). Further permission of Nature Publishing Group. processing by Dicer and TAR RNA-binding 5.3 Regulation by RNA Silencing 237

Figure 5.13 Cleaving and noncleaving RISCs. formation results in the cleavage of the target Depending on the extent of base RNA or in the stopping of translation. The complementarity between endogenous siRNAs mechanism of translational arrest is unknown. or miRNAs and the target RNA, RISC mismatches and bulges (Figure 5.13), although a key feature of recognition involves the Watson–Crick base-pairing of miRNA nucleotides 2 to 8, representing the seed region. The degree of miRNA–mRNA complementarity has been considered a key determinant of the regulatory mechanism. Perfect complementarity allows Ago- catalyzed cleavage of the mRNA strand. One of the Ago proteins, Ago2, carries an 238 5 RNA Processing, Translational Regulation, and RNA Interference

RNase activity (also termed “slicer” activity) that becomes active only in cases of perfect hybridized duplexes between the miRNA and the mRNA. However, this is a rare event in animal miRNAs, and the vast majority of animal miRNAs will form an imperfect hybrid with the target site on the mRNA, excluding cleavage by the Ago2 protein. The formation of an imperfect duplex on the mRNA has two observable consequences:

Translation is inhibited at some point and the production of protein is stopped. The miRNA directs the mRNA to the cellular 30 ! 50 degradation pathway involving deadenylation, decapping, and degradation by cytoplasmic RNase.

The mechanism of translational repression has long been a matter of debate, and has centered around the question of whether repression occurs at translation initiation or at another step after initiation, and whether mRNA decay is a consequence of a primary effect on translation. Moreover, it has been difﬁcult to discern the interdependence of these events. Genome-wide studies on miRNA-mediated decreases of protein production (i.e., translation inhibition) and mRNA levels now suggest a major role for mRNA degradation in miRNA-silencing effects. The rapid mRNA degradation that appears to occur before or immediately after a translational block provides an explanation for the majority of the miRNA regulation processes occurring in animal cells. How the translational block is mediated and linked to mRNA decay is not completely understood. One well-accepted model suggests that miRISC interferes with an early step in translation initiation, namely the circularization of mRNA (reviewed in Ref. [19]). This proposal is based on the observation that GW182 protein, which is an essential component of the miRISC, interacts with PABP. As outlined in Section 5.2.1, PABP is involved in a circularization of the mRNA by binding both the poly(A) tail and the elongation factor eIF-4G. The model proposes that the miRISC, composed of miRNA, Ago and GW182, binds to target sequences near the 30 end, allowing an interaction of GW182 with PABP bound to the poly(A) tail (see Figure 5.2). The assembly of this complex on the mRNA is thought to interfere with mRNA circularization and to trigger the deadenylation and decapping of the mRNA; the degradation of mRNA by cytoplasmic RNase is then initiated as a consequence (see also Ref. [20]).

5.3.2.4 Regulation of miRNAs The function of miRNAs is tightly regulated, and the misregulation of miRNAs can have severe consequences for the cell. miRNA genes are transcribed by RNA polymerase II and are subject to the same regulatory principles of RNA Pol II genes (as discussed above). Furthermore, miRNA levels can be regulated at several posttranscriptional steps, including processing by Drosha and editing of the mature miRNA. Overall, the miRNAs form part of a complex regulatory network that uses many links to maintain appropriate miRNA functions. The following regulatory mechanisms have been shown to inﬂuence levels and functions of miRNAs [21]. 5.3 Regulation by RNA Silencing 239

Transcriptional Regulation The transcription of miRNAs is subject to control by transcription factors and by chromatin structure in a developmental and tissue- specific manner. Many pri-miRNA promoters contain binding sites for well-known transcription factors, such as p53, Myc and muscle-specific myogenin, resulting in the expression of these miRNAs only under specific conditions.

& Regulation of miRNAs: — Transcription by common mechanisms of transcription regulation — Processing via regulation of Drosha and Dicer activity — Editing of mature miRNA.

Some autonomously expressed miRNA genes have promoter regions that allow miRNAs to be highly expressed in a cell type-speciﬁc manner. Chromosomal translocation can place the miRNA gene and its promoter in close vicinity to an oncogenic transcription factor, driving a strong upregulation of the transcription factor. One example is the translocation of Myc transcription factor gene close to the gene for miRNA miR-142 during B-cell leukemia, leading to aberrant expression of Myc.

Regulation of miRNA Biogenesis The processing of pri-miRNA by Drosha and of pre-miRNA by Dicer is subject to stringent control. The regulation of processing plays a crucial role in developmental and tissue-specific signaling, whilst inhibition of the miRNA biogenesis pathway leads to severe developmental defects and is lethal in many organisms. A major regulatory point appears to be processing by Drosha. Several proteins, such as the helicase p68, interact with Drosha and can either promote or inhibit the cleavage of specific pri-miRNAs [17]. Furthermore, the processing of pri-miRNA by Drosha appears to be coupled to transcription by RNA polymerase II, while many of the proteins involved in the regulation of Drosha also participate in splicing of nuclear mRNA. This indicates that Drosha is subject to the general regulations active in the transcription and processing of mRNA. External signaling pathways have been also shown to stimulate pri-miRNA processing. In response to DNA damage, activated p53 binds with Drosha complexed with helicase p68 and enhances the processing efficiency of several pri- miRNAs. This indicates that the regulation of pri-miRNA cleavage by Drosha is a component of the p53-induced DNA damage response.

Editing of Mature miRNAs The base-pairing and decoding functions of RNA can be modiﬁed in a process termed RNA editing, which implies the deamination of adenosine to inosine [22]. RNA editing is mediated by a family of enzymes referred to as “adenosine deaminases acting on RNA” (ADAR). The miRNAs are also targets of RNA editing, and the post-transcriptional regulation of miRNAs through RNA editing has been shown to alter miRNA targeting, processing and stability, and is considered to be potentially of a regulatory nature. 240 5 RNA Processing, Translational Regulation, and RNA Interference

5.3.2.5 Regulatory Functions of miRNAs Today, the sequences of almost 1000 human miRNA genes have been deposited in the miRNA databank, accounting for about 3% of all human protein-coding genes. Together with transcription factors, the miRNAs form a transcriptional regulatory network that shapes gene expression in a developmental- and cell type-specific manner. Importantly, transcription factors and miRNAs are often part of the same regulatory circuit. The expression of many transcription factors is subject to miRNA regulation, and the cell type-specific expression profiles of miRNAs are brought about by the conventional transcription factor-dependent control elements discussed in Chapter 4. These links place transcription factors and miRNAs in regulatory networks that include feed-forward and feedback loops.

Features of Regulation by miRNA As compared to transcription factors, miRNA- mediated gene regulation shows some unique features [23]. Whereas, transcription factors are known to regulate gene expression either positively or negatively, miRNAs appear to regulate gene expression mostly through repression. This provides the miRNAs with a specific role in the regulatory programs of the cell by allowing a dampening of the action of transcription factors and setting up specific gene expression profiles. Other particular features of miRNA regulation include the speed and reversibility of regulation. Typically, miRNAs can rapidly turn off protein production at the site of site of protein synthesis, the ribosome. Likewise, a miRNA-repressed target can be activated more rapidly than a transcriptionally repressed target, simply by replacing the target with another mRNA. Compartmen- talization is another specific property of miRNA regulation, providing for the compartment-specific regulation in specialized cells. Ribosomal protein synthesis is found in various subcellular compartments (e.g., the synapse), which allows miRNAs to exert a site-specific control of protein production.

& Functions of miRNAs: — Regulation of gene expression by rapid translational repression of target genes — Dampening of transcription factor functions — Repression can be location-specific — Repressive function of intron-derived miRNAs is coupled to cognate exon- derived mRNA — A particular miRNA may target more than 100 mRNAs — Different miRNAs may cooperate in silencing a target mRNA.

Basic Concepts of miRNA Regulation Genetic deletions of miRNAs and studies on target identiﬁcation and occupation have revealed the following basic features of miRNA regulation (Figure 5.14; for a review, see Ref. [24]):

Integration of miRNAs into introns of protein-coding genes: Many miRNAs are generated by the processing of introns of protein-coding genes providing for a link 5.3 Regulation by RNA Silencing 241

Figure 5.14 Basic concepts of miRNA single miRNA. This mechanism reduces the regulation. The potential modes of miRNA- dependence on a single miRNA---mRNA based regulation of gene expression are interaction and increases the robustness of the shown. (a) Intronic miRNAs are encoded gene-regulatory network. TF, transcription within an intron of a host gene. mRNA splicing factor; (c) Many miRNAs may cooperatively or generates a protein-coding transcript and an redundantly regulate a single biological miRNA stem---loop. Intronic miRNAs often process, by individually targeting many regulate similar processes to that of the components of that process or by protein encoded by the host gene. AAA, synergistically repressing a crucial component polyadenylated tail of the transcript; (b) A of a pathway. Small 2011 [24], figure 1. common mechanism of miRNA function Reproduced with permission of Nature involves the modest repression of several Publishing Group. mRNAs in a common biological process by a

between miRNA and the exon-coded protein. The integration of miRNAs into introns of protein-coding genes serves to coordinate the expression of the miRNA with the mRNA encoded by that gene, without the necessity for a separate set of cis- regulatory elements to drive expression of the miRNA. Multiplicity of miRNA targets: miRNAs typically exert modest inhibitory effects on many mRNAs, which often encode proteins that govern the same biological process. Computational predictions and genome-wide identiﬁcation of miRNA targets estimate that a single miRNA may target, on average, more than 100 different mRNAs. This allows for a modest repression of many mRNAs acting in a 242 5 RNA Processing, Translational Regulation, and RNA Interference

common biological process. The cumulative reduction in expression of several components of a molecular pathway reduces the importance of a single miRNA– mRNA interaction to elicit a biological response, and adds robustness to gene- regulatory networks. Cooperativity and redundancy: A given mRNA may harbor several binding sites for the same or different miRNAs. In the latter case, the synergistic action of different miRNAs is required to induce an efﬁcient repression of the mRNA. The multiplicity of miRNA targets may promote combinatorial regulation by miRNAs that individually target various mRNAs whose protein products contribute to one particular regulatory axis.

General Importance of miRNA-Mediated Regulation Over 60% of human protein- coding genes have been predicted to contain miRNA binding sites within their 30- UTRs, and thus may be subject to miRNA control. These estimates illuminate the huge regulatory potential of miRNAs, and it is now assumed that miRNAs form a second regulatory level operating in cooperation with transcriptional regulation, providing for a fine-tuning and modulating effect on gene expression. Regulation by miRNAs has been shown to play a critical role in all major cellular processes in eukaryotes, as miRNAs have been implicated in development, differentiation, cell proliferation and apoptosis, while the dysregulation or malfunction of miRNAs has been associated with many diseases. Together with transcription factors, the miRNAs appear to be the most numerous gene regulatory factors in multicellular genomes. Important extracellular signal molecules such as growth factors have been found to influence the level of distinct miRNA species modulating the expression of specific genes. For example, a genome-wide approach has shown that epidermal growth factor (EGF) initiates a coordinated transcriptional program of miRNAs and transcription factors. Activation of the EGF receptor by EGF initiates, in an early step, a decrease in the abundance of a subset of 23 miRNAs which then permits the rapid induction of oncogenic transcription factors [25].

& miRNAs regulate: — Development — Differentiation — Cell proliferation — Tumorigenesis by: Downregulation of tumor suppressor proteins Downregulation of oncogenes Aberrant miRNA production by oncogenic transcription factors.

Most information on the cellular function of miRNAs has been obtained from developmental studies, and from studies of the involvement of miRNAs in the genesis of cancers. Many vertebrate miRNAs are expressed in a developmentally regulated or tissue-speciﬁc manner, indicating an essential role in the control of the 5.3 Regulation by RNA Silencing 243 developmental program of an organism. For example, the involvement of a distinct miRNA (mi-R181) in the differentiation of hematopoietic cells has been shown by overexpression of this miRNA.

5.3.2.6 miRNAs and Cancer A large number of miRNAs appear to be deregulated in primary human tumors, and many human miRNAs are located at genomic regions and fragile sites involved in cancer [26]. Furthermore, the aberrant production of miRNAs under the control of oncogenic transcription factors has been frequently observed [15]. Examples include p53- regulated miRNAs (Figure 5.15) and the Myc-regulated miRNAs (see Figure 16.1). These miRNAs function mostly as tumor-suppressors, but may also have oncogenic properties. In the case of p53, a complicated network of miRNA regulation exists that includes positive and negative feedbacks. The tumor- suppressing activity of p53 is enhanced by p53-activated miRNAs and can be inhibited by oncogenic miRNAs. Furthermore, p53 directly activates the Drosha complex which enhances the formation of tumor-suppressing miRNAs. Another part of the network includes Mdm2, an E3 ligase that is involved in p53 downregulation and is negatively regulated by p53-induced miRNAs. Overall, it is difficult to attribute a distinct phenotype to the action of a particular miRNA, as any given miRNA may control up to 100 different genes and malfunction of that miRNA may therefore influence many processes in the cell. This makes it difficult to link a disease-associated phenotype with the action of a particular miRNA on a distinct mRNA target. Furthermore, the rules governing the binding of miRNAs to target sites are not well defined, which makes it difficult to identify and predict binding sites on mRNA targets.

5.3.3 siRNAs

The siRNAs repress translation in a pathway that is also termed RNA interference (RNAi). Moreover, siRNAs can also induce changes in chromatin structure and transcriptional repression. Whilst the miRNA and siRNA pathways have many steps in common, both rely on the action of Dicer endonuclease and Ago proteins as effectors.

5.3.3.1 Sources and Processing of siRNAs Typical siRNAs are derived from long, perfectly base-paired dsRNA precursors that are introduced directly into the cytoplasm or are taken up from the environment. siRNAs originating from exogenous sources are also termed exo-siRNAs. siRNAs are also known to be formed from endogenous sources; these are called endo- siRNAs. 244 5 RNA Processing, Translational Regulation, and RNA Interference

miR-380, miR-504

Mdm2 p53 DROSHA p68 p53

miR-192 miR-194 miR-34 family miR-107 miR-200 miR-192 miR-215 miR-605

Processing CDK6 BCL2 HIF1A ZEB1/2 of tumor suppressor miRNAs

´Proliferation Survival Angiogenesis Epithelial Tumor to mesenchymal suppression transition

Figure 5.15 p53 and miRNA regulation. This the same time, p53 can be negatively regulated diagram illustrates the complex network of by oncogenic miRNAs (in red). In addition, miRNA regulation of major regulatory proteins p53 is involved in the biogenesis of several such as the tumor suppressor p53 tumor suppressor miRNAs, via activation of (Section 16.7). p53 can regulate several tumor miRNA processing by the DROSHA complex. suppressor miRNAs (blue), activating different For CDK6, see Chapter 14; for BCL2, see antitumoral pathways. The regulation of Chapter 17; HIF1A, hypoxia-induced MDM2 (see also Section 16.7.5) by some of transcription factor 1A; ZEB1/2, transcription these miRNAs leads to feed-forward loops. At factor. Reproduced from [26].

The dsRNAs are processed by the RNA endonuclease Dicer into double-stranded siRNAs of 21–23 nt length that must be converted into a single-stranded form in order that they can bind to the target RNA. Similar to miRISC assembly, the production of single-stranded siRNA (Figure 5.16) relies on siRISC assembly pathways that vary in details among different organisms.

& siRNAs: — Derived from endogenous or exogenous long dsRNAs — Maturation by RNase Dicer — Incorporation into siRISC. 5.3 Regulation by RNA Silencing 245

Figure 5.16 Overview of RNA silencing by contain the siRNA bound to an Ago protein, endogenous siRNAs. Several different and many (if not most) forms of siRISC categories of transcripts can adopt dsRNA contain additional factors. Target RNAs are structures that can be processed by Dicer into then recognized by base-pairing, and silencing siRNAs. These duplexes can be either ensues through one of several mechanisms. In intracellular or intermolecular, and although many species, the siRNA populations that most are perfectly base-paired, some (e.g., engage a target can be amplified by the action hairpin RNAs and gene/pseudogene duplexes) of RNA-dependent RNA polymerase (RdRP) are not. An siRNA consists of a guide strand enzymes, strengthening and perpetuating the (red), which assembles into a functional silencing response. Carthew 2009 [16], siRISC, and a passenger strand (blue) which is figure 2. Reproduced with permission of ejected and degraded. All forms of siRISC Elsevier.

In mammals, a complex consisting of Dicer, Ago2 and the helper protein TRBP (see Section 5.3.2.2) has been identiﬁed as a RISC loading complex that binds dsRNA, cleaves it into a double-stranded siRNA, loads it into Ago-2, and then discards the passenger strand to generate a functional siRISC. Additional proteins associate with Ago complexes from human cells, but they do not appear to be essential for RISC loading or target cleavage. The selection of which of the two strands of ds siRNA becomes the passenger strand and is discarded has been shown to depend on the relative thermodynamic stabilities of the two duplex ends: whichever strand has its 50 terminus at the less stably base-paired end will be 246 5 RNA Processing, Translational Regulation, and RNA Interference

favored as the guide strand. This thermodynamic asymmetry is graded rather than being all-or-none, and siRNAs with equal base-pairing stabilities at their ends will incorporate either strand into RISC with approximately equal frequency.

5.3.3.2 Posttranscriptional Silencing by siRNAs Similar to target selection in the miRNA pathway, the guide strand of siRNA directs the siRISC to perfectly complementary target sites on mRNA, which are then degraded. The RNase activity (also called slicer activity) responsible for the initial cutting of the target is located in the PIWI domain of the Ago components of the siRISC. Once this initial cut has been made, cellular exonucleases attack the fragments to complete the degradation. The target dissociates from the siRNA after cleavage, freeing the siRISC to cleave additional targets. Based on this property, the siRISCs are multiple-turnover enzymes; the siRNA guide strand delivers the RISC to the RNA target, the target is cleaved, after which the siRNA can depart intact with the siRISC and is able to cleave another mRNA.

& siRISC: — “Slicer” activity cuts mRNA — Guide strand of siRNA directs siRISC to perfect complementary site on target mRNA — Multiple-turnover complex.

Another striking feature of silencing by siRNAs is its potency: only a few molecules can induce a robust and widespread response. In some organisms, such as Caenorhabditis elegans, the siRNA trigger may be ampliﬁed by the action of RNA- dependent RNA polymerases that generate a secondary pool of siRNAs by copying the siRNA guide strand bound to the target site (see Figure 5.16). However, such an activity has not been found in insects and vertebrates.

5.3.3.3 Functions and Applications of siRNAs The siRNAs are produced from dsRNAs that may be derived from either exogenous or endogenous sources. One important biological function of RNAi appears to be protection against the unprogrammed and potentially deleterious accumulation of dsRNA in the cell. Thus, siRNAs have been proposed to function in: (i) antiviral defense; (ii) silencing mRNAs that are overproduced or translationally aborted; and (iii) guarding the genome from disruption by transposons. A key distinction between miRNAs and siRNAs from transgenes and repeat sequences/transposons is that miRNAs target genes other than those that give rise to the miRNAs (i.e., they act in “trans”), whereas siRNAs target the very sequences that generate them (i.e., they act in “cis”). Cleavage of the target mRNA is the major mechanism by which siRNAs induce gene silencing, and RNA interference triggered by exogenous siRNAs has now become a widely used tool for the knock-down of genes. Furthermore, RNAi and siRNAs are expected to be used for applications in human disease, such as cancer Questions 247 and viral infections. By introducing synthetic RNAs with stem–loop structures into cells, or by transcribing specifically designed siRNA genes from viral vectors, the expression of genes can be selectively inhibited and information on gene function can be obtained. The vast field of biotechnological applications of siRNAs cannot be covered here, and the reader is referred to reviews (e.g., Ref. [27]). Endogenous siRNAs have been also shown to perform important biological roles. The results of studies in flies and mice have indicated that endo-siRNAs may be derived from transposons, hairpin RNAs within introns and pseudogenes revealing a significant regulatory potential of the endo-siRNAs. The circumstances leading to an induction of the endogenous siRNA pathway have been only partially characterized, however, and will not be discussed further at this point.

Questions 5.1. Describe the reactions involved in processing the primary transcript of RNA polymerase. 5.2. Which factors are assumed to regulate splice site selection? 5.3. How can pre-mRNA processing couple to transcription and translation? 5.4. What function can you ascribe chromatin structure in splice site selection? 5.5. Summarize the major steps and protein factors involved in translation initiation. 5.6. Describe the control mechanisms by which iron controls its own uptake. 5.7. How does Bicoid protein regulate translation? 5.8. Describe the major pathway that links insulin with translation stimulation. 5.9. How can a lack of amino acids trigger translation inhibition? 5.10. Which protein kinases target eIF-2, and under which circumstances do these kinases become activated? 5.11. Explain the role of heme in regulating translation in reticulocytes. 5.12. Name the types of noncoding RNAs. What are their main biological functions? 5.13. Describe the biogenesis of mature miRNAs and the protein components involved. 5.14. What is the minimal structure of miRISC, and how are target miRNAs selected for silencing? 5.15. Explain the mechanisms by which miRNAs can lead to mRNA silencing. 5.16. Describe the mechanism for the control of miRNAs. 248 5 RNA Processing, Translational Regulation, and RNA Interference

5.17. What are the basic concepts of miRNA regulation? 5.18. How can aberrant miRNA function participate in tumor formation? Give at least two examples.

References

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20 Djuranovic, S., Nahvi, A., and Green, R. 24 Small, E.M. and Olson, E.N. (2011) (2012) miRNA-mediated gene silencing by Pervasive roles of microRNAs in translational repression followed by mRNA cardiovascular biology. Nature, 469 (7330), deadenylation and decay. Science, 336 336–342. PubMed PMID: 21248840. (6078), 237–240. PubMed PMID: 22499947. Pubmed Central PMCID: 3073349. Epub 2012/04/14. eng. 25 Avraham, R. and Yarden, Y. (2011) Feedback 21 Farazi, T.A., Spitzer, J.I., Morozov, P., and regulation of EGFR signalling: decision Tuschl, T. (2011) miRNAs in human cancer. making by early and delayed loops. Nat. Rev. J. Pathol., 223 (2), 102–115. PubMed PMID: Mol. Cell Biol., 12 (2), 104–117. PubMed 21125669. Pubmed Central PMCID: PMID: 21252999. Epub 2011/01/22. eng. 3069496. 26 Lujambio, A. and Lowe, S.W. (2012) The 22 Maas, S. (2012) Posttranscriptional recoding microcosmos of cancer. Nature, 482 (7385), by RNA editing. Adv. Protein Chem. Struct. 347–355. PubMed PMID: 22337054. Biol., 86, 193–224. PubMed PMID: Pubmed Central PMCID: 3509753. 22243585. Epub 2012/01/17. eng. 27 Lares, M.R., Rossi, J.J., and Ouellet, D.L. 23 Hobert, O. (2008) Gene regulation by (2010) RNAi and small interfering RNAs in transcription factors and microRNAs. human disease therapeutic applications. Science, 319 (5871), 1785–1786. PubMed Trends Biotechnol., 28 (11), 570–579. PMID: 18369135. Epub 2008/03/29. eng. PubMed PMID: 20833440. Pubmed Central PMCID: 2955826.

251

6 Signaling by Nuclear Receptors

Summary Nuclear receptors (NRs) regulate gene expression in response to binding small lipophilic molecules of which many are known as classical hormones. These proteins are ligand-activated transcription factors that are localized in the cytoplasm and/or in the nucleus. The ligands may be of extracellular origin or may be derived from intracellular metabolites. There are two types of NR signaling, termed genomic and non-genomic signaling. In genomic signaling, the ligand-bound receptor functions as a sequence-specific transcription factor that binds to DNA elements (hormone-responsive elements; HREs) in the control regions of target genes and influences the transcription of these genes. Ligand binding activates the transcription regulation function of the NR which is localized in the cytosol and/or in the nucleus. Receptors which are found predominantly in the cytosol translocate to the nucleus in response to ligand binding,bindtothecognateHRE,andactivate transcription of the target gene. Other receptors are permanently associated with the HREs of target genes, and hormone binding then activates transcription. The non-genomic response involves a direct interaction of the NR with components of other signaling pathways, independent of transcription.

The classical mode of nuclear receptor (NR) signaling is based on the ability of NRs to regulate transcription in response to cognate ligands. By binding to DNA- recognition elements located in the control region of target genes, the NRs typically function as transcriptional activators. The number of genes regulated by NRs is very large; for example, the glucocorticoid receptor alone affects the transcription of more than 500 target genes in human cells. The genomic responses trigger often long-term changes in the protein pattern of a cell, and accordingly, many hormones of the signal transduction pathways involving NRs participate in the development and differentiation of organs. Examples include the sexual hormones, the thyroid hormone T3, the D3 hormone, and retinoic acid. In non-genomic signaling (Section 6.7), NRs receive signals from and transmit signals to other signaling paths. This crosstalk occurs mainly in the cytosol, and may result in a broad spectrum of signaling outcomes. By contrast to the genomic

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 252 6 Signaling by Nuclear Receptors

functions of NRs and their ligands, the non-genomic functions are very rapid and effects can be observed within seconds or minutes. & NRs transmit: — Genomic responses By delivering signals to the level of gene expression — Non-genomic responses By crosstalk with other signaling pathways

6.1 Ligands of Nuclear Receptors (NRs)

The naturally occurring ligands of NRs are low-molecular-weight, lipophilic compounds that may be of extracellular or intracellular origin. The most important natural ligands and the cognate receptors are listed in Table 6.1.

Table 6.1 Ligands of selected nuclear receptors from mammals.

Hormone Receptor

Estrogen receptor (ER) OH

HO Estradiol Androgen receptor (AR) OH

O Testosterone Progesterone receptor (PR) H3C C O OH

O Progesterone Glucocorticoid receptor (GR) CH2OH C O HO OH

O Cortisol 6.1 Ligands of Nuclear Receptors (NRs) 253

Mineralocorticoid receptor, MR CH2OH OH C O O CH OH

O Aldosterone Vitamin D receptor (VDR)

HO OH 1, 25-Dihydroxycholecalciferol (from Vit D3) T hormone receptor (T R) I I 3 3 NH2

HO O CH2 CH COOH

I 3, 5, 3' -L-triiodothyronine 9-cis-retinoic acid receptor (RXR)

COOH 9-cis-retinoic acid All-trans- retinoic acid receptor (RAR) COOH

All-trans-retinoic acid Oxidized sterols, Drug metabolites Liver X receptor, LXR Heterodimer with RXR Farnesoids Farnesoid X receptor, FXR Sterol derivatives Pregnane X receptor, PXR Drug metabolites Phospholipids, Peroxisome proliferator-activated receptor (PPAR) Fatty acids Prostaglandins Drug metabolites (continued) 254 6 Signaling by Nuclear Receptors

Table 6.1 (Continued)

Hormone Receptor

Drug metabolites Constitutive androstane receptor (CAR). ? Chicken ovalbumin upstream promoter transcription factor (COUP-TF) Orphan receptor ? Nerve growth factor-induced receptor B (NGFI-B) Orphan receptor

Among the extracellular ligands, the steroid hormones, the thyroid hormone T3, and derivatives of vitamins A and D have long been known as central regulators. These hormones play a signiﬁcant role in metabolic regulation, organ function, and development and differentiation processes. Following their formation and secretion in speciﬁc tissues – the endocrine organs – the hormones are distributed within the organism via the circulation and enter cells passively by diffusion. & Ligands of NRs are small lipophilic molecules: — Steroid hormones: Estradiol, progesterone, testosterone, cortisol, aldosterone — Amino acid derivatives: cis trans T3 hormone, retinoic acid derivatives, 9- -retinoic acid, all- - retinoic acid — Prostaglandins: Prostaglandin J2 — Others: Fatty acids, oxidized cholesterol, phospholipids, farnesoids, xenobiotics

During recent years it has been recognized that intracellularly formed lipophilic metabolites can also serve as ligands of NRs, and function as sensors of the metabolic state of the cell. By binding to NRs, these compounds can elicit genomic and/or non-genomic responses. The endogenous ligands for NRs include prostaglandins, leukotrienes, fatty acids, oxidized fatty acids, cholesterol derivatives, bile acids, phospholipids, xenobiotics, and drugs such as phenobarbital. The cognate receptors – for example, the peroxisome proliferator-activated receptor (PAR) and the farnesoid X receptor (FXR) – are quite promiscuous with respect to the nature of the ligand, and have been shown capable of binding to a broad range of lipophilic ligands. This type of receptor is thought to be involved in the homeostasis of metabolism and in the detoxiﬁcation of foreign substances.

6.2 Principles of Signaling by Nuclear Receptors (NRs)

The process of signal transduction by NRs, following the classical and the new nonclassical pathways, is shown schematically in Figure 6.1. 6.2 Principles of Signaling by Nuclear Receptors (NRs) 255

Figure 6.1 Genomic and nongenomic signaling involves binding of the hormone to signaling by nuclear receptors (NRs) and their another type of receptor and crosstalk with ligands. In the genomic path, NRs function as other intracellular signaling proteins as for ligand-controlled transcription factors that example, protein kinases. These pathways can bind cognate DNA sequences, or as hormone- trigger a rapid, transcription-independent responsive elements (HREs) leading to response. H, hormone; Hsp, heat shock transcription activation. The nongenomic protein. 256 6 Signaling by Nuclear Receptors

& Coactivators and corepressors: — Cooperate with NRs in transcription regulation

In the classical genomic pathway, the NRs convey signals directly to the level of transcription. Many of the natural ligands of NRs are lipophilic hormones that enter the cell either in a passive manner or by active transport mechanisms. Once inside the cell, the hormone ligand binds the cognate receptor which is localized in the cytosol and/or in the nucleus. Such hormone binding activates the transcription regulation function of the receptor.

& Hormone binding to the receptor induces transcription activation by: — Nuclear translocation of receptor — Activation of DNA-bound receptor

One class of receptors, which includes most of the steroid hormone receptors, is located predominantly in the cytosol. Here, hormone binding induces translocation into the nucleus where the hormone–receptor complex binds a cognate DNA element termed the hormone-responsive element (HRE), and alters the transcription of the target gene. The HREs are located in the control regions of genes, and their presence determines whether a gene is subject to control by NRs at all. Very often, the target genes are far away from the HRE where the receptor binds, and the regulatory inﬂuence of the receptor is exerted by long-range interactions between distant chromosomal regions. Those receptors which are localized predominantly to the nucleus are found permanently associated with their HRE in the control regions of genes. The cognate hormones enter the nucleus, bind to the DNA-associated receptor, and thereby trigger transcription activation. In the absence of the hormone, the DNA- bound receptor often has a repressive effect on transcription, and ligand binding to the DNA-bound receptor then relieves the repression. Rare cases are known where ligand binding induces a repression of transcription. Overall, the functions of NRs in transcription regulation are subjected to the same principles that have been discussed in a general sense in Section 4.3. As part of the transcriptional regulatory network of the cell, the genomic functions of NRs depend on, and are regulated by:

the sequence of the HRE; the availability and chemical nature of the ligand; the cooperation of coregulators (coactivators and corepressors); the status of chromatin (e.g., nucleosome modiﬁcation) in the vicinity of the HRE; posttranslational modiﬁcation (PTM) of the receptor; nucleocytoplasmic shuttling of the receptor; and interaction with neighboring transcription factors in composite clusters of DNA elements.

In the newly discovered, non-genomic modes of action, the NRs perform functions that are not linked to DNA-binding events. These novel functions have been clearly 6.3 Structure of Nuclear Receptors (NRs) 257 established for many NRs, a pool of which can be found in close association with the cell membrane and with cell organellesasforexample,inthemitochondria.

& Non-genomic mode: — NRs receive signals from other signaling paths, and transmit signals to other signaling paths

This pool of receptors conveys signals to protein components of other signaling pathways, so as to allow crosstalk between different signaling paths (Section 6.7). The direct coupling to other signaling paths provides for a diverse set of rapid reactions as, for example, the production of second messengers, the activation of þ protein kinases, and Ca2 inﬂux. Another twist to the non-genomic functions of NR ligands relates to the ability of, for example, estrogen to bind to a transmembrane receptor of the GPCR class (see Chapter 7), triggering a set of signaling events that has hitherto not been linked to NR functions.

6.3 Structure of Nuclear Receptors (NRs)

Summary Nuclear receptors are of a modular structure, comprising domains responsible for DNA binding, dimerization, ligand binding, and transactivation. The DNA- binding domain interacts with the HREs which are composed typically of two hexamer sequences, and homo- or heterodimers form on the HREs. Binding of the ligand to the ligand-binding domain induces a conformational change in this domain and in the transactivation domain, which leads to the exposure of binding surfaces for coactivators and transactivation. Results of structural studies have suggested that NRs are very flexible proteins which can exist in various conformational states, each with different regulatory properties. The various domains of NRs interact with each other, so as to allow coupling between DNA binding, ligand binding, and transactivation.

The NRs are grouped into a large superfamily comprising at least 48 members in humans. Some 25 members of this superfamily are currently considered as “orphan receptors,” because they have no known ligand. As with other transcriptional regulators, the NRs exhibit a modular structure with different regions corresponding to distinct functional domains. At the level of the primary structure the NRs can be subdivided into ﬁve different domains (Figure 6.2). A typical NR contains a variable N-terminal A/B region, a highly conserved domain responsible for DNA-binding (region C), a linker region D containing nuclear localization signals, the ligand-binding and dimerization domain (region E), and an F region which is not found in all receptors. 258 6 Signaling by Nuclear Receptors

Figure 6.2 Domain structure of the nuclear receptors (NRs). Functional domains of NRs are portrayed in a one-dimensional, linear fashion. Not all NRs carry two transactivation domains.

& Domains of NRs: — DNA binding — Ligand binding — Transactivation: AF-1, AF-2 — Variable domains

Nuclear receptors are regulatory transcription factors that receive and transmit signals and bind to distinct DNA elements of the target genes. In order to fulfill their regulatory function, they need to communicate with the transcription machinery in a process called transactivation (Section 4.3.6). Two types of transactivation functions have been identified on NRs: activation function 1 (AF-1), which is located in the A/B region; and activation function 2 (AF-2), which is located in the E region and sometimes in the F-region. Until now, most information on the functions of the different domains has been obtained from studies on isolated domains that fold independently of the remainder of the protein. However, the first structure of a complete heterodimer of two intact NRs in complex with DNA has revealed distinct interactions and conformational coupling between the domains (Section 6.3.5). These structural studies can be taken as exemplary for other NRs, and it is now generally accepted that NRs are very flexible proteins that can exist in a variety of conformational states, each with different regulatory properties. Which conformation is predominant may be modulated by many factors, such as the PTMs of the receptor, the exact sequence of the HRE, the chemical nature of the ligand, and the nature of associated coregulators.

6.3.1 DNA-Binding Elements of NRs: HREs

Thesteroidhormonereceptorsaresequence-speciﬁcDNA-bindingproteinswhose cognate DNA elements are termed HREs. All HREs known to date possess a common structure, being composed of two hexamer sequences – the half-sites of the HRE – each providing a binding site for a receptor monomer. As a consequence, receptor dimers form on HREs, either as homodimers ofthesamereceptor,oras heterodimers between different receptors (see Figure 6.3a). The receptors for steroid hormones, for example, bind as a homodimers to the twofold symmetrical recognition sequence, whereby the receptor is already dimerized in solution. 6.3 Structure of Nuclear Receptors (NRs) 259

Figure 6.3 Structure of hormone recognition GR, glucocorticoid receptor; (c) Binding of a elements (HREs) and binding of receptor heterodimeric receptor to a DNA element with dimers. (a) Shown is the consensus sequence direct repeats of the recognition sequence, of the HREs of the RXR heterodimers and whereby the 50 side of the HRE is occupied by different arrangements of the hexameric half- the receptor for 9-cis-retinoic acid (RXR). RAR, site sequences. n indicates the number of base receptor for all-trans-retinoic acid, T3R, pairs that lie between the two hexamers; receptor for the T3 hormone; PPAR, (b) Binding of a homodimeric receptor to a peroxisome proliferating activated receptor; twofold symmetric palindromic DNA element. VDR, receptor for vitamin D3.

& NRs bind to HREs as: — Homodimers — Heterodimers

Currently, very few NRs are known for which the HRE contains only a single copy of the receptor binding sequence; these receptors bind as monomers to the cognate HRE.

The NRs for all-trans-retinoic acid, for 9-cis retinoic acid, for the T3 hormone and for the vitamin D3 hormone usually bind as heterodimers on the DNA. One of the partners in the heterodimer is in most cases the receptor for 9-cis-retinoic acid (RXR). The identity of an HRE is determined by the sequence, polarity, and distance of the hexamers. The mutation and duplication of an ancestral recognition sequence have allowed the creation of many and various DNA elements during the course of 260 6 Signaling by Nuclear Receptors

Figure 6.4 Consensus sequence of the glucorticoid receptor response element. So 2007 [2] figure 7. Reproduced from Pubmed Central. evolution, the sequence, polarity and distance of which are characteristic for a given hormone receptor or receptor pair. The half-site of an HRE is often arranged as a palindrome or a direct repeat (Figure 6.3a); furthermore, everted repeat arrange- 0 0 ments of half sites such as 5 -ACTGGA(N)68AGGTCA-3 are also known. For a given receptor dimer, optimal spacings of the half-sites exist, while the number of base-pairs between the half-sites is another characteristic feature of the HRE. Regions adjacent to the core HRE can also be involved in binding of the receptor dimer on the HRE. The structure of the complete PPARc-RXR heterodimer in complex with DNA has shown that the A/B region of PPARc makes contacts with a 50 extension of the core HRE. These contacts appear to be required for the polar arrangement of the heterodimer on the direct repeat of the HRE [1]. The sequence requirements for the recognition of a half-site by a given receptor are quite complex. The NR typically regulates a large number of genes each associated with the HRE; as an example, more than 500 different target genes have been identified in a human cell line that contain binding sites for the glucocorticoid receptor (GR). Sequence comparisons of the GR binding sites have shown that only five positions among the 15-bp GR-responsive element are absolutely conserved (Figure 6.4) [2], and therefore only consensus sequences can be proposed for the half-sites. Interestingly, certain HREs are strictly conserved among species and do not tolerate variations in either HRE sequence and/or arrangement (see also Section 6.5.6). A detailed structural and biochemical analysis of GR bound to different GR-responsive elements has revealed a novel aspect of the half-site sequence specificity [3]. Variations of half-site sequence by as little as a single base- pair can differentially affect the conformation of the bound receptor and its interaction with coregulators. Accordingly, the DNA of the HRE is ascribed the function of an allosteric modulator of the NR. Different HRE sequences can induce different conformational states of the receptor, and thereby induce distinct regulatory effects on the transcription of the associated target gene. One further complication of the interpretation of HRE specificity and in vivo function arises from the fact that the HREs typically are found in composite regulatory regions, either close to promoters or in enhancers.

6.3.2 The DNA-Binding Domain of NRs

Within the family of NRs, the DNA-binding domain is the most conserved structural element and is located in region C of the primary structure. The DNA- binding domain possesses structural elements that mediate the speciﬁc recognition 6.3 Structure of Nuclear Receptors (NRs) 261 of the HRE, and contribute to the dimerization of the receptor on the HRE. The core of the DNA-binding domain includes a span of 70–80 amino acids, in which all information for the speciﬁc recognition of the cognate half-site is contained. & DNA-binding domain: — Independently folding — Two Zn2Cys4 motifs — a-Helices for readout of HRE

Within the core of the DNA-binding domain are two Zn2Cys4-motifs which serve to position a recognition helix in the major groove of the DNA. Via the recognition helix, speciﬁc contacts are formed with the hexamer half-site of the HRE, and the two Zn- motifs then assume nonequivalent positions in the DNA-binding domain. Whilst the N-terminal Zn-motif participates in positioning of the recognition helix and interactions with the sugar-phosphate backbone, the C-terminal Zn-motif serves to position the dimerization region and contact the phosphate backbone of the DNA (Figure 6.5). An important regulatory function has been ascribed to a sequence region named the “lever arm,” which connects the recognition helix with the dimerization region. The position of the lever arm of the GR appears to be highly sensitive to the exact sequence and spacing of the recognition element [3]. In this way, the identity of the HRE can inﬂuence receptor dimerization, which in turn is proposed to affect the interaction with coregulators and the transcriptional regulatory activity.

6.3.3 Ligand-Binding Domain

The region E with the ligand-binding domain harbors three important functions:

Homo- and heterodimerization Binding of ligands, both agonists and antagonists Activation function-2 (AF-2): binding of coactivators and corepressors, contacts to mediator and to RNA Pol II

Figure 6.5 Structure of a fragment of a RXR- are contacted by an a-helix of each of the

T3R-heterodimer bound to the HRE AGGTCA receptors in a nearly identical manner. (N)4AGGTCA.). The DNA-binding domains of Different structural elements of each of the the RXR-T3R heterodimer bind in a polar monomeric receptors are involved in the manner on the HRE, with RXR occupying the 50 dimerization process, leading to a polar side of the HRE. Both hexameric sequences lie configuration of the monomers on the DNA. on the same side of the DNA double helix and 262 6 Signaling by Nuclear Receptors

& Ligand-binding domain: — Variable size — Variable specificity — Couples ligand binding to transactivation — Ligand induces conformational change of helix 12

6.3.3.1 Ligand Binding The ligand-binding domain has been at the focus of structural studies of NRs, as it is the site on which the ligands bind and coregulator-derived motifs interact. The crystal structures of many ligand-binding domains have been resolved and have demonstrated a similar overall structure [4]. A schematic representation of the canonical ligand-binding domain of NRs, with an example of the receptor for 9-cis- retinoic acid (RXR) is shown in Figure 6.6. The ligand-binding domain is formed from 12 a-helices numbered from H1 to H12 that are arranged around a central hydrophobic pocket, with helices 3, 7, and

Figure 6.6 Structural changes in the ligand- E of apo-RXRa is depicted in green and yellow, binding domain of RXR on binding of 9-cis- and domain E of the binary complex in blue retinoic acid. The models of domain E of apo- and red. The gray arrows indicate the structural RXRa and the binary complex of RXRa and rearrangements of helices 11, 12, and the 9-cis-retinoic acid were superimposed. Domain N-terminus of helix 3. Reproduced from [5]. 6.3 Structure of Nuclear Receptors (NRs) 263

10 providing amino acid residues that shape the pocket. A ligand-binding pocket which accommodates the ligand is located in the bottom half of the structure. Thesizeoftheligand-bindingpocketcanvaryconsiderablyamongdifferent receptors. Notably, receptors with specific ligands (e.g., T3R) bind the ligand in a small pocket that is tailored to bind only the specificligand,theT3 hormone, and only small deviations from the T3 structure will be tolerated. In contrast, the ligand- binding pocket of the peroxisome proliferator activator receptor (PPAR), is of a much larger size. This receptor binds a wide variety of endogenous ligands such as fatty acids, with rather low affinities. Apparently, the ligand-binding cavity of PPAR has been adopted to bind hydrophobic ligands of different size. The many structures of ligand-binding domains with bound agonists, semi-agonists and antagonists have revealed a high conformational flexibility and variability of this domain. Notably, different ligands can induce distinct conformations of the binding pocket, with the shapes and sizes of the binding pocket being highly inducible, both in size and in receptivity, to molecules that are unrelated to endogenous ligands.

6.3.3.2 Ligand Binding and AF-2 Function Ligand binding induces a conformational change in the ligand-binding domain that is crucial to the AF-2 function of the receptors [4]. The C-terminal-most helical segment, helix 12 (H12), is the major architectural feature associated with AF-2 function, and can undergo dramatic shifts in position in response to the molecule in the pocket. Other a-helices in the ligand-binding domains also shift in positions in subtle, but still meaningful, ways that can impact on receptor activation. The ligand-induced conformational change of H12 is illustrated in Figures 6.6 and 6.7). H12 is amphipathic, and possesses a hydrophobic and a hydrophilic face. Whereas, in the unliganded RXR, H12 projects away from the body of the

Figure 6.7 Conformational changes in ligand-activated receptor uses H12, as well as retinoid X receptor (RXR) associated with additional features in H3 and H4, to promote ligand binding and receptor activation. The the binding of short helical LXXLL (where L is unliganded RXR-ligand-binding domain (LBD) leucine and X is any residue) elements from changes its protein conformation, including coactivator protein (shown in purple) de Lera helix 12 (H12) (shown in red) positioning 2007, figure 2. Reproduced with permission of upon binding to an agonist (SR11237). The Nature Publishing Group. 264 6 Signaling by Nuclear Receptors

ligand-binding domain, in the liganded structure the helix reorients such that the hydrophobic residues face inward and form part of the ligand-binding pocket. In contrast, the conserved polar residues face outwards and form a surface for the binding of coactivators. Both, structural data and mutational analyses have shown that H12 is directly involved in transcriptional activation. Indeed, it is at the center of AF-2 function and thus plays a key role in transactivation and transrepression. The ligand- induced structural rearrangements of H12 provide a binding platform for the association of coregulators that form essential parts of the transcription regulation function of the NRs.

6.3.3.3 Switch Function of Ligand Binding The NRs are involved in the regulation of numerous physiological processes and are therefore important medical targets. An enormous number of synthetic compounds are available that act as agonists, mixed agonist/antagonists or as pure antagonists of the natural ligands. The ligands of NRs exert their regulatory function by recruiting distinct coregulators, classiﬁed as coactivators or corepressors, to the DNA-bound receptor, and it is now well established that the ligand can be ascribed a switch function for coregulator binding. As illustrated in Figure 6.8, the structure of the ligand-binding domain of ER in the agonist- and antagonist-bound forms shows distinct conformations of helix H12 that dictate which type of coregulator can bind. The coregulators bind to nuclear receptors typically using one or more LXXLL motifs within their polypeptides, and associate with H12 via a charge clamp that supports this motif’s binding as a short a-helix to the surface of the ligand-binding domain. The

Figure 6.8 H12 geometry and activation PPARa LBD, some antagonist ligands, such function-2 (AF-2) function is ligand-dependent. as GW6471, prevent the AF-2 helix (H12, The ligand-mediated exchange of coactivators shown in red) from assuming the active and corepressors on NRs. The peroxisome conformation, resulting in a larger crevice that proliferator-activated receptor a (PPARa) can accommodate a corepressor (SMRT, binding to coactivator versus corepressor shown in blue) segment with the LXXXIXXXL segments can be governed by specific ligands. motif. Adapted from Steinmetz AC, Renaud JP, Whereas, agonist ligands such as GW731 Moras D. Binding of ligands and activation of recruit coactivator NR-box elements from transcription by nuclear receptors. Annu Rev coactivators (SRC-1, shown in purple) to Biophys Biomol Struct.2001;30:329-59. 6.3 Structure of Nuclear Receptors (NRs) 265 structural studies suggest that an agonist can be viewed as a molecule that enhances the interactions of ligand-binding domains with one or more coactivator LXXLL motifs and therefore leads to transcriptional activation in a cell-based assay. By contrast, antagonists position H12 to physically block the binding site on the coactivator, or otherwise do not support this binding. Antagonist-function may be also mediated by the ability of ligands to recruit corepressors, such as NcoR or SMRT (Section 6.4.2), to ligand-binding domains via their related leucine-rich motifs. The binding of coactivator and corepressor peptides to PPARc is shown in Figure 6.8.

6.3.3.4 Promiscuous Ligand Binding A number of NRs are known that are highly promiscuous in ligand binding, or for which ligands have not yet been identified. Some of these receptors show constitutive activity; examples include receptors involved in drug disposition such as liver X receptor (LXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR). An analysis of the ligand-binding domain and the AF-2 domain of these receptors show a similar overall structure of the LBD. However, the AF-2 surface and the ligand-binding pocket show great variations. For receptors involved in detoxification, such as PXR and CAR, the ligand-binding pocket is very large and can accommodate a variety of hydrophobic compounds. Furthermore, the ligand- binding pocket is flexible and contains some polar residues.

6.3.4 Transactivation Functions of the NRs

Most NRs harbor two regions with potent transcription activation function. The activation function 1 (AF-1) is located in the highly variable A/B region and mediates a ligand-independent transactivation. It harbors sites for PTMs (e.g., phosphorylation) and interaction sites for coactivators. The AF-1 domain appears to be intrinsically disordered [1], and may become structured only upon interaction with coregulators or other components of the transcription machinery. The AF-2 function is located within the ligand-binding domain. This function refers to the recruitment of coactivators in a ligand-dependent fashion, and it is associated with structural rearrangements of helix H12 as described above, as well as other structural elements of the E region. In some receptors, ligand-dependent rearrangements within the ligand-binding domain provide interaction sites for transcriptional repressors. Both types of coregulator link the nuclear receptors directly to large chromatin-modiﬁcation complexes.

& AF-1: — Located in A/B-region — Mediates ligand-independent transactivation — Contains multiple phosphorylation sites — Contains interaction sites with other proteins — Interacts with E/F-region 266 6 Signaling by Nuclear Receptors

AF-2: — Part of E-domain — Undergoes ligand-dependent conformational change — Contains binding sites for coactivators and corepressors

6.3.5 Structure of an Intact NR Complex on DNA

Most structural studies on NRs have employed isolated domains to obtain insights into the structure–function relationships of this receptor class. However, the NRs are multidomain transcriptional regulators that have many different interaction partners and appear to exist in different allosterically controlled conformations. For a deeper understanding of the regulatory functions of NRs, it is necessary to realize how the different domains of NRs interact within the homo- or heterodimer, and how the various ligands affect these interactions. The ﬁrst information on these crucial questions can be obtained from the structure of an intact, heterodimeric PPARc–RXR complex on a cognate DNA element [1]. The structure of this complex is shown in Figure 6.9. PPARc is activated by a variety of lipophilic metabolites, including drugs such as thiazolidi- nedion. This subclass of NR regulates adipocyte differentiation, lipid storage and release, and it is linked to insulin signaling pathways. The retinoid X-receptor is activated by 9-cis-retinoic acid and is a common partner in heterodimeric

complexes with, for example, T3R, PPAR, and RAR. The DNA response element used for structure determination is composed of two copies of the same hexamer AGGTCA which are separated by a single nucleotide spacer and arranged as a direct repeat. Interestingly, both receptor monomers bind to the same hexamer sequence. The following features of the intact structure of the heterodimer bound to DNA are important for structure–function relationships of NRs:

PPARc and RXRa display half-site selective binding that results in a polar arrangement of the DNA-binding domains. PPARc occupies the 50 part of the HRE arranged as a direct repeat. The C-terminal extension of PPARc makes signiﬁcant contacts to a 50-extension site on the HRE, and the two DNA binding domains interact within the minor groove of the spacer nucleotide. The HRE appears to function as a clamp that allosterically induces interactions between the two DNA-binding domains. There is an intense cooperation between the receptor domains. Heterodimeriza- tion is mediated by interactions between the two DBDs, between the LBDs and – as a completely novel feature – between the LBD of PPARc and the DBD of RXR. The latter feature shows that the LBD of PPARc can affect the DNA-binding properties of the other monomer through domain–domain interactions. RARa and PPARc with agonistic ligands bound assume the active conformation of the ligand-binding domain. This conformation consists of both receptors having their H12 appropriately positioned to allow the binding of LXXL motifs of 6.3 Structure of Nuclear Receptors (NRs) 267

Figure 6.9 Overall structure of the PPAR-c-- and the coactivator LXXLL peptides are in light -RXRa complex on the PPAR-c recognition blue and purple; (b) Sequence of the DNA element. (a) Orthogonal views are shown in element used for crystallization. Chandra 2011 which RXR-a is blue and PPAR-c is red. The [1], figure 1. Reproduced with permission of ligands rosliglitazone and 9-cis-retinoic acid are Nature Publishing Group. shown in green, the Zn(II) ions are light gray,

coactivators. The two individual LXXL motifs appear to bind independently to the LBDs of PPARc and RXRa. The A/B segment of PPARc, which harbors a potentAF-2 function, could not be visualized in the complex. This region of PPARc appears to be highly mobile and may be intrinsically unstructured. Overall, the structural studies show that there is a tight coupling of the ligand- binding domain to other receptor segments, both within its own polypeptide and within the heterodimeric partner. Importantly, the ligand-binding domain may modulate the DNA-binding properties of the receptor dimer, depending on the manner in which the complex is organized. Distinct NR ligands could modulate the response element afﬁnities in a graded manner, which may contribute to selective gene regulation. 268 6 Signaling by Nuclear Receptors

6.3.6 Orphan Nuclear Receptors

A NR is considered to be an orphan as long as a natural ligand has not yet been identified that binds in a functionally relevant manner [6]). The binding of fatty acids has been demonstrated for some orphan receptors, such as hepatocyte NR-4, but the relevance of these interactions remains uncertain. Most of the NRs classified to date as orphan receptors adhere to the classical domain structure discussed in Section 6.3.4, with only few exceptions (e.g., the small heterodimerization partner; SHP) that lack a DNA-binding domain and cannot act alone to bind DNA. In contrast to ligand-regulated NRs, the orphan NRs often display a constitutive transcriptional activity. Even with an empty ligand- binding domain or with a nonspecific ligand bound, Helix H12 – which is crucial for activation – is prepositioned for maximal activation. The mechanisms of regulation by orphan NRs are not well characterized, and it is assumed that these receptors function in cooperation with other NRs or with other transcription factors. One recent example of cooperation with other transcriptional regulators is the retinoic acid-related orphan receptor a,RORa [7]. This receptor exerts a repressive effect on the transcription regulatory activity of b-catenin which is an essential component of the Wnt signaling pathway (see Section 16.9). The transrepression of b-catenin by RORa is dependent on the phosphorylation of RORa on Ser35, which is catalyzed by protein kinase Ca.ThePKCa-catalyzed phosphorylation of RORa enhances the transrepression of b-catenin and leads to an attenuation of Wnt signaling. This example illustrates how a PTM in the form of phosphorylation is used to regulate NR function, and also shows how orphan NRs canreceiveandtransmitsignalswithouttheneedofligandbinding.

6.4 Transcriptional Regulation by NRs

Summary The genomic functions of NRs can be described in terms of transcription activation and transcription repression. In transcription activation, ligand-bound NRs recruit chromatin-modifying and chromatin-remodeling complexes to relieve repressive structures. Furthermore, the receptors participate at the start of transcription by communicating with chromatin structures at the promoter, with Mediator, and with the transcription apparatus. Chromatin modification and restructuring includes the recruitment of coactivators such as the SRC/p160 family of coactivators that recruit further chromatin-modifying complexes with histone acetyltransferase and lysine methyltransferase activities, among others. Transcription repression is exerted primarily through an association of corepressor complexes with unliganded NRs. These complexes often harbor histone deacetylase activity, and ligand binding to the receptor then induces dissociation of the corepressor. 6.4 Transcriptional Regulation by NRs 269

Most of the functions of NRs can be described in terms of the activation and repression of transcription. The outcome of these “genomic” actions of NRs is determined in a gene- and cell type-speciﬁc fashion by a series of variables:

The exact sequence of the HRE; the neighborhood of the HRE; the cooperation with other transcriptional activators; the availability of other NRs to form heterodimers; the availability of coregulatory proteins, mainly coactivators and corepressors; PTMs of the receptors; and a dynamic distribution of the receptors between the nucleus and the cytoplasm.

Overall, NRs must fulﬁll the following main tasks during transcription activation:

The selection of target genes by speciﬁc binding to the HRE. Recruitment of chromatin-modifying and chromatin-remodeling complexes to relieve repressive structures. Participation at the start of transcription by communication with chromatin structures at the promoter and with the transcription apparatus.

The genomic functions of the NRs are governed by the same processes that have been recognized as being essential for other transcriptional activators or repressors. The basic steps required for transcriptional activation have been outlined in Chapter 4, and the same types of proteins are necessary in order for the NRs to perform their regulatory function. The functions of NR domains in the major steps of transcription regulation are summarized in Figure 6.10.

6.4.1 Coregulators in NR Function

Transcriptional regulation by NRs requires the recruitment of protein complexes that mobilize and reorganize chromatin, both around the HRE and at the promoter of target genes. Furthermore, NRs recruit enzyme complexes that introduce covalent modiﬁcations into histones, preparing the chromatin at target sites for deposition of the transcription machinery and the onset of transcription. The proteins directly recruited by NRs to target genes are termed coregulators and include coactivators and corepressors (Section 4.3.3). Often, the DNA-bound NR and its associated coregulator impacts chromatin structures at promoters of target genes that are located far away from the HRE. Coregulators are found as part of large protein complexes associated with a variable set of proteins that do not directly contact the NRs but rather contribute to the activating or repressive function of the coregulator. In the case of coactivators, the associated proteins have also been named secondary coactivators. To a large part, the subunit association of coregulators is regulated by PTMs. To date, more than 350 coregulators have been identiﬁed, attesting to their central role in 270 6 Signaling by Nuclear Receptors

Figure 6.10 Functions of nuclear receptor (HDAC) and histone acetyltransferase (HAT) (NR) domains. The domains A/B, C, E and F of activities, respectively, to the HRE region. Red the NRs are involved in multiple arrows indicate intramolecular interactions. protein---protein interactions and are subject to TRAP, thyroid hormone receptor activating regulatory modifications, as indicated. Most protein; DBD, DNA-binding domain; LBD, important are the corepressor and coactivator ligand-binding domain; PTM, posttranslational complexes that direct histone deacetylase modification. transcriptional regulation. Many of these coregulators have been identiﬁed from their inﬂuence on NR-mediated regulation, and most of them are also involved in transcriptional regulation by other transcription factors. The variable subunit composition and PTM patterns of these complexes allow for a huge variability and complexity of coregulators [8]. Typical coregulator complexes contain about six different subunits, each of which may contain six to eight different PTMs. Therefore, coregulators provide a deep reservoir of potential complexity to accommodate a broad range of distinct biological states and functions. & NRs interact with and recruit: — Mediator — Coactivators of the SRC-1/p160 and/or TRAP family — Corepressors: NcoR, SMRT — Proteins of the SWI/SNF family — Histone acetylases: GCN5, CBP/p300 — Histone deacetylases — Arginine methylases: CARM — Lysine methylases — Protein kinases — Ubiquitin ligases: E6AP 6.4 Transcriptional Regulation by NRs 271

6.4.2 NR Coactivators

Coactivators are proteins that are directly recruited to target genes by NRs and enhance gene expression. Recruitment of the coactivator is typically mediated by the receptor’s AF-1 or AF-2 domain, and it is usually – but not always – ligand- dependent.

& Coactivators Src1/p160; TRAP220: — Contain LXXLL motif — Are required for transactivation — Interact with ligand-bound receptor

Two examples of coactivators that bind to the AF-2 domain will be discussed in the following, namely the SRC/p160 family of coactivators and the TRAP complex.

6.4.2.1 SRC/p160 Family of Coactivators This coactivator family comprises, among others, the steroid receptor coactivators 1 to 3 (SRC-1, SRC-2, and SRC-3) as well-characterized members. The SRC coactivators are multifunctional proteins that are central regulators not only in NR signaling but also in the signaling pathways of other transcription factors such as NFkB, AP-1, E2F1, and the tumor suppressors p53 and Rb (see Chapter 16). SRCs have been shown to function in transcription initiation, elongation, RNA splicing, receptor and coregulator turnover, and even mRNA translation. The members of the SRC/p160 coactivator family fulﬁll two main functions during NR-mediated transcriptional activation:

Interaction with the receptor in a ligand-dependent fashion. Recruitment of chromatin-modifying enzymes such as histone acetylases (CBP/ p300), lysine methyltransferases and arginine methyltransferases (e.g., CARM1/ PRMT1).

The domain structure and interaction partners of SRC-3 are shown in Figure 6.11. The most important function for NR regulation is located in the central receptor interaction domain (RID), which carries three LXXLL (where X is any amino acid) motifs. These motifs form amphipathic helices and are essential for NR interaction and activation, both in ligand-dependent and ligand-independent receptor activation. In ligand-dependent activation, agonist binding to the ligand-binding domain induces a conformational change that includes the reorganization of H12 (see Figures 6.6 and 6.7), thus creating a binding surface for the RID with its LXXLL motifs. In contrast, antagonists stabilize a conformation of the ligand-binding domain where H12 occupies a position that covers the coactivator binding surface. 272 6 Signaling by Nuclear Receptors

Figure 6.11 Schematic representation of SRC/ bHLH---PAS domain that functions as a DNA- p160 coactivator family and NCoR corepressor binding or dimerization surface in many family. Domain structure and interactions. transcription factors; (b) NcoR: the nuclear (a) SRC: the location of the receptor receptor (NR) interaction region that contains interaction domain (RID) in the SRC is the “CoRNR box” motifs near the C-terminal indicated. RID and activation domain 1 (AD1) region is indicated. Near the amino-terminal each contain three LXXLL motifs. The specific region is the deacetylase activation domain domains for interaction with P/CAF, CBP/ (DAD) that interacts with and activates p300, as well as the histone acetyltransferase HDAC3, required for repression by T3R. domain, are indicated. Located near the Reproduced from [9]. amino-terminal region is the highly conserved

Another essential function of SRC coactivators is the recruitment of enzymes that catalyze covalent modifications of chromatin components. The C-terminal region of SRC coactivators harbor two domains – termed activation domains 1 and 2 (AD1, AD2) – that are responsible for the coactivation function. AD1 contains binding sites for histone actetylases such as CBP/p300 (see Section 4.5.2.1) whereas AD2 is responsible for recruitment of the arginine methylase PRMT1 (see Section 4.5.3.2). Furthermore, the C-termini of SRC coactivators harbor intrinsic histone acetylase activity. A large number of PTMs have been reported for SRCs, including phosphorylation, methylation, acetylation, ubiquitination, and sumoylation. As an example, more than 50 unique sites for PTMs have been identified on SRC-3 that form a type of modification code which specifies, in dynamic fashion, the complement of proteins associated with the coactivator. This in turn sets the parameters for the gene-specific and timely variable function of the coactivator [10]. Coactivators typically exist as multisubunit complexes that can transmit signals from the environment by supplying a variety of enzymatic activities to the genes they regulate. For instance, the SRC-3 coactivator exists in a complex that includes protein kinases, ATPases, methyltransferases, lysine acetyltransferases and for the E3 ligase E6-AP. 6.4 Transcriptional Regulation by NRs 273

6.4.2.2 TRAP Complex, Mediator Interaction studies have identiﬁed another type of coactivator that has been located to another protein complex, then named the TRAP complex (TRAP; thyroid hormone receptor-activating protein). Later, this complex was shown to represent a distinct type of type of Mediator complex (Section 4.2.6). The TRAP/Mediator complex has the task of forming a bridge between the DNA- bound transcription factors and the core transcription apparatus. It contacts the core transcription complex and interacts with the AF-2 domain in a ligand- dependent manner via a LXXLL motif that is found in the TRAP220 component.

6.4.2.3 Variability of Coactivator Recruitment A medically important aspect of coactivator recruitment is the ability of distinct synthetic agonists to recruit a speciﬁc set of coactivators leading to distinct physiological responses (Figure 6.12). This is based on the ability of synthetic

Figure 6.12 Ligand-selective recruitment sensitivity and adipogenesis. In the presence of different coactivators. The PPAR---RXR of the ligand 9-fluorenylmethyloxycarbonyl heterodimer recruits the coactivator SRC-2 in (FMOC)-L-leucine, the coactivator SRC-1 is response to binding the synthetic ligand recruited, resulting in insulin sensitivity only. thiazolidinedione (TZT), inducing insulin 274 6 Signaling by Nuclear Receptors

agonists to induce distinct conformations of the ligand-binding domain that allow the binding of distinct coactivators. The concept of tissue- and gene-specificNR ligands has been developed as an important new step towards a specific modulation of NRs. For estrogen receptors, these compounds have been termed collectively as SERMs (selective estrogen receptor modulators) that demonstrate both organ- and tissue-specific activities. For example, the estrogen analogs tamoxifen and raloxifene behave as estrogen receptor antagonists in breast tissue, and as agonists in bone. 6.4.3 Corepressors of NRs

The corepressors of NRs serve an important role in negatively regulating receptor- dependent gene expression. The repressive function of corepressors is exerted primarily through interaction with unliganded NRs. The current understanding of how NR corepressors mediate transcriptional repression derives mostly from repressors that are recruited by the T3 receptor (T3R) and the retinoic acid receptor (RAR).

& Corepressors: — Recruit HDACs and lysine methylases to induce a silent chromatin state.

In the absence of a ligand, these receptors recruit two large proteins – nuclear corepressor (NcoR), and its homolog, “silencing mediator for retinoic and thyroid hormone receptors” (SMRT). Both corepressors bind to the surface of the ligand- binding domain via the repeated LXXLL motifs in the C-terminal region. Upon

ligand binding, the corepressors dissociate from the receptor and enable T3R and RAR to associate coactivators and stimulate gene expression (Figure 6.13). Both corepressors form part of large repressive complexes, as for example, the mSin3 or NuRD complex that contain histone deacetylase subunits. The histone deacetylase then helps to maintain the repressed state of the chromatin.

6.5 Regulation of Signaling by Nuclear Receptors

Summary The function of NRs in transcription regulation is regulated primarily by the concentration of the cognate ligand, the nature of the HRE, the availability of coregulators, and by PTMs such as phosphorylation, acetylation, methylation, ubiquitination, sumoylation, and lipidation. The PTMs have been shown to modulate the signaling function of NRs in terms of recruitment of coregulators, DNA binding and subcellular distribution, among others. Furthermore, the PTMs mediate crosstalk with other signaling pathways. 6.5 Regulation of Signaling by Nuclear Receptors 275

Figure 6.13 Model of repression and are removed and coactivators (e.g., the activation of T3R. In the absence of the T3 SRC/p160 complex) bind to the receptor hormone, a heterodimeric RXR---T3R receptor heterodimer. The histone acetylase activity of is bound at the T3-responsive element (TRE), the associated proteins helps to induce a establishing a basal repressed state by the transcription competent state around the recruitment of corepressor complexes promoter. In collaboration with the mediator containing histone deacetylase activity. In the complex (TRAP complex) and chromatin- repressed state, the promoter region is remodeling enzymes, nucleosomes are thought to be covered by nucleosomes which removed from the promoter region, which prevents the binding of RNA polymerase. In allows the binding of RNA polymerase and the presence of T3 hormone, the corepressors transcription initiation.

In the classical, genomic pathway, NRs receive signals and pass them on to the level of transcription. The steps involved in this signaling process are subject to a variety of regulatory mechanisms that ultimately determine the magnitude and dynamics of transcriptional activation and its cell type speciﬁcity.

6.5.1 Regulation at the Level of Ligand Concentration

One main determinant of NR signaling is the concentration of the ligand available for binding, and which can be regulated in many ways (for details, see textbooks on 276 6 Signaling by Nuclear Receptors

hormone action). For example, a feedback regulation via the circulating hormone concentration is important in the hypothalamus–pituitary system of the brain, where feedback inhibition at various levels is used to prevent overproduction of, for

example, steroid hormones or the T3 hormone. & Regulation of ligand concentration of NRs occurs via: — Secretion, transport and storage — Modification — Feedback regulation of biosynthesis

6.5.2 Regulation by PTM

Nuclear receptors are the targets of multiple PTMs that serve to modulate both their genomic and nongenomic functions in response to hormonal and growth factor signals, and to link NR signaling to other signaling pathways. Furthermore, PTMs serve to regulate the subcellular distribution of NRs. The following PTMs have been found to regulate NR functions:

Phosphorylation on Ser/Thr and Tyr residues Acetylation, methylation on Lysine residues Ubiquitination Sumoylation Palmitoylation

Most PTMs are found in the AF-1 region and in the hinge region that separates the DNA-binding domain from the ligand-binding domain. An example, of estrogen receptor alpha (ERa) on the multiplicity of NR modiﬁcations is illustrated in Figure 6.14.

6.5.2.1 Regulation by Phosphorylation The phosphorylation sites comprise not only Ser/Thr sites but also Tyr sites, and are mainly found in the AF-1 region of the receptors. The consequences of phosphorylation for the receptor proteins are varied and have been shown to inﬂuence many aspects of nuclear hormone signaling. One of the best-studied examples is that of ERa and ERb, which can be phosphorylated at multiple sites throughout the whole protein and within all major structural domains [11]. The exact role of phosphorylation at individual or multiple sites is not well known, but it is possible that a phosphorylation code exists which is used in a dynamic and combinatorial fashion to regulate the different receptor functions. A broad spectrum of protein kinases, including MAPK kinases, cyclinA-CDK2, Akt kinase, PI3 kinase, JNK and Src kinase, has been implicated in ERa phosphorylation, indicating that many different signals may induce ER phosphorylation and modulate receptor function. 6.5 Regulation of Signaling by Nuclear Receptors 277

Figure 6.14 Post-translational modifications region, and ligand-binding domain (LBD). For (PTMs) of ERa. Multiple phosphorylated sites the expanded hinge region, various PTM target in ERa have been identified by a variety of residues are shown to illustrate the multiplicity approaches (for details see Ref. [11]). These of PTMs in that region. The consensus are shown schematically in the figure, which sequence for Lysine methylation by SET1 depicts different structural and functional (Section 2.7.1) is boxed in yellow. Reproduced domains of human ERa: activation function 1 from [12]. (AF-1), DNA-binding domain (DBD), hinge

& Phosphorylation affects NR signaling via modulation of: — DNA binding and transactivation function — Ligand requirement — Nuclear–cytoplasmic distribution — Membrane localization

The most extensively studied phosphorylation site of ER is Ser118. Such phosphorylation results in a ligand-independent activation of the receptor and allows for the transcription of ER-target genes in the absence of estrogen. Furthermore, Ser118 phosphorylation has been implicated in ER protein stability. The protein kinases involved appear to be kinases of the MAPK cascade (Erk1/2) and Cdk7. The MAPK cascade (see Chapter 12) transmits signals from growth factor-stimulated pathways and from Ras proteins, providing a link between growth factor signaling and NR signaling.

Regulation by Lysine Acetylation and Methylation Nuclear receptor acetylation by histone acetyltransferases has been located mostly to the hinge region. Lysine acetylation of ERa by the acetyltransferase p300 (Section 4.5.2) appears to increase estrogen-dependent activation, suggesting a role for ERa acetylation in ligand sensitivity. Since NR coactivators such as SRC contain binding sites for 278 6 Signaling by Nuclear Receptors

acetyltransferases, the recruitment of these enzymes to the receptor may be mediated by the associated coactivator. Nuclear receptor methylation at lysine residues [12] is found mostly in the hinge region. Methylation of ERa at Lys302 is catalyzed by the histone methyltransferase Set7. This modiﬁcation is suggested to increase sensitivity to estrogen and to enhance transcription activation.

Lipidation of Nuclear Receptors Distinct fractions of NRs have been found to be associated with the cell membrane, allowing interactions with other membrane- associated signaling proteins. Both, N-myristoylation and S-palmitoylation of NRs have been reported [13], and these modiﬁcations are involved in the triggering of nongenomic responses at the cell membrane (Section 6.7.1).

6.5.3 Ubiquitination and Sumoylation of NRs

A critical factor for NR signaling is the steady-state concentration of the receptor, which is determined by its rate of expression and ubiquitin-mediated degradation.

— Levels of NRs are regulated by ubiquitin-dependent proteolysis

Ubiquitination regulates NRs in various ways. One main path includes the ubiquitin-dependent proteolysis of NRs by allowing for the downregulation of receptors under long-term hormone treatment. A ligand-dependent ubiquitination and subsequent proteasomal degradation has been described for example, for ERa, PR, VDR, T3R, and RARa. It has been proposed that the proteasomal degradation process provides a mechanism to control the magnitude and duration of ligand- mediated transcription regulation. In another pathway, the ubiquitination of NRs is required to activate transcription. This effect is explained in terms of the dynamic removal and reassembly of receptor complexes at promoters during the course of ongoing transcription cycles.

Sumoylation Modiﬁcation by sumoylation is a widely used tool to modulate the function of NRs [14]. In most described cases, sumoylation inhibits the transcriptional activity of the modiﬁed NRs and even triggers repression of the target gene. The inhibitory mechanisms of sumoylation appear to be varied, including changes in intranuclear localization, crosstalk with other PTMs, as well as interference with dimerization and DNA binding.

6.5.4 Composite DNA Elements, Interaction with Other Transcription Factors, and Long-Range Effects

The genome-wide analysis of NR target sites revealed that the hormone-responsive elements typically form part of the composite regulatory regions that may harbor 6.5 Regulation of Signaling by Nuclear Receptors 279 several copies of the HRE as well as DNA-binding elements of other transcription factors. This organization results in an extensive crosstalk between NRs and other transcription factors. Furthermore, the clusters often function as enhancers that regulate gene promoters located far away from the NR binding site. The following examples may serve to illustrate the complexity of NR function in the context of the cellular network of transcription regulation.

6.5.4.1 Crosstalk with Other Transcription Factors Nuclear receptors and their binding sites form part of a regulatory network where several transcription factors using composite DNA elements cooperate to generate distinct patterns of gene regulation. In composite regulatory elements, the regulatory functions of the proteins involved are assumed to be interdependent, which results in mutually activating or inhibiting effects. For example, the ER, T3R, RAR and GR proteins have been shown to act as transrepressors of the transcription factor AP-1, which is a heterodimer composed of c-Jun and c-Fos proteins. Reciprocally, AP-1 can inhibit transactivation by these receptors.

& NRs modulate activity of other transcription factors: — AP-1 — Sp-1 — STAT proteins

The suppression of transactivation of other transcription factors through protein–protein interactions appears to be particularly important in the suppression of immune function and inﬂammation by glucocorticoids. For example, glucocorticoid receptors have a mutually antagonistic effect on the function of the transcriptional activator NFkB (Section 2.8.5.3), a major player in inﬂammatory responses. The mechanistic basis of these cross-regulatory effects is largely unknown. Other mechanisms of direct crosstalk between NRs and other transcription appear to use direct protein–protein interactions. For example, ERs and progesterone receptors can stimulate gene expression without binding to DNA by associating with other transcription factors bound to the regulatory regions of responsive genes. Protein–protein interactions between ERs and the transcription factors Sp-1, AP-1 as well as the Stat proteins (Section 13.2.2), have been reported for a variety of genes that do not contain the canonical ER-responsive DNA elements.

6.5.4.2 Long-Range Actions of NRs Earlier studies of the location of HREs in relation to the cognate target genes had concentrated on promoter-proximal control regions located 100–200 bp upstream of the promoter region. However, the development of techniques for studying long- range interactions between DNA-bound transcription factors and promoter regions revealed that most of the NR DNA-binding sites are far away (>100 kbp) from the 280 6 Signaling by Nuclear Receptors

promoter of the target gene. A genome-wide analysis of the chromatin interaction sites of ER bound to its DNA elements showed that the majority of receptors is involved in long-range interactions with promoters, implicating an extensive looping of chromatin [15]. These long-range interactions are used to bring together DNA-bound ER with remote promoters, allowing coordinated transcriptional regulation.

6.5.5 Determinants of Cell- and Gene-Specificity of NR Action

One major issue in NR function relates to the question how the large number of NR target genes can be activated in a gene- and cell-speciﬁc fashion. Some crucial aspects of NR selectivity have been revealed by a genome-wide analysis of the genes that respond to glucocorticoids [3]. In the human cell line investigated, more than 500 glucocorticoid target genes were shown to exist, each regulated via a cognate glucocorticoid response element (GRE). The analysis of GRE architecture, GR binding sequence and GR responsiveness at distinct loci revealed the following determinants of glucocorticoid selectivity on a genomic scale:

Binding of the GR to the GRE is the primary determinant of the glucocorticoid responsiveness of target genes. The DNA sequences bound by GRs vary widely around a consensus; however, the precise sequences of individual GREs are highly conserved. This finding suggests a decisive role of the DNA sequence in gene-specific GR regulation.As outlined in Section 6.3.1, the sequence of the HRE can modulate ligand binding and the recruitment of distinct coregulators. The exact sequence of an individual HRE appears to be a major determinant of the cell type-specific sensitivity to ligands and recruitment of the associated coregulator. Native chromosomal GREs are generally composite elements, comprised of multiple transcription factor binding sites that are highly variable in composition. As with the GREs, the composition of the regulatory regions is highly conserved at individual loci.

These ﬁndings illustrate the complexity of transcriptional regulation by NRs when considered on a genomic scale. There is intensive crosstalk of NRs with other transcription factors, but how this crosstalk is tuned in a gene- and cell-speciﬁc fashion is not well understood. It appears that the sequence of the HRE and its surrounding regions dictate the local chromatin composition and structure, allowing interactions with promoters that are located more than 100 kbp away from the HRE.

6.6 Subcellular Localization of NRs

The signaling functions of NRs and their ligands are intimately linked to the subcellular compartment where the receptor resides. There is a highly variable 6.6 Subcellular Localization of NRs 281 pattern of subcellular distribution of NRs that depends on receptor type, ligand binding, and PTMs.

6.6.1 Nuclear and Cytoplasmic Pools

Transcriptional regulation by NRs is exerted in the nucleus. In this classical, genomic mode of action, DNA-bound receptors either activate or repress transcription in response to ligand binding. Two major subcellular pools of NRs are linked to genomic signaling:

One group, which includes most of the steroid hormone receptors, localizes to the nucleus and to the cytoplasm, and there is active shuttling of these receptors between the two compartments. The distribution of these receptors between cytoplasm and nucleus is mainly regulated by the ability of these receptors to undergo ligand-dependent translocation from the cytosol to the nucleus. A second group of receptors, including those for the derivatives of retinoic acid, the T3 hormone and vitamin D3, are predominantly localized in the nucleus, and ligand-binding to the DNA-bound receptor activates transcription. In the absence of ligand, these receptors may have corepressors associated to inhibit transcription activation.

6.6.2 Other Subcellular Pools

In addition to the cytoplasmic and nuclear pools, distinct NR pools are associated with other subcellular compartments as for example, the cell membrane, mitochondrial compartments and the endoplasmic reticulum, where nongenomic actions of the receptors become active (see Section 6.7.1). The localization of nuclear receptors to speciﬁc subcellular compartments is for the most part regulated by PTMs and may be either ligand-dependent or ligand-independent.

6.6.3 Ligand-Dependent Translocation of Nuclear Receptors

Genomic signaling by steroid hormones (glucocorticoids, mineralocorticoids, androgens,) is distinguished by the fact that the receptor receives the hormonal signal in the cytosol and becomes activated by hormone binding, at which point it enters the nucleus to regulate the transcription initiation of cognate genes. The processes involved in nuclear translocation have been best studied for the GR. The most important steps in the nuclear translocation of the steroid hormone receptors are shown in Figure 6.15. The steroid hormones are distributed throughout the entire organism by means of the circulatory system. Transport often occurs in the form of a complex with a speciﬁc binding protein, an example of which is transcortin, responsible for the 282 6 Signaling by Nuclear Receptors

Figure 6.15 Principle of signal transduction by can be transported into the nucleus where it steroid hormone receptors which, in the can bind to its cognate HRE. Currently, it is cytosol, are found in the form of an inactive still not well understood, in which form the complex with the heat shock proteins Hsp90 receptor is transported into the nucleus, and and Hsp56 and with protein p23. Binding of to which extent the associated proteins are the hormone activates the receptor so that it involved in the transport.

transport of corticosteroids. The steroid hormones enter the cell by diffusion and activate the cytosolic receptors. However, in the absence of steroid hormones the receptors remain in an inactive complex, designated the aporeceptor complex, which is transformed into an active complex by hormone binding.

& Aporeceptor complexes of steroid hormone receptors contain: — Receptor protein — Hsp90 chaperone complex — Cochaperones — Immunophilins — p23 6.6 Subcellular Localization of NRs 283

Various proteins belonging to the chaperone class participate in the maturation of the inactive aporeceptor complex. The multistep process of receptor maturation involves a complicated cooperation between various chaperones and cochaperones [16]. The proteins include the chaperones Hsp90, Hsp 40 and Hsp 70, the cochaperone Hop, immunophilins of the FKBP family (FKBP51, FKBP52) and another protein factor, p23. Chaperones are proteins that assume a central function in the folding process of proteins in the cell. They aid proteins in avoiding incorrectly folded states, and thereby participate in the folding of proteins during and after ribosomal protein biosynthesis, during membrane transport of proteins, and in the correct assembly of protein complexes. The term heat shock protein (Hsp) is derived from the observation that these proteins were produced at higher levels following heat treatment. The immunophilins are proteins that carry peptidyl-prolyl cis–trans isomerizing activity and associate with Hsp 90 through distinct domains, named tetratricopeptide repeats. The chaperones are used as tools in this system for regulating the activity of the steroid hormone receptors. It is believed that Hsp90 holds the receptor in a partially unfolded conformation that is competent for ligand binding, and that the cochaperones facilitate the correct association of Hsp90 with the receptor.

& Heat shock proteins (Hsp): — Proteins produced at higher levels following heat treatment. Chaperones: — Aid proteins in avoiding incorrectly folded states and thereby participate in the folding of proteins. Immunophilins: — Proteins that carry peptidyl-prolyl cis–trans isomerizing activity and associate with Hsp90 through distinct domains, named tetratricopeptide repeats.

6.6.4 Nuclear Translocation

Binding of the hormone to the aporeceptor complex leads to an activation of the receptor and initiates translocation of the receptor into the nucleus; this occurs under the participation of motor proteins (e.g., dynein), which are guided by microtubules to the nuclear pores. The activated receptor possesses an accessible nuclear localization sequence and is also capable of DNA-binding and transactivation. An ability to transactivate implies that the transactivating domain is correctly positioned, as a result of the hormone binding, so as to allow stimulatory interactions with the transcription apparatus. 284 6 Signaling by Nuclear Receptors

It is now well established that NRs are ﬂexible molecules that can exist in different conformational states, each with distinct functions. It may be a function of the chaperones to stabilize the particular conformation that is optimal for hormone binding. Inactive, chaperone-bound NR complexes may also be found in the nucleus; this is certainly the case for ER, which is sequestered in the nucleus within a large inhibitory Hsp complex. Binding of estrogen to the ER enables displacement of the Hsps and facilitates DNA binding, the formation of receptor dimers, and also interactions with coactivators and the transcription apparatus.

6.7 Nongenomic Functions of NRs and their Ligands

Summary The NRs and their hormone ligands have been shown to elicit fast responses that are not dependent on transcription and are termed nongenomic functions.These responses are mediated by actions of the receptors at sites outside of the nucleus. The nongenomic functions include direct interaction with other signaling proteins such as scaffold proteins, protein kinases and transmembrane receptors. In this way, signals can be transmitted to and received from other signaling pathways. Furthermore, lipidation may serve to recruit the NRs to the cell membrane, allowing for crosstalk with other membrane-associated signaling proteins.

It is now well established that NRs perform distinct functions in the cytoplasm and at speciﬁc subcellular structures and organelles, such as the cell membrane, the endoplasmic reticulum and mitochondrion. Importantly, some hormone

ligands of NRs, such as estradiol, progesterone and T3, can bind to other proteins to trigger responses that are not mediated by the cognate NR. Both modes of action link the NRs and their hormone ligands directly to cytoplasmic signaling pathways that can induce a broad range of biochemical events on a fast time scale.

& Nongenomic actions of NRs and their ligands: — Linkage to other signaling pathways — Binding to G protein-coupled receptors

6.7.1 Nuclear Receptor Functions Outside of the Nucleus

6.7.1.1 Cytoplasmic Functions Some cytoplasmic-localized NRs can interact directly with other signaling proteins

to initiate rapid nongenomic responses. For example, T3R, when complexed with ﬁ the T3 hormone, interacts speci cally with the p85 subunit of PI3 kinase (see 6.7 Nongenomic Functions of NRs and their Ligands 285

Section 9.4), leading to an activation of PI3 kinase at the cell membrane. As a consequence, the formation of phosphoinositides is stimulated and the Akt kinase pathway is activated. Targets of this pathway include endothelial nitric oxide (NO) synthase, which is rapidly induced upon estradiol administration in endothelial cells. Furthermore, the transcription of speciﬁc genes may be stimulated via the PI3K/Akt pathway. In a similar way, ER can activate the PI3 kinase/Akt pathway.

6.7.1.2 ER Actions at the Cell Membrane Distinct pools of NRs are associated with the cell membrane where they can initiate rapid nongenomic functions by interacting with other membrane-associated signaling proteins. The membrane targeting of NRs is mediated by N-terminal myristoylation and S-palmitoylation, and also by a direct interaction with membrane-associated proteins. The nongenomic functions of membrane-associated NRs may be illustrated by the example of ERs, of which the extranuclear activities have been studied in detail, fueled by the involvement of estrogen and its receptors in the development of cancers of the breast and uterus. Two isoforms of ER, namely ERa and ERb, have almost the same DNA binding domain and a similar ligand-binding domain, whereas there is a wide divergence in the N-terminal AF-1 domain.

& ERs can receive signals from other central signaling pathways in a ligand-independent fashion.

Both ER isoforms act through the same general mechanism in target cells, with distinct biological actions on speciﬁc gene promoters and in response to synthetic ligands. ERa is more highly expressed and plays a major role in steroid-dependent cancers of the breast and uterus. Currently, it is commonly realized that ERs receive and transmit a multitude of signals that are not directly related to the classical steroid hormone pathway. These signaling processes are associated to a large part with the cell membrane, and involve PTMs of the NR that link NR signaling to other signaling pathways. The ERs are phosphoproteins (see Section 6.5.2), and multiple Ser and Thr residues are phosphorylated basally and in response to ligands, second messengers and growth factors via, for example, MAPK, PKA, PKC and CDK pathways (Figures 6.16 and 6.17). In addition, Tyr-phosphorylation is observed on ERs. These phosphorylations mediate the ligand-independent stimulation and modulation of ERs, and link the receptors to major signaling pathways of the cell, notably to growth factor signaling.

& Activated ERs can transmit signals directly to other signaling paths via interaction with: — Adapter proteins — Protein kinases — Transmembrane receptors 286 6 Signaling by Nuclear Receptors

Figure 6.16 Ligand-independent linkage of the does not require estrogen binding to ER is estrogen receptor (ER) to the major signaling ascribed to Src kinase. Phosphorylated ER can pathways of the cell. ER is phosphorylated by thus stimulate transcription in a ligand- protein kinases in response to signals that independent fashion. PKA, protein kinase A start from receptor tyrosine kinases (RTKs), (Section 9.3); PKC, protein kinase C (Section via Ras protein, or from G protein-coupled 9.5); ERK, extracellular regulated kinase receptors. A central role in this crosstalk which (Section 12.2); Src, src kinase (Section 10.3.2).

The direct and indirect linkage of estrogen signaling to the central signaling pathways of the cell appears to involve membrane-associated and cytoplasmic pools of ERs. A dynamic exchange must be assumed between the different compartments, and it is therefore difﬁcult to differentiate between both pools. Membrane targeting of ERs may involve several mechanisms, such as through lipidation or through interaction with transmembrane receptors or other membrane proteins [17]. The ERs have been shown to contain myristinic and palmitoic membrane anchors that promote membrane association. Furthermore, membrane- bound fractions of ER are found in large transmembrane signaling complexes at distinct sites, the caveolae.

& Some functions of ERs are linked to the cell membrane

The major constituent of these membrane domains is caveolin-1, a protein which has been identiﬁed as a binding partner of the ER during membrane association. 6.7 Nongenomic Functions of NRs and their Ligands 287

Figure 6.17 Nongenomic functions of ER and proteins Shc or MNAR. ERs can directly estrogen. The steroid hormone 17-b-estradiol activate PI3 kinase and thereby transmit a (E2) can initiate signaling via the G protein- signal to Akt kinase. One effect of Akt kinase coupled receptor Gp30, or via its receptor (Section 9.4) activation is stimulation of (ER). Binding of E2 to ER can stimulate endothelial NO synthase (NOS) and the transcription or activate (in a nongenomic production of the second messenger NO. Akt mode) other central signaling proteins of the kinase also mediates an anti-apoptotic and cell cell. The crosstalk with other signaling proliferation-promoting effect. pathways may require the presence of adapter

Estrogen binding to the membrane-bound ER has been reported to weaken membrane association and to allow for interaction with other important signaling enzymes, as for example c-Src kinase (Section 10.3.2) and PI3-kinase (Sec- tion 9.4.1). It is not yet clear whether these responses are activated while the receptor is still associated with the cell membrane or has already been released from the cell membrane. The interaction with c-Src kinase may occur directly via the binding of ER to the SH2 domain of c-Src. Phosphorylation at Tyr537 in the ligand-binding domain of the ER has been implicated to be involved in this interaction. The bindingofc-Srcmaybemediatedalsobyascaffoldingmoleculetermed “moderator of nongenomic activity of ER” (MNAR). The interaction of Src kinase with ER or MNAR may relieve Src from the autoinhibited state and may activate – via Shc, Grb2-mSos and Ras – the MAPK pathway and thus stimulate the transcription of a variety of genes important for cell growth and cell proliferation (see Chapters 11 and 12). 288 6 Signaling by Nuclear Receptors

6.7.1.3 NR Action at the Mitochondrion The regulation of mitochondrial functions by NRs may occur via two approaches: (i) by a direct action of the receptors in the mitochondrion; and (ii) by the transcription of target genes that affect mitochondrial functions in either a direct or indirect manner. Thyroid hormones and their receptors represent an example of the ﬁrst approach.

It has long been known that the thyroid hormones T3 and T4 are directly involved in mitochondrial numbers and energetics, though the biochemical basis of these ﬂ in uences remains largely unknown. The involvement of T3/T4 hormone and its receptor (T3R) in regulating mitochondrial functions appears to depend on the presence of the A/B domain of the receptor. For example, T3Ra1, which lacks the A/B domain, is imported into the mitochondrion and impacts mitochondrial functions in several ways, by:

increasing transcription in mitochondria; triggering changes in proton gradient; þ enhancing IP3-mediated Ca2 signaling.

Interestingly, a TR-responsive element has been found on mitochondrial DNA [9].

6.7.2 Hormone Binding to Other Receptor Types

b For some NR ligands, such as 17- -estradiol, progesterone and the T3 hormone, nonclassical modes of action have been discovered that do not involve the cognate NRs. Rather, these hormone ligands can bind with high afﬁnity to distinct receptor proteins that form part of other signaling pathways. Two examples involving b 17- -estradiol and the T3 hormone are highlighted here:

In a nonclassical mode of action, 17-b-estradiol binds to and activates a transmembrane receptor termed GPR30, which belongs to the class of G protein-coupled receptors [18]. GPR30 has been located to the cell membrane and to the endoplasmic reticulum, and binds the physiological ligand of classical ER, 17-b-estradiol with nanomolar afﬁnity, which is close to the afﬁnity of 17-b-estradiol for ERa. Other 17-b-estradiol analogs, such as tamoxifen, can also bind to and activate GPR30. Via a heterotrimeric G protein, a multitude of downstream signals can be produced, including the stimulation of transcription. As outlined in Chapter 7, the G protein-coupled receptors stimulate the synthesis and release of a number of small signaling molecules, the second messengers. Furthermore, they can directly activate ion channels leading, for example, to an þ increase in cellular Ca2 levels. Such an effect is observed upon the administration of 17-b-estradiol. The nonclassical action of the T3/T4 hormone [9] is mediated, at least in part, by b binding to and activating a transmembrane protein, integrin av 3. Integrins are References 289

receptors at the cell membrane that can receive and transmit both extracellular ﬁ and intracellular signals (see Chapter 13). High-af nity binding of T3/T4 b hormone to integrin av 3 at the outside of the cell membrane has been shown to activate the MAPK pathway with broad physiological effects, including the stimulation of angiogenesis and cell proliferation.

& Estradiol binds and activates GPR30, a G protein-coupled receptor.

Questions 6.1. Name at least six different chemical classes of nuclear receptor ligand, and give their chemical structures. 6.2. Describe the domain structure of nuclear receptors and the function of the domains. What is the common structural motif of the DNA-binding domain? Do you know any structurally related motifs? 6.3. What are the structural features of HREs? What may be the advantage of homodimeric or heterodimeric nuclear receptors instead of monomers? How would you change the sensitivity of a glucocorticoid receptor target gene from cortisone induction into progesterone induction? 6.4. How is ligand binding coupled to transcription activation? What is the structural basis of this coupling? 6.5. Give examples of coregulators of nuclear receptors. What functions have these complexes in transcription regulation? 6.6. Describe the processes that are triggered following the passage of cortisone across the cell membrane. 6.7. What are SERMs, and what is the structural basis of their function? 6.8. Which variables determine the genomic function of nuclear receptors? 6.9. Which processes contribute to the regulation of nuclear receptor signaling? 6.10. Give examples of nongenomic actions of nuclear receptors. Which reactions are involved in the nongenomic actions of estrogen?

References

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7 G Protein-Coupled Signal Transmission Pathways

7.1 Transmembrane Receptors: General Structure and Classification

During intercellular communication, extracellular signals are registered by the cell and converted into intracellular reactions. Signal transmission into the cell interior takes place by reaction chains, which involve many signal proteins. The nature of the extracellular signal can be very diverse and may include extracellular signal molecules, such as low-molecular-weight messenger substances or proteins, or sensory signals such as light signals. The cell employs two principal methods to transduce signals into the interior of the cell: (i) nuclear receptor signaling, where the signaling molecule crosses the cell membrane and activates the receptor in the interior of the cell; and (ii) the signal is registered at the cell membrane and transduced into the cell by transmembrane proteins. Two different types of transmembrane proteins also participate in this mode of signaling, namely signaling via transmembrane receptors and/or via ligand- or voltage-gated ion channels.

7.1.1 Signaling via Transmembrane Receptors

Transmembrane receptors are proteins that span the phospholipid bilayer of the cell membrane. The signaling molecule binds on the extracellular side to the receptor, which is thereby activated. Reception of the signal is synonymous with activation of the receptor for transmission of the signal across the cell membrane.

& Transmembrane signaling uses: — Transmembrane receptors activated by: Extracellular ligands Sensory signals — Voltage-gated ion channels — Ligand-gated ion channels

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 292 7 G Protein-Coupled Signal Transmission Pathways

Transmission of the signal implies a specific communication with the effector protein, the next component of the signal transmission pathway on the inner side of the cell membrane. In this process enzymatic activities can be triggered and/or the activated receptor engages in specific interactions with downstream signal proteins. An intracellular signal chain is set in motion, which finally triggers a defined biochemical response of the target cell (Figure 7.1a). Sensory signals (light, pressure, odor, taste) can also be received by transmembrane receptors and can be transmitted into intracellular signals. One example of a transmembrane receptor that registers sensory signals is rhodopsin, a sensory receptor that plays a role in the vision process by receiving light signals and converting them into intracellular signals.

7.1.2 Signaling via Ligand- or Voltage-Gated Ion Channels

One simply designed path of signal transmission is found in neuronal communication. Likewise, transmembrane receptors can also be used for signal transmission, and these can have the character of a ligand-gated ion channel (Figure 7.1b).

& TM receptors may be activated by intracellular signals

The binding of a ligand (neurotransmitter or neurohormone) to a transmembrane receptor leads to a conformational change of the receptor that enables a ﬂow of ions through the membrane. In this case, the receptor presents itself as an ion channel with an open state controlled by ligand binding to the outer side (or also to the inner side). Another mechanism of signaling across the cell membrane involves changes in membrane potential that induce the opening of an ion channel, allowing ions to cross the membrane. In this case, the change of the ion’s milieu is the intracellular signal. Ion channels with an open state regulated by changes in membrane potential are known as voltage-gated ion channels (Figure 7.1c). The potential-driven passage of ions through ion channels forms the basis for stimulation in nerves. Transmembrane proteins are also known for which reception of the signal and activation take place on the inner side of the membrane. The cGMP-dependent ion channels involved in signal conduction in the vision process are ligand-regulated ion channels with an open state controlled by intracellularly created cGMP. Another example is the receptors for inositol triphosphate which are localized in þ the membrane of Ca2 storage organelles and also have the character of ligand- controlled ion channels. Inositol triphosphate is an intracellular messenger substance that binds to the cytosolic side of the corresponding receptor located in the membrane of cell organelles. Ligand binding leads to an opening of the ion þ channel via a conformational change and thus leads to inﬂux of Ca2 ions from the storage organelle into the cytosol (Section 8.5). 7.1 Transmembrane Receptors: General Structure and Classification 293

Figure 7.1 Mechanism of signal transduction channels. The ligand binds to the extracellular at membranes. (a) Signal transmission via side of a receptor that also functions as an ion ligand-controlled transmembrane receptors. channel. Ligand binding induces the opening The ligand L binds to the extracellular domain of the ion channel, and there is an ion efflux of a transmembrane receptor, whereby the and a change in the membrane potential; receptor is activated for signal transmission to (c) Signal transduction via voltage-gated ion the cytosolic side. The cytosolic domain of the channels. A change in the membrane potential activated receptor R transmits the signal to DV is registered by an ion channel which signal proteins next in sequence; (b) Signal transitions from the closed to the open state. transduction via ligand-controlled ion 294 7 G Protein-Coupled Signal Transmission Pathways

7.2 Structural Principles of Transmembrane Receptors

Transmembrane receptors are integral membrane proteins; that is, they possess a structural portion that spans the membrane. Moreover, an extracellular domain,a transmembrane domain and an intracellular or cytosolic domain can be differentiated within the structure (Figure 7.2a) .

& Structural parts of transmembrane receptors: — Extracellular domain — Transmembrane domain — Intracellular domain

In general, these receptors function as dimers or higher oligomers composed of identical or different transmembrane subunits (Figure 7.2b). Furthermore, non-transmembrane subunits can associate at either the extracellular or intracellular side.

7.2.1 The Extracellular Domain of Transmembrane Receptors

In many receptors, the extracellular domain contains the ligand-binding site. Glycosylation sites, that is, attachment sites for carbohydrate residues, are also located nearby in the extracellular domain.

& Extracellular domain: — Ligand binding — Glycosylation — Association of further subunits

The structure of the extracellular domain can be very diverse and is determined by the number of transmembrane sections as well as the subunit structure of the receptor. The extracellular localized protein portion may be formed from a continuous protein chain and may include several hundred amino acids. If the receptor crosses the membrane with several transmembrane segments (Figure 7.2c), the extracellular domain is formed from several loops of the protein chain that may be linked by disulﬁde bridges. Receptors are also known in which only one subunit spans the membrane, while other subunits are bound to this subunit on the extracellular side via protein– protein interactions or via disulﬁde bridges (Figure 7.2b; see also examples in Chapter 13). 7.2 Structural Principles of Transmembrane Receptors 295

Figure 7.2 Structural principles of transmembrane domain may be composed of transmembrane receptors. (a) Representation an a-helix (1) or several a-helices linked by of the most important functional domains of loops at the cytosolic and extracellular side (2). transmembrane receptors; (b) Examples of The seven-helix transmembrane receptors are subunit structures. Transmembrane receptors a frequently occurring receptor type (see can exist in a monomeric form (1), dimeric Section 7.3). Several subunits of a form (2) and as higher oligomers (3, 4). transmembrane protein may associate into an Further subunits may associate at the oligomeric structure (3), as is the case for þ extracellular and cytosolic domains, via voltage-controlled ion channels (e.g., K disulfide bridges (3) or via noncovalent channel) or for receptors with intrinsic ion interactions (4); (c) Examples of structures of channel function. the transmembrane domains of receptors. The 296 7 G Protein-Coupled Signal Transmission Pathways

7.2.2 The Transmembrane Domain

The transmembrane domains have different functions, according to the type of receptor. For ligand-controlled receptors, the function of the transmembrane domain is to pass the signal on to the cytosolic domain of the receptor, whereas for ligand- or voltage-controlled ion channels the transmembrane portion forms an ion pore that allows a selective and regulated passage of ions. The transmembrane receptors span the approximately 5 nm-thick phospholipid bilayer of the cell membrane with structural portions known as transmembrane elements.

& a-helical transmembrane domains: — Contain one to seven a-helices each of 20–30 amino acids — Are often arranged in bundles.

The inner region of a phospholipid layer is hydrophobic and, correspondingly, the surface of the structural elements that come into contact with the inner side of the phospholipid double layer also has a hydrophobic character. The polypeptide chain of the vast majority of transmembrane receptors contains a single a-helical transmembrane element or seven a-helical elements, organized as a bundle in the membrane. In the latter case the transmembrane elements are linked by both extracellular and intracellular loops. Generally, the transmembrane elements include 20–30 mostly hydrophobic amino acids. At the interface with aqueous medium, hydrophilic amino acids are often found in contact with the polar head groups of the phospholipids, and these also mediate a distinct ﬁxing of the transmembrane section in the phospholipid double layer.

7.2.2.1 Structure of Transmembrane Elements High-resolution structural information about the transmembrane elements of transmembrane receptors could be obtained using rhodopsin, the light-activated G protein-coupled receptor of the vision process (Figure 7.3). These data have conﬁrmed that a-helices are the principal structural building blocks of the transmembrane elements of membrane receptors. The transmembrane helices are composed of 20–30 hydrophobic amino acids with some polar amino acids interspersed between or located at the helix ends. Most of the helices are arranged almost perpendicular to the phospholipid bilayer and form bundle-like structures in which the helices are linked by loops of variable size. Ligand binding or the reception of a sensory signal triggers a change in the mutual orientation of the helices that is transmitted into conformational changes of the cytoplasmic loops. These are sensed by the next components of the signaling chain, or they result in the activation of an enzyme at the cytoplasmic side of the membrane.

& Some transmembrane domains are composed of b-elements 7.2 Structural Principles of Transmembrane Receptors 297

Figure 7.3 Three-dimensional structure of (bottom) to the cytoplasm (top). The rhodopsin. Two views of rhodopsin. (a) The chromophore 11-cis-retinal (yellow) is nestled seven a-helices of the G protein-coupled among the transmembrane helices; (b) View receptor rhodopsin weave back and forth into the membrane plane from the cytoplasmic through the membrane lipid bilayer (yellow side of the membrane. Roman numerals lines) from the extracellular environment indicate numbered helices.

A signal is thereby generated in the interior of the cell and is propagated further. In addition to a-helices, proteins also use b-structures to cross the membrane. The transmembrane domain of the bacterial OmpF porin is made up of b-elements (Figure 7.4). The b-elements, in this case, are not mostly made up of hydrophobic amino acids and form a barrel-like structure. 298 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.4 The OmpF porin from Escherichia with the individual subunits in green, red, and coli. OmpF is an integral membrane channel- blue. In total, 16 b-bands are configured in the forming protein which spans the outer form of a cylinder and form the walls of a pore membrane in Gram-negative bacteria. The through which the selective passage of ions structure of a trimer of the OmpF is shown takes place.

7.2.3 The Intracellular Domain of Membrane Receptors

Two basic mechanisms are used to conduct the signal to the inner side of the membrane (Figure 7.5):

& Activated transmembrane receptors transmit signals mainly via: — Activation of downstream effectors — Triggering of enzyme activity.

7.2.3.1 Recruitment of Downstream Signaling Proteins The next protein component in the signal transmission pathway, the effector protein, is activated via speciﬁc protein–protein interactions. The conformational change that accompanies the perception of the signal by the receptor creates a new interaction surface for proteins located downstream of the receptor. In the absence of a signal, this interaction surface is not available, and therefore signal transmission will depend strictly on signal perception by the receptor, while activation of the effector molecule must be preceded by activation of the receptor via a signal. Mechanistically, the active and inactive states of the receptor may be 7.2 Structural Principles of Transmembrane Receptors 299

Figure 7.5 General functions of tyrosine kinases and of protein tyrosine transmembrane receptors. Extracellular signals phosphatases. The latter enzymes may be an convert the transmembrane receptor from the intrinsic part of the receptor or they may be inactive form R to the active form R. The associated with the receptor. The activated activated receptor transmits the signal to receptor may also include adapter proteins in effector proteins next in the reaction sequence. the signaling pathway to form larger signaling Important effector reactions are the activation complexes, or it may induce the opening of ion of heterotrimeric G proteins, of protein channels.

also regarded as fluctuations of receptor conformations. Agonists stabilize and fix the active conformation, whereas the inactive conformation predominates in the absence of the ligand and may be further fixed by antagonists.

7.2.3.2 Triggering of Enzyme Activity Arrival of the signal triggers enzyme activity in the cytosolic domain of the receptor which, in turn, pulls other reactions along with it. The enzyme activity of the cytosolic domain is often due to tyrosine kinase, although other examples are known where tyrosine phosphatase or Ser/Thr-speciﬁc protein kinase activity is activated. In all of these examples the cytoplasmic domain carries an enzyme activity which is regulated by ligand binding. This activity may form an integral part of the receptor, or it may be a separate enzyme associated with the receptor on the inner side of the membrane (see Chapters 10 and 13). Starting from the activated receptor, a large number of reactions can be set in motion (Figure 7.5). One main route of signal transmission is mediated by the activation of G proteins, another via the activation of tyrosine-speciﬁc protein kinases, and a further route is via the activation of ion channels. 300 7 G Protein-Coupled Signal Transmission Pathways

& Main downstream targets of activated transmembrane receptors: — G proteins — Protein kinases — Ion channels — Adapter proteins.

During the further course of G protein-mediated signal transmission, secondary diffusible signals are often formed, termed “second messenger” molecules (see Chapter 8), that function as effectors and activate further enzyme systems in the sequence, especially protein kinases. The activated receptor can also associate with adapter molecules, which serve as coupling elements for further signal proteins.

7.2.4 Regulation of Receptor Activity

One physiologically important aspect of signal transmission via transmembrane receptors is its regulation, for which the cell has various mechanisms available that allow the number and activity of transmembrane receptors to be regulated.

& Transmembrane receptors are regulated by: — Downregulation, desensitization — Crosstalk — Phosphorylation and recycling — Degradation

The regulation of transmembrane receptor activity serves two main goals:

Desensitization, attenuation and downregulation: A ubiquitous feature of signaling through transmembrane receptors is the loss of cellular sensitivity following the presentation of a stimulus. This attenuation or desensitization can occur on a long-term or short-term time scale, and is accompanied by a reduced response of the receptor or by a lowered number of receptor molecules. Crosstalk with other signaling pathways: Signaling through transmembrane receptors is subjected to crosstalk with other signaling pathways, allowing the coordination and modulation of signaling events. This crosstalk mostly involves phosphorylation of the cytosolic part of the receptor by protein kinases that have been activated through other signaling paths, and is also referred to as heterologous desensitization.

7.2.4.1 Receptor Phosphorylation and Receptor Recycling The structural elements involved in the regulation of receptor activity are generally located in the cytosolic domain. These are, above all, protein sequences that permit 7.3 G Protein-Coupled Receptors 301 phosphorylation of the receptor by protein kinases. Phosphorylation at Ser/Thr or Tyr residues of the cytosolic domain may lead to inactivation or activation of the receptor, and thus weaken or strengthen signal transmission. In this way, Ser/Thr- phosphorylation is used in the process of internalization of receptors in order to remove the receptor from circulation after it has been activated (Section 7.3.4). The protein kinases involved are often part of other signaling pathways and can link the activity of the transmembrane receptors to other signaling networks.

7.2.4.2 Ubiquitination and Degradation The targeted degradation of transmembrane receptors is another means of regulating receptor activity, and signals for ubiquitination and subsequent degradation in the proteasome have been identiﬁed in the cytosolic domain of transmembrane receptors. A major mechanism of targeting transmembrane receptors for proteolysis employs the signal-directed phosphorylation of the cytosolic domain (see Section 2.8.3.4).

7.3 G Protein-Coupled Receptors

Summary G protein-coupled receptors (GPCRs) constitute the majority of transmembrane receptors in humans. These receptors receive chemical and physical signals at the cell membrane, and then transmit them to intracellularly located downstream effectors, primarily heterotrimeric G proteins. The spectrum of stimuli that can activate GPCRs is very broad and comprises small-molecule hormones, protein hormones and odorants, as well as physical stimuli. The GPCRs are constructed from seven transmembrane helices arranged in a bundle and linked by loops on the extracellular and intracellular sides. The binding sites for ligands are located in the interior of the bundle or on the extracellular loops, depending on the ligand size. Ligand binding or the reception of a physical signal induces a rearrangement of the helices within the bundle, leading to activation of the GPCR and transmission of the signal to the intracellular heterotrimeric G protein that binds to the receptor via its Ga-subunit. The seven-helix bundle of GPCRs is a flexible entity that can assume many conformations. Different ligands can stabilize distinct conformations and thereby transmit the signal to distinct members of the family of heterotrimeric G proteins or other effectors. The signaling function of GPCRs is regulated by PTMs and protein binding. The phosphorylation of GPCRs on the intracellular loops by GPCR kinases serves to create a binding site for arrestin proteins that is involved in the downregulation and desensitization of the receptor, among others.

Of the transmembrane receptors that receive signals and conduct them into the cell interior, the G protein-coupled receptors (GPCRs) comprise about 60% and 302 7 G Protein-Coupled Signal Transmission Pathways

Table 7.1 Extracellular signals for the activation of GPCRs.

þ Biogenic amines Ca2 ions Phospholipids Light Amino acids Pheromones Fatty acids Odorants Peptides Prostanoids Nucleosides Bitter and sweet Nucleotides gustatory substances Glycoproteins

form the largest single family [1]. The human genome encodes about 720 different GPCRs, of which about half are thought to encode sensory receptors involved in the perception of light, taste, and smell.

& GPCRs: — Signal through G proteins — Encoded by ca. 720 genes — Include 60% of all transmembrane receptors.

For about two-thirds of the remaining receptors the ligand is known, while the remainder are so-called orphan receptors with no known ligand or function. As summarized in Table 7.1, the nature of stimuli that signal through GPCRs is very diverse; indeed, it has been estimated that about 80% of all known hormones and neurotransmitters signal through GPCRs. Interestingly, GPCRs are also known that are activated by proteases; one example of these is thrombin (see also Figure 7.8); in this case the protease cleaves off a peptide from the N-terminus, after which the newly created N-terminus serves as a tethered ligand for the activation of these protease-activated receptors. Ligand binding or the reception of a physical signal is linked to the activation of GPCRs and, as a consequence, the receptors undergo a conformational change that is transmitted to the inner side of the membrane; in this way, the next sequential member of the signal chain becomes activated and the signal is further conducted via other reaction pathways (see Figure 7.16). One characteristic structural feature of the GPCRs is the presence of seven transmembrane helices (Figures 7.3 and 7.6). I Figure 7.6 Classification of G protein-coupled terminus containing a series of cysteine receptors (GPCRs). The GPCRs can be divided residues that presumably form a network of into three major subfamilies. Family A disulfide bridges. Representative members of receptors are characterized by a series of highly family B receptors include calcitonin receptor, conserved key residues (black letter in white glucagon receptor and parathormone circles). In most family A receptors, a disulfide receptors. Family C receptors are characterized bridge is connecting the E-II and E-III loops. In by a very long N terminus forming the addition, a majority of the receptors have a extracellular ligand-binding site. There is only palmitoylated cysteine in the cytoplasmic C one putative disulfide bridge and the third terminus. Ligands include the biogenic amines cytoplasmic loop is very small. The taste (adrenaline, serotonin, dopamine, histamine), receptors, the metabotropic glutamate neuropetide Y, adenosine, chemokines and receptors, the c-aminobutyric acid (GABA) þ melatonin, among others. Family B receptors receptors and Ca2 -receptors belong to this are characterized by a long extracellular N class, among others. 7.3 G Protein-Coupled Receptors 303 304 7 G Protein-Coupled Signal Transmission Pathways

For the vast majority of GPCRs, the downstream located signaling protein is a heterotrimeric G protein.

& GPCRs are also termed: — Heptahelical receptors — Serpentine-like receptors — Seven-helix transmembrane receptors.

Because some GPCRs may employ both heterotrimeric G proteins and other cytoplasmic proteins in their signaling, while others may depend on non-G protein transducers only, alternative terms such as heptahelical receptors, seven-helix transmembrane receptors or serpentine-like receptors have been also coined for this receptor superfamily (although in the present book the term GPCR is preferred). The routes for signal transmission from activated G protein-coupled receptors that do not involve G proteins will be discussed in Section 7.8.

7.3.1 Classification of GPCRs

The current classification of GPCRs includes the A, B, C, Frizzled, and olfactory receptor families. This classification is based on sequence similarity, the size of the extracellular loops, the presence of key residues, and the formation of disulfide bonds (Figure 7.6).

& GPCR families: — Family A — Family B — Family C — Frizzled — Olfactory/gustatory.

Family A: This family is characterized by a conserved disulfide bridge and by a consensus motif DRY at the cytoplasmic side. The binding sites for all small- molecule ligands are buried in between the seven transmembrane helices. Rhodopsin and the b-adrenergic receptor are well characterized members of GPCR family A. Family B: These receptors have a large N- terminal domain and several disulfide bridges. Some members form heterodimers with a single-span transmembrane protein, named RAMP, which modifies either the ligand-binding or signaling properties of the receptor. Family C: Receptors of family C contain a large N-terminus. Both, homo- and heterodimerization has been demonstrated for receptors of this family. 7.3 G Protein-Coupled Receptors 305

Frizzled receptors: These receptors constitute a unique family among the GPCRs because receptor activity is modulated by interactions with additional plasma membrane receptors. Olfactory and gustatory receptors: This receptor family is the largest GPCR family in vertebrates, with about 400 members in human and 1200 in mouse. Much smaller numbers of this receptor type are found in invertebrates.

7.3.2 Structure of G Protein-Coupled Receptors

GPCRs constitute the major fraction of transmembrane receptors, and serve as the major drug target for commercially available medicines; indeed, approximately 30% of all marketed small-drug molecules are targeted at GPCRs. Consequently, intense research has been – and continues to be – devoted to elucidating the structure and function of this superfamily of transmembrane receptors. In the classical signaling pathway of GPCRs, ligand binding to the receptor triggers activation of the downstream effector – the heterotrimeric G protein – which is composed of a, b, and c subunits (see Section 7.5). In addition to this well- characterized signaling mode, a wealth of studies has shown that many GPCRs have much more complex signaling behavior. For example, receptors may show significant constitutive activity, and a given receptor may couple to different heterotrimeric G proteins. Furthermore, signaling may occur in a G protein- independent manner through arrestin (Section 7.8). Despite the large amount of information available regarding the functional characteristics of GPCRs, the molecular details of ligand-induced activation and communication with the cytosolic effector proteins have been revealed only recently. During the past few years, various three-dimensional (3D) structures of GPCRs in both inactive and active states, with and without bound ligand, have been determined [2]. Most importantly, the structure of an activated GPCR in association with the heterotrimeric G protein was resolved, and this proved to be major breakthrough in understanding the structural basis of GPCR signaling [3]. Bovine rhodopsin was the first GPCR for which the 3D structure could be directly visualized using high-resolution X-ray analysis [4]. This structure confirmed the overall building principle of GPCRs, namely the presence of a bundle of seven transmembrane helices. Further highly resolved structures b showing a very similar overall structure have since been reported for the 1- b and 2- adrenergic receptors (ARs) and the A2A adenosine receptor (A2AR). All 3D GPCR structures known to date have been obtained for members of the rhodopsin family (class A) of GPCRs. These receptors are activated by small ligands that bind to a site that is more or less buried in the interior of the seven-helix bundle. 306 7 G Protein-Coupled Signal Transmission Pathways

& Rhodopsin structure: — Bundle of seven transmembrane helices — H-bond network between helices — Retinal binding site within the bundle.

b The high-resolution structures of bovine rhodopsin, of A2AR and 2AR, are shown in Figure 7.7.

Figure 7.7 Comparison of GPCR structures. structures. The greatest diversity in these b (a) Bovine rhodopsin (purple), avian 1AR structures lies in the extracellular ends of the (orange) and human A2A adenosine receptors transmembrane helices and the connecting (green) are each superimposed on the human loops; (b) Extracellular views of rhodopsin, the b2AR structure (blue). The extracellular loop 2 b2AR and the A2A adenosine receptor. The (ECL2), intracellular loop 2 (ICL2), cytoplasmic ligands are shown as spheres. Rosenbaum helix 8 (H8) and several of the transmembrane 2009 [2], figure 2. Reproduced with permission segments are indicated on one of the of Nature Publishing Group. 7.3 G Protein-Coupled Receptors 307

7.3.2.1 Overall Structure The general architecture of the GPCRs shows a bundle of seven transmembrane helices that are connected by three alternating extracellular loops (ECL1–3) and three intracellular loops (ICL1–3). The amino terminus is located on the extracellular side, and the carboxy terminus on the intracellular side, while other small helices are found on the intracellular and extracellular sides, running parallel to the cell membrane. Packaging of the transmembrane helices is determined by multiple H-bond interactions between polar amino acids contained in the transmembrane a-helices, and it is thought that the hydrogen-bonding network is a critical determinant of the exact arrangement of the a-helices within the bundle.

Extracellular Loops The extracellular loops are highly variable in size and harbor sites for PTMs as, for example, in the form of the consensus sequence Asn-X-Ser/ Thr for an N-linked glycosylation. Furthermore, the extracellular parts frequently ﬁ b contain conserved Cys residues and disul de bridges. In the 2AR structures, the extracellular loop 2 contains a short a-helix that is stabilized by intra- and inter-loop ﬁ b disul de bonds. Overall, the extracellular loops of A2R and 2AR are arranged in a way that allows access of the diffusible ligand to its binding pocket. In the case of rhodopsin, the covalently bound ligand 11-cis-retinal is less accessible. The extracellular loop 2 of rhodopsin forms a short b-sheet that caps the covalently bound 11-cis-retinal, preventing hydrolysis of the Schiff base. Further shielding is provided by the N-terminus (for details, see Ref. [5]).

Ligand-Binding Site The location of the ligand-binding site is dependent on the size of the ligand and on the size of the extracellular N-terminus and loops. Small, diffusible ligands bind to the interior of the seven-helix bundle, whereas the binding of a large peptide or protein ligands is mediated mostly by the extracellular parts. A schematic representation of the ligand-binding sites for different receptor types is shown in Figure 7.8. The area of ligand binding has been particularly well deﬁned for class A GPCRs that have small, diffusible ligands (e.g., adrenaline, noradrenaline, dopamine, serotonin, histamine). In fact, targeted mutagenesis, biochemical and structural determinations have shown that these ligands are bound in a binding crevice formed by the transmembrane helices. Rhodopsin is a unique case, as the ligand 11-cis-retinal is covalently attached by Schiff base linkage to a Lys residue of transmembrane helix VII. In that case also, the binding site is deeply buried in the interior of the transmembrane segment. A comparison of the available GPCR structures shows that the binding pockets with bound ligands partly overlap. However, there are distinct differences in the binding modes of the various ligands studied, and there are different conformational couplings to the transmembrane helices and the residues that undergo key conformational transitions in GPCR activation. b For both 2AR and rhodopsin, ligand binding is mediated by polar and hydrophobic contact residues from transmembrane helices 3, 5, 6, and 7. In rhodopsin, the retinal chromophore (which mediates light activation) is covalently 308 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.8 Schematic models of ligand--- terminus. Thereafter, the new N termini receptor complexes for structurally diverse activate the receptor as a tethered agonist by ligands interacting with GPCRs. The binding interacting with residues in ECL2. Glycoprotein sites for all small-molecule ligands in family A hormones (e.g., luteinizing hormone, LH) receptors are buried in between the seven interact with the leucine-rich repeat region in transmembrane (7TM) helices. In many the N terminus and extracellular loops of their neuropeptide receptors, the binding sites for receptors. Family C contains a large Venus the native peptides involve residues from both flytrap module (VFM), which contains the extracellular regions and 7TM domains (e.g., binding site for competitive antagonists and angiotensin II), whereas the binding site for agonists in a cleft between two domains. A

substance P in the NK1 neurokinin receptor competitive antagonist-bonded complex is involves only extracellular domains. Thrombin shown here. The N-terminal CRD in Frizzled and serine proteases activate protease- receptors contains the binding site for their activated receptors by cleavage of the N native ligands, the Wnt proteins.

bound in the interior of the bundle. The retinal appears to be coupled more strongly to the conformation of the bundle helices than the GPCRs that have diffusible ligands, and this results in a higher basal activity relative to rhodopsin. A

different binding mode is observed for ligand binding to the A2A receptor, where interactions are mostly found for transmembrane helices 7 and 6, and the interactions extend to the extracellular loops. The comparison shows that GPCRs can support a wide variety of ligand-binding modes that have different degrees of interaction with regions involved in conformational switches that couple ligand- binding to G protein activation. 7.3 G Protein-Coupled Receptors 309

For those receptors that have peptides or proteins as ligands, the structural portions of the extracellular domain – in addition to areas of the transmembrane domain – are involved in ligand binding. The receptors of GPCR subfamily C, which contain a large extracellular N-terminal domain, bind their ligands (e.g., glutamate, c-aminobutyric acid) in this region. Structural information on ligand binding to these receptors is still lacking.

7.3.2.2 Ligand Binding and Effector Activation A key question in understanding the complex signaling behavior is how the binding of ligands leads to distinct rearrangements of the transmembrane helices and the subsequent changes in the cytoplasmic surface that ultimately determine the strength and specificity of effector protein binding and activation. Studies conducted with many different types of agonist and antagonist have shown there to be a wide spectrum of ligand efficacies for individual GPCRs [2]. The natural and synthetic ligands can be grouped into different efficacy classes (see also Section 1.3.2):

Full agonists are capable of maximal receptor stimulation. Partial agonists are unable to elicit full activity, even at saturating concentrations. Neutral antagonists have no effect on signaling activity, but can prevent other ligands from binding to the receptor. Inverse agonists reduce the level of basal or constitutive activity below that of the unliganded receptor.

The different influences of the various ligand types show that efficient conformational coupling between the binding pocket and the site of G protein interaction is dependent on multiple interactions between the receptor and hormone, and requires more than simply occupying the binding site. Ligands containing different subsets of functional groups stabilize distinct conformational states by engaging with distinct subsets of conformational switches in the receptor. These findings lead to a complex picture of GPCR activation in which a particular conformation stabilized by a ligand’s structure determines the efficacy towards a specific pathway.

& Ligand binding to GPCRs: — Via extracellular loops or in the interior of the bundle — Induces reorientation of bundle helices in a ligand-selective fashion — Creates binding sites for downstream effectors.

Ligand binding to the ligand-binding site triggers conformational changes at the cytoplasmic side of the receptor, thus creating a high-afﬁnity binding site for the effector protein. In the classical GPCR signaling mode, the effector protein is a heterotrimeric G protein that is bound to the GPCR via its Ga subunit. This 310 7 G Protein-Coupled Signal Transmission Pathways

binding event may couple back to the ligand-binding site, leading to an increased fi fi b af nity for the ligand. For example, the agonist-binding af nity to the 2-adrenergic receptor is increased almost 100-fold in the presence of the Ga,s subunit, which indicates a functional coupling between effector binding and the ligand binding site of the receptor. The series of conformational changes that lead to receptor activation appear to be specific for receptor subtype [6], and have been best characterized for the light-induced activation of rhodopsin. In rhodopsin, activation is triggered by a light-induced isomerization of the inverse agonist 11-cis-retinal to the full agonist all-trans-retinal. The binding of 11- cis-retinal into a tight binding pocket and the covalent Schiff base linkage to Lys296 are responsible for the exceptionally low basal activity of rhodopsin. A salt-bridge between the protonated Schiff base and the retinal counterion Glu113 represents a key restraint that appears to lock an inactive conformation of TM6. Upon light- induced isomerization of 11-cis-retinal to all-trans-retinal, the salt bridge is broken and a rotation of TM6 is induced that finally leads to conformational rearrangements at the cytoplasmic side of transmembrane helices 5 and 6, which creates a binding site for transducin, the effector G protein of rhodopsin (for details on rhodopsin activation, see Ref. [7]).

& Activation of GPCRs: — Reorientation of helical bundle — New interaction surface at cytosolic site.

7.3.2.3 Binding of Activated GPCR to G Protein The ﬁrst insights into the structural details of effector binding to the activated, ligand-bound GPCR were obtained on the example of the complex between the b 2AR and a nucleotide-free G protein heterotrimer [3]. In the ternary complex, the receptor can be assumed to exist in an activated state that couples to and stabilizes a nucleotide-free G protein.

Interaction surface: b The active state of the 2AR is stabilized by extensive a interactions with the G-domain of the G s-subunit. The interface is formed by transmembrane helices 5 and 6 and the intracellular loop 3 of the receptor, and by the a4 and a5 helices of the Ga-subunit. There are no direct interactions with the Gb- and Gc-subunits of the heterotrimer. The structural parts of the GPCR involved in binding of Gabc show signiﬁcant diversity among individual receptors, and the details of the binding process certainly differ among various GPCR types. Another feature of GPCRs, namely the ability to form dimers or even higher oligomers (see Section 5.3.4), may also inﬂuence the structural changes associated with activation. This point may be of relevance for the binding of the bc-subunits that are not in direct contact with the receptor in the GPCR–Gabc complex. Conformational changes upon activation: The structure of the complex reveals distinct conformational changes of the ligand-bound receptor and of the 7.3 G Protein-Coupled Receptors 311

Ga-subunit of the heterotrimer as compared to an inactive GPCR structure and the free G protein heterotrimer. The conformational changes on the receptor affect mostly transmembrane helices 5 and 6 and the second intracellular loop. The largest difference between the inactive and active structures is a large outward movement of helix 6. There is a smaller outward movement and extension of the cytoplasmic end of the helix 5 by seven residues. Another notable difference between inactive and active structures is the second b intracellular loop, which forms an extended loop in the inactive 2AR structure a b – abc and an -helix in the 2AR G complex.

A striking conformational change is observed for the G-domain of the Ga- subunit. As compared to the inactive, GDP-bound state of Ga, this domain undergoes a large rotation in the ternary complex. The a5-helix of the G-domain undergoes a signiﬁcant conformational change, and the carboxy-terminal end of Ga projects into the transmembrane core of the receptor.

7.3.2.4 Ligand-selective GPCR Signaling Studies on a large number of ligands for a particular GPCR have shown that different ligands can selectively recruit different intracellular signaling pathways to produce various phenotypic effects in cells. This phenomenon is explained by the conformational flexibility of the receptors, which can assume many conformations [8], each of which can potentially interact with a distinct ligand in a highly selective manner. In turn, a specific conformation selectively recruits a specific intracellular effector protein or signaling complex (Figure 7.9). As a consequence, the preponderance of a specific signaling complex in a particular cell will help to

Figure 7.9 Ligand-induced selective signaling active receptor states are selectively coupled to by GPCRs. Schematic depicting the concept of different signaling complexes (SC1---SC4) that multiple active states (R1---R4) of a single give rise to different effects in cells. Milar 2009 GPCR (gonadotropin-releasing hormone [8], figure 5. Reproduced with permission of (GnRH) receptor) that are selective for Elsevier. different agonist ligands (L1---L4). The different 312 7 G Protein-Coupled Signal Transmission Pathways

stabilize a certain GPCR conformation, thereby inducing selectivity for a certain ligand. For a particular GPCR, the chemical nature of the ligand may determine which downstream effector proteins and signaling paths will be activated. As outlined in Sections 7.4 and 7.8, GPCRs may signal through two major downstream effector proteins – the G- proteins and the arrestins – and ligands are known that are biased towards signaling through these two effectors. For example, G protein-biased agonists will stabilize a receptor conformation that favors the recruitment of a Ga-subunit, whereas arrestin-biased agonists will favor a receptor conformation that preferentially binds arrestins. Thus, these agonists may differentially activate distinct intracellular signaling paths.

7.3.3 Regulation of GPCRs

The GPCRs form part of signaling networks with links to several major signaling pathways of the cell. These links provide positive and negative signals to regulate the duration and intensity of GPCR signaling in coordination with other incoming signals, and they deliver distinct signals to other signaling paths. Aside of the classical signaling of GPCRs via G proteins, the GPCRs also mediate cell signaling by functioning as scaffolds for the recruitment of a large number of GPCR- interaction proteins, either transmembrane proteins or cytosolic proteins. These proteins play important roles in regulating receptor ligand speciﬁcity, receptor endocytosis and receptor recycling [9]. Another major function of GPCRs within the signaling network of the cell is to generate signals by G protein-independent mechanisms, the best-characterized of which is b-arrestin-mediated signaling (Section 7.8). The important mechanisms involved in the regulation of GPCRs are summarized in Figure 7.10. A large number of processes have been recognized to regulate GPCR functions. These include:

PTMs, mainly phosphorylation Association with ligand-binding and signal-modifying proteins Binding of distinct effector proteins Internalization, trafﬁcking Oligomerization.

How these different processes cooperate and interact is not well understood, and only selected aspects of GPCR regulation can be presented in the following sections. Given the large number of the GPCRs found in the human genome, and their very diverse signaling functions, it is not surprising that many proteins have been discovered which interact with the receptor in an agonist-dependent or an agonist-independent manner. Most regulatory inﬂuences are mediated by the cytoplasmic loops and the C-terminal tail of the receptor. 7.3 G Protein-Coupled Receptors 313

Figure 7.10 Potential mechanisms for the and, once at the cell surface, through regulation of GPCRs. Schematic describing oligomerization and interactions with various how GPCRs can be regulated at many levels: other nonreceptor proteins or through from their biosynthesis (gene transcription, modifications such as differential translation, and posttranslational processing) phosphorylation. through their trafficking to the cell membrane

& Proteins interacting with the C terminus of GPCRs: — Ga subunits of G proteins — RAMPS — Scaffolding proteins — GRKs — PDZ-containing proteins — Arrestins.

In addition to the canonical binding partner at the cytoplasmic side, the heterotrimeric G protein, a large number of other proteins have been shown to interact with cytoplasmic regions of the GPCR to modulate receptor function. The most important classes of GPCR-interacting proteins are:

G protein-coupled receptor kinases (GRKs) (see Section 7.3.3.3) Arrestins: See Sections 7.3.3.4 and 7.8 Receptor-activity modifying proteins (RAMPs): These are single-span transmembrane proteins that modulate diverse functions of the GPCRs belonging to class B, as for example the calcitonin receptor. RAMPS appear to regulate the ligand-binding properties, the intracellular trafﬁcking, and the PTM (e.g., glycosylation) of GPCR targets. 314 7 G Protein-Coupled Signal Transmission Pathways

Regulators of G protein signaling (RGS): These proteins are negative regulators of G protein signaling (Section 7.5.3.2). Members of this protein family have been shown to interact with ICL3 of GPCRs. Scaffolding proteins: GPCRs can form large signaling complexes where various types of signaling proteins cooperate. These large complexes are assembled with the aid of scaffolding proteins. Examples are members of the adapter protein family AKAP, the scaffolding proteins, Shank, and spinophilin. PDZ-containing proteins: Many GPCRs contain a PDZ-binding domain in the C terminus that recognizes and binds to PDZ modules (Section 10.2.1). These modules are found on a large number of signaling proteins and serve as scaffolding or interaction modules for the formation of higher protein complexes. For example, subtypes of PLC-b are recruited to GPCRs via speciﬁc PDZ proteins. Furthermore, GPCR–PDZ protein interactions serve to regulate intracellular trafﬁcking and the subcellular distribution of GPCRs.

As illustrated by these examples, protein binding to the C terminus serves mainly the formation of higher signaling complexes and the trafﬁcking and speciﬁc localization of the receptor (for a review, see Ref. [9]).

7.3.3.1 Phosphorylation of GPCRs Typically, GPCRs are subjected to various PTMs such as phosphorylation, acetylation, palmitoylation, myristoylation and ubiquitination that modify and regulate their functional properties. One common modification is phosphorylation of the cytoplasmic region of GPCRs [10]. Following agonist occupation, GPCRs become multiply phosphorylated, mostly at the C-terminal tail and ICL3. Whereas, the majority of the receptor phosphor- acceptor sites are in Ser/Thr-rich regions of the intracellular domains, phosphorylation of Tyr-sites has also been observed, generating P-Tyr protein-interaction motifs (see Section 10.1). This diversity of phosphorylations is mediated by various protein kinases, such as G protein-coupled receptor kinase (GRK), protein kinase A (PKA), protein kinase B (Akt kinase), protein kinase C (PKC), members of the casein kinase family (e.g., CK2), and receptor tyrosine kinases as for example, the insulin receptor. The phosphorylation patterns generated by the protein kinases are assumed to represent a bar code that dictates which effector protein binds to the GPCR and which type of signaling complex forms. Characteristic fingerprints of receptor phosphorylation have been detected in different cells, each with its own spectrum of protein kinases. This observation suggests that the differential phosphorylation of GPCRs is an important tool for generating cell type-specific responses to GPCR activation.

& Phosphorylation of GPCRs: — In the cytoplasmic region — Catalyzed by multiple protein kinases — Regulates binding of effectors — Involved in signal switching. 7.3 G Protein-Coupled Receptors 315

The biochemical functions of receptor phosphorylation appear to be manifold. A major function, mediated mostly by GRKs, is receptor desensitization (see the following section). Other functions include the use of alternative effector proteins as for example, arrestin (Section 7.8) and coupling to distinct downstream signaling pathways. Phosphorylation may occur in an agonist-dependent fashion (e.g., binding of arrestin), and may occur also independently of the bound agonist. For example, agonist-independent Ser/Thr phosphorylation can be used as a switch for coupling a given receptor to different Ga subunits. PKA-mediated phosphoryla- b tion of the 2AR has been shown to switch coupling of the receptor from Gs to Gi.

7.3.3.2 Desensitization and Downregulation of GPCRs One phenomenon often seen in transmembrane receptors in general – and in GPCRs in particular – is desensitization (Figure 7.11), which involves a weakening and downregulation of the signal transmission under conditions of long-lasting stimulation by hormones, neurotransmitters, or sensory signals. GPCRs are dynamically regulated to adapt responses to the intensity of extracellular signals, such as ligand concentration or the strength of a physical stimulus. Despite the persistent effect of extracellular stimuli, the signal is no longer passed into the cell interior, or is passed only in a weakened form, during conditions of desensitization and downregulation.

& Homologous desensitization: — Agonist-dependent Heterologous desensitization: — Agonist-independent

Figure 7.11 General principle of signal chain, triggering inhibition of the signal desensitization of GPCRs. Desensitization of a chain. Receptor systems may also mutually hormone-bound receptor can occur via two influence one another, in that a signal protein principal routes, shown schematically in the X formed in one signal chain mediates the figure. A suppressing influence may be exerted desensitization of another receptor system R, on the receptor system via proteins (X) of a and vice versa. 316 7 G Protein-Coupled Signal Transmission Pathways

This is associated with a decrease of receptor molecules caused by an uncoupling of the receptor from downstream signaling molecules, and by increased endocytosis/internalization and the degradation/recycling of receptors. Desensitization may occur in either a homologous or in a heterologous fashion, and typically uses phosphorylation as a ﬁrst step. In the former case, agonist- dependent phosphorylation of GPCRs by GRKs initiates desensitization. In the latter case, the GPCR is modulated by signals derived from other signaling pathways, for example, from nuclear receptor signaling (Chapter 6) or receptor tyrosine kinase signaling (Chapter 10). Heterologous desensitization is independent of agonist binding and leads to the phosphorylation of both active and inactive receptors. A major path of desensitization uses the binding of arrestin to the phosphorylated regions of the GPCR, followed by further processes that occur on short and long timescales. During short-term desensitization, receptor signaling is blocked by the binding of arrestin to the phosphorylated sites. A long-term downregulation is achieved by arrestin-mediated internalization and recyclization of the receptor.

7.3.3.3 GPCR Phosphorylation by GRKs A major pathway for receptor desensitization is initiated by phosphorylation of GPCRs mediated by GRKs [11]. Desensitization consists of a two-step process in which the agonist-bound receptor is ﬁrst phosphorylated by a GRK and then binds an arrestin protein which interrupts signaling to the G protein.

& Homologous desensitization: — By GRKs, as for example, Rhodopsin kinase bARK.

The GRKs recognize and phosphorylate only the active, agonist-occupied form of the GPCR. Typically, ICL3 and/or the C-terminal tail are multiply phosphorylated and the presence of the phosphate residues serves as major recognition element for arrestin binding. GRKs belong to the AGC family of protein kinases (Section 9.3) and seven GRKs (GRK1–7) have been identiﬁed in humans. The well-characterized GRK for b ﬁ rhodopsin, rhodopsin kinase, belongs to the GRK1 subfamily, while 2AR-speci c GRK is a member of the GRK2 subfamily. This kinase is also known as the b-adrenergic receptor kinase (bARK). Some of the GRKs shuttle between the cytoplasm and the membrane in an activation-dependent manner. During translocation of GRKs to the membrane- localized receptor, the bc-subunit of the G protein, as well as the binding of phosphatidylinositol trisphosphates (see Section 8.6), plays an important role. 7.3 G Protein-Coupled Receptors 317

& Membrane attachment of GRKs is enhanced by: — bc dimer — PtdIns compounds — PtdIns compounds being bound to PH domain.

When an agonist binds to a GPCR, it causes the receptor to associate a heterotrimeric G protein, leading to dissociation into its a- and bc-subunits. The bc dimer, which carries a prenyl membrane anchor, binds to the GRK and thereby promotes its membrane association. In a cooperative way, the binding of phosphatidyl inositol-messengers to the PH-domain of GRKs enhances binding of the GRK to the membrane. Membrane association of some GRKs is also mediated by farnesyl or palmitoyl anchors (Section 2.9.3). For many GPCRs, GRK-mediated phosphorylation on its own is insufﬁcient to mediate receptor desensitization. Instead, the recruitment of arrestin proteins to agonist-occupied and phosphorylated receptors enhances an uncoupling of the receptor from the heterotrimeric G protein. However, for some receptors, phosphorylation-independent desensitization has been reported [9].

7.3.3.4 Binding of Arrestin Receptor phosphorylation by GRKs triggers several reactions (Figure 7.12), of which high-afﬁnity binding of arrestin proteins is most important. The family of arrestins includes the visual arrestins (arrestin1 and arrestin4) that are speciﬁc for rhodopsin and the arrestins2 and 3. The latter two isoforms are most important for the desensitization of activated receptors, as for example, the bAR.

& Arrestin: — Binds to the phosphorylated C terminus of GPCR — Mediates GPCR desensitization — Triggers the internalization of GPCRs — Couples GPCRs to other signaling paths.

The arrestins are multifunctional proteins that function as scaffolds for the formation of signaling complexes at GPCRs, and a large number of proteins have been shown to interact specifically with the arrestin isoforms [9,12]. Arrestin binding has been shown to involve mainly two sites on the receptor: one site that is revealed upon agonist binding (i.e., receptor activation), and a second site that requires receptor phosphorylation. These sites are assumed to contribute differentially to the recruitment of arrestins to distinct GPCR members. Overall, the arrestins are conformationally flexible proteins that are capable of adopting multiple conformations which in turn reveal a specific set of interaction domains that specify the nature of the GPCR bound and the nature of further signaling proteins. Most importantly, arrestins can bind differentially to distinct 318 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.12 Receptor desensitization: protein kinases of the MAPK cascade phosphorylation, arrestin binding and associated. This serves as a trigger for internalization. The activated, agonist-bound internalization of the receptor to endosomes. receptor is phosphorylated on the cytoplasmic The receptor may now be dephosphorylated region by a G protein-coupled receptor protein and transported back to the cell membrane kinase (GRK). The phosphate residues serve as (not shown in the figure). attachment sites for b-arrestin which has

active conformations of a given GPCR, which allows for agonist speciﬁc interactions between arrestins and GPCRs. The recruitment of arrestins to GPCRs serves several functions:

Desensitization: Arrestin binding leads to decoupling of the phosphorylated receptor from the interaction with the heterotrimeric G protein next in the sequence, so that signal transmission is suppressed. In subsequent steps, the GPCR is dephosphorylated by phosphodiesterases and becomes resensitized. Arrestin binding serves, for example, to rapidly weaken signal transmission during the vision process, during conditions of longlasting light stimulus. Internalization, trafﬁcking, and recycling: Following b-arrestin binding, the phosphorylated receptor can be translocated into the cell interior. In this process, 7.3 G Protein-Coupled Receptors 319

which occurs on a longer time scale, b-arrestin serves as an adapter that targets the receptor for internalization via endocytosis. In addition to receptor binding, the b-arrestins bind to clathrins and other proteins that form the clathrin-coated vesicle machinery. The receptor is thereby internalized in the membrane- associated form, dephosphorylated, and then transported back to the cell membrane. Translocation into the cell interior serves, in particular, to weaken signal transmission during conditions of longlasting hormonal stimulation.

7.3.3.5 Signal Switching In another reaction, the Ser/Thr phosphorylation of GPCRs can be used as a switch for coupling a given receptor to different Ga subunits, the downstream effector protein. For example, the PKA-mediated phosphorylation of bAR has been shown to switch coupling of the receptor from Gs to Gi and to trigger a new set of downstream signaling reactions.

7.3.3.6 Coupling to other Signaling Pathways Arrestin has been now recognized as an important component for the coupling of GPCRs to other signaling pathways in a manner that is independent of G proteins. This aspect of GPCR signaling will be discussed in Section 7.8.

7.3.4 Oligomerization of GPCRs

Although the GPCRs were generally believed to function as monomeric entities, there is now abundant evidence that they form functional oligomers in vivo. Both, biochemical and biophysical studies have shown that GPCRs can exist as homodimers, as heterodimers, or as higher oligomers. Oligomerization has been shown to affect receptor function in various ways (reviewed in Ref. [13]). Receptors that are inactive in binding or signaling can become active as oligomers, whereas receptors that are intrinsically active as monomers can acquire new activities as oligomers. Oligomerization has been demonstrated for a large number of GPCRs. Indeed, for class C GPCRs homodimerization and the formation of heterodimers with related members of the same subfamily appears to be a common property [14]. The structural, functional and mechanistic consequences of oligomeric receptor complex formation appear to be manifold, with ligand binding, effector interaction, phosphorylation and trafﬁcking/endocytosis each being modulated by oligomerization. For example, changes in effector interaction and signal switching have been demonstrated for dopamine D1 and D2 receptors (Figure 7.13) which, as monomers, signal through Ga,s and Ga,i, respectively, whereas the D1/D2 heteromers utilize another Ga subunit for signaling, namely Ga,q.

& GPCRs may function as either homodimers or heterodimers 320 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.13 Signal switching by dopamine through coupling to Ga,s and Ga,i, respectively. receptor heteromers. When individually However, SKF 83959-activated D1/D2 receptor expressed, dopamine D1 and D2 receptors are heteromers couple to Ga,q, which equates to activated by the agonist SKF 83959 and signal in vivo signaling. Adapted from Ref. [8].

7.4 Regulatory GTPases

Summary The heterotrimeric G proteins, the major effector proteins of the GPCRs, belong to the superfamily of regulatory GTPases. Members of this superfamily perform a general switch function that is based on a cyclical, unidirectional transition between an active, GTP-bound state and an inactive, GDP-bound state. The transition between the active “on state” and inactive “off-state” states occurs in a cyclic process that can only run in one direction because of the irreversible hydrolysis of GTP. In both inactive and active states, the proteins of the GTPase superfamily possess a specific affinity to other signaling proteins that are upstream or downstream parts of the reaction chain. When in the GTP-bound state, the regulatory GTPases interact with downstream effectors to transmit the signal further. The switch function of the GTPases is regulated mainly by two types of protein: (i) guanine nucleotide exchange factors (GEFs) that promote the exchange of bound GDP for GTP, which leads to activation of the GTPase for further signal transduction; and (ii) proteins that activate the GTPase activity (GAPs) and function as negative regulators of the GTPases by decreasing the amount of the active GTP-bound state. A common structural domain of the GTPases is the G-domain (also called Ras-domain), which undergoes distinct conformational changes in switching from the inactive to the active state, and vice versa. 7.4 Regulatory GTPases 321

The heterotrimeric G proteins – the major effector proteins of the GPCRs – belong to the large family of regulatory GTPases that bind and hydrolyze GTP and thus function as a switch in central cellular processes. The family of regulatory GTPases is also referred to as the GTPase superfamily.

7.4.1 The GTPase Superfamily: General Functions

Proteins of the GTPase superfamily are found in all plant, bacterial, and animal systems. The following examples illustrate the central functions of the regulatory GTPases in the cell, and their involvement in:

protein biosynthesis on ribosomes; signal transduction at membranes; visual perception; senses of smell and taste; control of differentiation and cell division; translocation of proteins through membranes; and transport of vesicles in the cell.

The members of the GTPase superfamily show an extensively conserved reaction mechanism. One common trait is a switching function that enables a reaction chain to be switched on or off.

7.4.2 Switch Functions of GTPases and the GTPase Cycle

The regulatory GTPases are involved in signaling chains by functioning as a switch. Incoming signals from activated upstream signaling components are received by the GTPases and passed on to downstream components of the signaling chain that are themselves activated for transporting the signal further.

& Regulatory GTPases act as switches: — Off-state: GDP-bound state — On-state: GTP-bound state.

The switch function of GTPases is based on a cyclical, unidirectional transition between an active, GTP-bound form, the “off state”,andaninactive, GDP-bound form, the “on-state” (Figure 7.14). In both inactive and active forms, the proteins of the GTPase superfamily possess a speciﬁcafﬁnity to other signaling proteins that are either upstream or downstream parts of the reaction chain. The GTPases bring about the transition between the active and inactive states in a cyclic process that can only run in one direction because of theirreversiblehydrolysisofGTP. 322 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.14 The switch function of the nucleotide dissociation inhibitors (GDIs). The regulatory GTPases. The GTP form of the regulatory GTPases run through a GTPase regulatory GTPases represents the “switched- cycle, the signals of which flow into via GEFs on” form of the GTPase, while the GDP form, and are conducted further in the form of the in contrast, represents the “switched-off” GTPase---GTP complex to effector molecules form. The switch function of the regulatory further down the sequence. Hydrolysis of the GTPases may be controlled by guanine bound GTP ends the activated state. The rate nucleotide exchange factors, by GTPase- of GTP hydrolysis is either intrinsically activating proteins (GAPs), and by G- determined or may be accelerated via GAPs.

The upstream signaling component must be in the active state in order to interact with the inactive GDP-state of the GTPase. This interaction triggers an exchange of bound GDP for GTP and induces thereby the transition into the “on state,” the GTPaseGTP form. From the GTP-bound state, the signal is passed on to the effector molecule next in sequence, and this in turn is activated for further signal transmission. Hydrolysis of the bound GTP by an intrinsic GTPase activity converts the GTPase from the “on state” into the “off state,” and disrupts the signal transmission. The system is now again in the inactive GTPaseGDP ground state and is ready to receive a further signal. 7.4 Regulatory GTPases 323

7.4.2.1 Modulation and Regulation of the Switch Function There are two processes in the GTPase cycle where the cycle can be switched on and switched off, and where the main regulation of GTPase signaling occurs: GDP–GTP exchange and GTP hydrolysis. For most GTPases these reactions are intrinsically very slow, and their acceleration by speciﬁc proteins is used by the cell to switch signaling on and off, or to modulate it. The following types of protein serve as the main regulators of information ﬂow through regulatory GTPases.

Guanine Nucleotide Exchange Factors: Signal Input The receipt of a signal by the regulatory GTPases manifests itself as an increased rate of dissociation of GDP. Most GTPases bind GDP very strongly, and hence the intrinsic rate of GDP dissociation is very low.

& Guanine nucleotide exchange factors (GEFs) deliver input signals by promoting GDT/GTP exchange

The rate of dissociation of GDP may be increased by speciﬁc proteins known as guanine nucleotide exchange factors (GEFs), which function as the upstream signaling protein for the GTPases. For the heterotrimeric G proteins, the agonist- bound, activated receptor is the exchange factor.

GTPase Activating Proteins: Signal Attenuation and Termination Most GTPases show a very slow intrinsic rate of GTP hydrolysis which is equivalent to a long lifetime of the activated state. GTPase activating proteins (GAPs) increase the rate of GTP hydrolysis and thereby reduce the lifetime of the active, GTP-bound state. The GAP protein class is an important instrument for controlling the rate of signal transmission.

& GAPS negatively regulate G protein signaling by enhancing GTPase activity up to 105-fold

The activation of GAPs leads to a weakening of signal transmission, and the GAPs mostly function as negative regulators for signaling by GTPases. For example, the small regulatory GTPase Ras has a low intrinsic GTPase activity (Chapter 11), but this may be increased about 105-fold by the corresponding GTPase-activating protein. Often, the activity of the GAPs is regulated by other signaling pathways. Thus, a regulatory inﬂuence on signal transmission via G proteins can be achieved from other signaling pathways.

Guanine Nucleotide Dissociation Inhibitors (GDIs) The dissociation of GDP or of GTP may be inhibited by speciﬁc proteins known as guanine nucleotide dissociation inhibitors (GDIs). Proteins with this function act on members of the superfamily of Ras proteins (see Section 11.4). 324 7 G Protein-Coupled Signal Transmission Pathways

& GDIs inhibit GDP or GTP dissociation from the complex with Ga

The GDIs have the function, above all, to provide a cytosolic pool of inactive, GDP-bound proteins.

7.4.3 Inhibition of GTPases by GTP Analogs

Nonhydrolyzable GTP analogs are an indispensable tool in the identiﬁcation and structural and functional characterization of GTPases. The GTP analogs shown in Figure 7.15, GTPcS, b,c-methylene GTP and b,c-imino GTP, are either not hydrolyzed by GTPases or are hydrolyzed only very slowly. The addition of these analogs ﬁxes the G-protein in the active form, where it is permanently switched on.

& Nonhydrolyzable GTP analogs fix GTPase in GTP-state: — GTPcS, — b,c-methylene GTP — b,c-imino GTP.

Figure 7.15 Examples of nonhydrolyzable GTP analogs. 7.4 Regulatory GTPases 325

For cellular signal transduction, this means a permanent activation of the signal transmission pathway. In many cases, a role for G proteins in a signal chain was inferred from the observation that nonhydrolyzable GTP analogs bring about a lasting activation of signal transmission. The GTP analogs were equally important for structural determination of the activated form of GTPases. The formation of a stable complex between the nonhydrolyzable GTP analog and different GTPases has enabled the crystallization and structure determination of the complex in its activated form.

7.4.4 The G-Domain as a Common Structural Element of the GTPases

One common property of the GTPases is the enzymatic activity of GTP hydrolysis. GTP binding and hydrolysis take place in a domain of the GTPases known as the G- domain or Ras domain. The G-domain is found in all GTPases; the G-domain of the bacterial elongation factor EF-Tu is shown in Figure 7.16. In all of the GTPase structures known at present, the G-domain has a very similar architecture and a very similar means of binding the guanine nucleotide. The sequence element GX4GK(S/ T) is a consensus sequence for guanine nucleotide binding; this sequence is involved in binding the b- and c-phosphate of GTP and GDP, and is also known as the P-loop.

Other consensus sequences, such as RX2T and DX2G, are involved in both binding the c-phosphate and in the GTPase reaction (X ¼ any amino acid). A further consensus sequence (N/T)(K/Q)XD and SA interacts with the guanosine.

Figure 7.16 Structure of the G-domain of the and the switch regions I and II are shown, elongation factor EF-Tu from Thermus which play an important role in transition from thermophilus with bound GppNH. The the inactive GDP form to the active GTP form. nonhydrolyzable analog GppNHp, the P-loop 326 7 G Protein-Coupled Signal Transmission Pathways

7.4.5 The GTPase Families

The superfamily of GTPases, with over 100 members, is subdivided according to sequence homologies, molecular weight and subunit structure into further (super) families. These are the families of the heterotrimeric G proteins, the Ras superfamily of small GTPases, and the family of initiation and elongation factors (Figure 7.17).

& Major GTPase families: — Heterotrimeric G proteins — Ras superfamily — Initiation, elongation factors.

The heterotrimeric G proteins are built up of three subunits, with the GTPase activity localized on the largest a-subunit (Section 7.5). The members of the Ras family of GTPases, in contrast, are monomeric proteins with a molecular weight of about 20 kDa (Chapter 11). A further functionally diverse class is composed of the proteins involved in protein biosynthesis and membrane transport. GTPases with functions in protein biosynthesis include the elongation factors, termination factors and peptide translocation factors. These are mostly monomeric proteins with molecular weights of 40–50 kDa. GTPases of this class are also found in protein complexes such as the “signal recognition particle” (SRP) and the corresponding receptor.

Figure 7.17 The superfamily of regulatory GTPases. SRa,SRb and SR54 are GTPases involved in protein translocation at the endoplasmic reticulum. 7.5 The Heterotrimeric G Proteins 327

Both protein complexes are needed during ribosomal protein biosynthesis, for the transport of newly synthesized proteins through the endoplasmic reticulum.

7.5 The Heterotrimeric G Proteins

Summary The heterotrimeric G proteins are constructed from three subunits: a large a-subunit of 39–46 kDa; a b-subunit of 36 kDa; and a c-subunit of 8 kDa. The a-subunit has a binding site for GTP or GDP and carries the GTPase activity. The b- and c-subunits exist as a tightly associated complex and are active in this form. Most signaling functions of G proteins are realized by the a-subunits that

are divided into four families, the Gs,Gi,Gq, and G12. Like all regulatory GTPases, the heterotrimeric G proteins run through a cyclical transition between an inactive, GDP-bound form and an active, GTP-bound form. Thereby, the activated GPCR functions as a nucleotide exchange factor, GEF, to promote formation of the active GaGTP state. The signaling function of the G proteins is downregulated by proteins with GAP activity, such as the RGS proteins and downstream effectors. The Gbc complex performs its own signaling functions, not only in pathways associated with activated GPCR but also in pathways that are independent of GPCRs. Additional negative regulators of G protein signaling are the phosducin proteins that bind to the Gbc complex. Signaling by G proteins is intimately linked to the inner side of the cell membrane due to lipid modification of the bc- and a-subunits. The most important downstream effectors of G proteins are adenylyl cyclase, phospholipases, ion channels and cGMP-specific phosphodiesterases. The activation of these enzymes leads to concentration changes of diffusible signal molecules such as cAMP, cGMP, þ diacylglycerol, inositol triphosphate, and Ca2 , which trigger further signaling reactions.

The heterotrimeric G proteins are the most prominent reaction partners of receptors composed of seven transmembrane helices, which is why these receptors are also known as GPCRs. From the G protein, the signal is then passed on to the effector protein next in the sequence.

& Heterotrimeric G proteins of mouse/human: — a-subunit: 39–46 kDa, more than 16 genes — b-subunit: 36 kDa, five genes — c-subunit: 8 kDa, 12 genes.

One common structural feature of the G proteins is their construction from three subunits (Figure 7.18): a large a-subunit of 39–46 kDa, a b-subunit of 36 kDa, and a 328 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.18 Structure and activation of the effector molecules. The a- and c-subunits are heterotrimeric G proteins. Reception of a associated with the cell membrane via lipid signal by the receptor activates the G protein, anchors. Signal reception and signal which leads to exchange of bound GDP for transmission of the heterotrimeric G proteins GTP at the a-subunit and to dissociation of the take place in close association with the cell bc-complex. Further transmission of the signal membrane. This point is only partially shown may take place via Ga-GTP or via the in the figure. bc-complex, which interact with corresponding

c-subunit of 8 kDa. The a-subunit has a binding site for GTP or GDP and carries the GTPase activity. The b- and c-subunits exist as a tightly associated complex and are active in this form. All three subunits show great diversity. In the human and mouse genomes there are at least 16 genes for the a-subunits, five genes for b-subunits, and 12 genes for c-subunits. Specificity of the switch function is mostly determined by the a-subunit: the a-subunit carries out the specific interaction with the receptors preceding it in the signal chain, and with the subsequent effector molecules. The bc-complex is also involved in signal transmission to the effector proteins.

7.5.1 Classification of the Heterotrimeric G Proteins

Most functions of signal transmission by G proteins are realized by the a-subunit. As different G proteins interact with very different partners, there are signiﬁcant 7.5 The Heterotrimeric G Proteins 329

Table 7.2 Classification of the heterotrimeric G proteins according to the a-subunits.

Subunit Tissue Examples of receptors Effector protein, function

Gs a b " s Ubiquitous -adrenergic Adenylyl cyclase þ receptor, glucagon Ca2 channels " receptor aolf Nasal epithelium Olfactory receptor Adenylyl cyclase "

Gi a a a a þ " i1, i2, i3 Mostly ubiquitous 2-adrenergic K channels þ receptor Ca2 channels # þ aoA Brain a2-adrenergic K channels " þ receptor u.a. Ca2 channels # at1 transducin Retina Rhodopsin cGMP-speciﬁc phosphodiesterase " ag Taste buds Gustducin cNMP-speciﬁc phosphodiesterase " a " z Brain Adenylyl cyclase Gq a a b " q Ubiquitous 1-adrenergic Phospholipase C- receptor u.a. a11, a14, a15, a16,

G12 a12, a13 Ubiquitous Thromboxane GEFs and GAPs receptor for Rho GTPase

differences in the structures of the a-subunits. Because of the common GTPase domain and the common interaction with the bc-subunits, however, there are also considerable sequence homologies. Based on a comparison of the amino acid sequences, the Ga-proteins may be divided into four families of Gs,Gi,Gq, and G12, the details of which are summarized in Table 7.2, together with representative members and their characteristic properties.

& Gafamilies: — Gs — Gi — Gq — G12

An overview of the main patterns of signaling through the various Gs families is shown in Figure 7.19. 330 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.19 Common patterns of coupling inositol-1,4,5-trisphosphate (Section 8.4); between GPCRs and G proteins. PLC-b, DAG, diacylglycerol (Section 8.8); Rho, Rho phospholipase C-b (Section 7.7.2); PI3-K, guanine nucleotide exchange factor (Section phosphatidyl-inositide-3-kinase (Section 9.4); 11.3); GABA, c-aminobutyric acid; 5-HT, 5- PKC, protein kinase C (Section 9.5); Ip3, hydroxytryptamine. 7.5 The Heterotrimeric G Proteins 331

7.5.1.1 Gs Subfamily a A characteristic of -subunits of the Gs subfamily is that they are inhibited by cholera toxin (Section 7.5.2). Members of the Gs subfamily are activated by hormone receptors, by odor receptors, and by taste receptors. Gs-proteins mediate, for example, signal transmission by b-adrenergic receptors and by glucagon receptors.

& Gs: — Activation of adenylyl cyclase.

When percepting taste, the taste receptors are activated and pass the signal on via “ ” the olfactory G protein, Golf. The perception of sweet taste is also mediated via Gs- proteins. Transmission of the signal further involves an adenylyl cyclase in all cases, the activity of which is stimulated by the Gs-proteins.

7.5.1.2 Gi Subfamily ﬁ The rst members of the Gi subfamily to be discovered displayed an inhibitory “ ” effect on adenylyl cyclase, hence the name Gi for inhibitory G proteins. Further b members of the Gi subfamily have phospholipase C (PLC)- (Section 7.7.2) as the corresponding effector molecule. Signal transmission via PLC-b ﬂows into the inositol triphosphate and diacylglycerol pathways (see Chapter 8).

& Gi: — Inhibition of adenylyl cyclase — Activation of PLC-b

The Gt- and Gg-proteins are also classed as Gi-proteins, based on sequence homologies. The Gt- and Gg-proteins are involved in transmitting sensory signals. Signal transmission in the vision process is mediated via G proteins known as transducins (Gt). The Gt-proteins are activated by the photoreceptor rhodopsin and are located in the rods and cones of the retina. The sequential effector molecules of fi the Gt-proteins are cGMP-speci c phosphodiesterases. a Perception of bitter taste can take place via -subunits of the Gi class; the a-subunit of these G proteins, known as gustducin, is highly homologous with transducin. The corresponding receptors are for example, the taste receptors T2R [15]. A phosphodiesterase with specificity for cyclic nucleotides and a cyclic nucleotide-gated ion channel have been identified as downstream components of the signaling cascade. Signal transmission evidently takes place here in a similar fashion to the vision process.

Apart from a few exceptions (Gz), the members of the Gi family are characterized by their inhibition by pertussis toxin (Section 7.5.2). 332 7 G Protein-Coupled Signal Transmission Pathways

7.5.1.3 Gq Subfamily ﬁ The members of the Gq subfamily are not modi able by pertussis toxin or cholera toxin. The signal protein next in the reaction sequence is generally the b-type of PLC.

& Gq: — Activation of PLC-b.

7.5.1.4 G12 Subfamily The G12 subfamily has been implicated in cellular processes such as reorganization of the cytoskeleton, activation of the c-Jun N-terminal protein kinase (Sec- þ þ tion 12.4.2), and stimulation of Na /H exchange.

& G12: Activation of GAP for Rho GTPase

Activation by thromboxane and thrombin receptors has been described for

members of the G12 subfamily. Examples of effector molecules include nucleotide exchange factors for Rho proteins and the GTPase-activating protein Ras-GAP (see Chapter 11).

7.5.2 Toxins as Tools in the Characterization of Heterotrimeric G Proteins

Two bacterial toxins, namely pertussis toxin and cholera toxin, were of great importance in determining the function of G proteins. Both toxins catalyze ADP ribosylation of proteins, during which an ADP-ribose residue is transferred from þ NAD to an amino acid residue of a substrate protein (Figure 7.20). Cholera toxin is composed of A and B subunits [16]. The A1 domain of the A

subunit catalyzes the ADP ribosylation of an arginine residue (Arg174 in Gt, Arg201 in Gs) in various Ga-subunits.

& Cholera toxin:

Catalyzes ADP ribosylation of Arg on Ga,s thereby fixing the GTP-state

The Arg174 residue of Ga,t contacts the phosphate group of the bound GTP and is involved in stabilization of the transition state of GTP hydrolysis. Modiﬁcation of Arg174 by ADP ribosylation interferes with this function and inactivates the GTPase activity of the G protein. Consequently, the intrinsic deactivation

mechanism of the Gs-protein is suspended. The G protein is constitutively activated and the downstream effector molecules are – without any hormonal stimulation – permanently activated. 7.5 The Heterotrimeric G Proteins 333

þ Figure 7.20 ADP-ribosylation of the Ga- the reaction, the ADP-ribose residue of NAD subunit of transducin by cholera toxin. Cholera is transferred to Arg174 of Ga,t, which toxin catalyzes the ADP-ribosylation of the inactivates the GTPase activity of Ga,t. a-subunit of the G protein transducin. During

The constitutive activation of Gs-proteins by cholera toxin is the cause of the devastating effect of the cholera bacterium, Vibrio cholerae, on the water content of the intestine. Because of the lack of deactivation of the Gs-protein, adenylyl cyclase which is next in the reaction sequence is constantly activated, so that the level of cAMP in the cells of the intestinal epithelium is greatly increased. This, in turn, leads to an increased active transport of ions such that an excessive efﬂux of water þ and Na takes place in the intestine. Pertussis toxin, formed by Bordetella pertussis, the causative organism of whooping cough, carries out an ADP ribosylation at a cysteine residue close to the C terminus of the a-subunits. The modiﬁcation prevents activation of the G protein by the receptor, whereby the signal transmission is blocked.

& Pertussis toxin:

Catalyzes ADP ribosylation of Cys on Gas, which prevents activation 334 7 G Protein-Coupled Signal Transmission Pathways

7.5.3 The Functional Cycle of Heterotrimeric G Proteins

Signal transmission via G proteins takes place in close association with the inner side of the cell membrane. Both, the a-subunit and the bc-complex are associated with the membrane via membrane anchors (Section 7.5.6). Like all regulatory GTPases, the heterotrimeric G proteins run through a cyclical transition between an inactive, GDP-bound form and an active, GTP-bound form. Thereby, the activated GPCR functions as a nucleotide exchange factor, GEF. A general model of the different functional states and the role of the individual subunits is sketched in Figure 7.21. Details of this model may differ among the various types of GPCRs and heterotrimeric G proteins.

7.5.3.1 Inactive Ground State In the inactive ground state, the G proteins exist as a GaGDP(bc)-heterotrimer that is either free or already receptor-bound. The binding sites on Ga for the downstream effectors are blocked by the b and c subunits, and the heterotrimeric complex is tethered to the inner face of the membrane via its lipid anchors.

& Inactive ground state:

GaGDP(bc)

7.5.3.2 Activation The binding of extracellular signal molecules (hormones, neurotransmitters) to the GPCR initiates conformational changes of the receptor that lead to high-affinity binding to the heterotrimeric G protein. In subsequent reactions, GDP dissociates, GTP binds, and dissociation of the heterotrimer into the Ga- and Gbc-subunits is triggered. In an intermediary step, the heterotrimer transits into an “empty” state, in which it possesses a high affinity for the activated receptor. The free nucleotide- binding site is immediately occupied by GTP, since GTP exists in large excess compared to GDP in the cell and since the Ga-subunit binds GTP more strongly than GDP. By catalyzing an expulsion of the bound GDP from the GaGDP(bc) complex, the activated receptor functions as a nucleotide exchange factor, GEF. It is the agonist-bound, activated receptor that represents the incoming signal in G protein signaling. The signal is then passed on by GaGTP and the bc-subunits to downstream targets of the signaling cascade. As a consequence of GTP binding, the bc-complex dissociates. The Ga-subunit may remain associated with the receptor, or binding to the activated receptor may be canceled. In the latter case, the receptor released from the complex can activate other G proteins, enabling amplification of the signal, as for example, during the vision process. For many GPCRs, it is assumed that the GaGTP subunit remains associated with the receptor for some time. In this case, larger signaling complexes 7.5 The Heterotrimeric G Proteins 335

Figure 7.21 Functional cycle of the effector molecule in the sequence E1 and heterotrimeric G proteins. (a) The G proteins activates the latter for further signal exist in the ground state as a heterotrimeric transmission. The released bc-complex may complex (GaGDP)(bc); (b) The activated also take part in signal conduction by binding receptor binds to the inactive heterotrimeric to a corresponding effector molecule E2 and complex of the G protein, which leads to activating the latter for further signal dissociation of the bound GDP and the conduction; (d) Hydrolysis of the bound GTP bc-complex; (c) Binding of GTP to the “empty” terminates the signal transduction via the Ga-subunit transforms the latter into the active a-subunit. GaGTP state. GaGTP interacts with an 336 7 G Protein-Coupled Signal Transmission Pathways

form at the membrane where receptor, G protein subunits and effector proteins cooperate in signaling.

& Activated GPCR: — Functions as GEF for G protein by triggering dissociation of GDP

& Active state:

— GaGTP, activates effectors

7.5.3.3 Transmission of the Signal The free a-subunit with bound GTP represents the activated GaGTP form of the G protein, and its interaction with the corresponding effector molecule initiates the next step in the signal transmission chain. The bc-complex released during activation can also perform a signal-mediating function (Section 7.5.5).

7.5.3.4 Termination of the Signal and GTPase-Activating Proteins Hydrolysis of GTP by the intrinsic GTPase activity of the a-subunit ends signal transmission at the level of the G proteins.

& GAPs enhance intrinsic GTPase activity of Ga

The rate of GTP hydrolysis functions as an inner clock for signal transmission; it determines the lifetime of the activated state and the extent of the reactions next in sequence. The intrinsic GTPase activity of GaGTP is rather low, and is accelerated by the action of GTPase-activating proteins (GAPs), which thereby mostly act as negative regulators of G protein signaling. Two classes of proteins participate in GTPase activation:

i) Proteins with GAP activity, named regulators of G protein signaling (RGS) (Section 7.5.7). The RGS proteins can increase the GTPase activity by up to three orders of magnitude, thereby attenuating signal transmission by G proteins. ii) Effector proteins of the G protein signaling cascade. In addition to RGS proteins, downstream effector proteins can act as GAPs. Examples include phospholipase C-b, which stimulates the intrinsic GTPase activity of the

corresponding Gq-11 by close to two orders of magnitude. A further effector molecule, isoform V of adenylyl cyclase (AC V), has been shown to function as a GAP for the monomeric GaGTP state. Another example is the c-subunit of cGMP phosphodiesterase, which is required for the GTPase-stimulatory action of a speciﬁc RGS protein in the vision process.

& GAPs of Ga: — RGS — Downstream effectors 7.5 The Heterotrimeric G Proteins 337

7.5.3.5 Heterotrimeric G Proteins in Supramolecular Complexes There is increasing evidence that the interactions of G proteins with their upstream and downstream effectors occur in specific membrane microdomains, where several signaling proteins are assembled in multiprotein complexes and free diffusion of the signaling proteins is not relevant. One such microdomain may be the lipid rafts in the plasma membrane that are formed by a coalescence of sphingolipid and cholesterol (Section 3.2.3). A subset of lipid rafts, the caveolae, are characterized by the presence of the protein caveolin at the cytoplasmic side. By assembling various components of the GPCR signaling path within such microdomain as supramolecular complexes, a high local concentration of the reaction partners of the signaling chain is achieved. Such supramolecular assemblies are thought to ensure a high efficiency and specificity of signaling. For example, c-aminobutyric acid (GABA) receptors (which are GPCRs) have been shown to exist in higher complexes comprised of receptor oligomers, Gi subunits, þ adenylyl cyclase and K channels [17].

7.5.4 Structural and Mechanistic Aspects of the Switch Function of G Proteins

The reaction cycle of the heterotrimeric G proteins involves the formation and breaking of numerous protein–protein contacts. In a dynamic way, protein–protein interactions are formed and resolved during the cycle, deﬁning distinct states of the G protein and leading to new functions and reactions. Currently, a wealth of structural information is available for most of the distinct functional states of the heterotrimeric G proteins.

7.5.4.1 Coupling of the Activated Receptor to the G Protein How an activated receptor activates the downstream G protein can be inferred b mainly from the recent studies on the 2AR in complex with a Gabc heterotrimer (Section 7.3.2.3). These studies indicate a major involvement of the transmembrane helices TM5, TM7 and the intracellular loop ICL3 in the recognition and stabilization of the complex with Gabc. In this complex, the heterotrimer contacts the GPCR via the Ga-subunit.

& Rhodopsin: — GPCR activated by light in the vision process — Light-induced activation of rhodopsin triggers reorientation of TM helices 3, 6, and 7.

7.5.4.2 Structure of the Ga-subunit The switch function of the a-subunit of the heterotrimeric G proteins is founded on the change between an active GaGTP conformation and an inactive GaGDP conformation. In this process, interaction sites with downstream effector proteins are exposed that are not available in the inactive GDP-state. The structures of both 338 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.22 GTP and GDP structures of domain with b2, switch II affects in particular transducin. In contrast to Ras protein and helix a2, and switch III, the b3---a3 loop. Switch other small regulatory GTPases, the Ga,t III includes a sequence that is characteristic for subunit of transducin possesses an a-helical the a-subunits of G proteins. The domain that hides and closes the G-nucleotide conformational changes of switches II and III binding pocket. The conformational changes affect structural sections that are assumed to that accompany transition from the inactive be binding sites for the effector molecule c Ga,t GDP form (a) into the active Ga,t GTP adenylyl cyclase (AC) and the -subunit of form (b) are restricted to three structural cGMP-dependent phosphodiesterase (PDEc), sections that are known as switches I, II, and based on mutation experiments and III. Switch I includes the link of the a-helical biochemical investigations.

forms of Ga are shown in Figure 7.22, based on the example of Gat, the Ga protein activated by rhodopsin in the vision process.

Gat is made up of two domains: the GTPase domain (also named G domain or Ras domain); and a helical domain. 7.5 The Heterotrimeric G Proteins 339

& Transducin: — G protein activated by rhodopsin

Ga,tstructure: — G domain — Helical domain.

The GTPase domain indicates that Gat is a member of the superfamily of regulatory GTPases. In addition, Gat possesses a helical domain, which represents a characteristic feature of the heterotrimeric G proteins. The nucleotide-binding site is in a cleft between the two domains. It is assumed that the presence of the helical domain is the reason that bound nucleotide dissociates only very slowly from transducing, and that the activated receptor is therefore necessary to initiate the GDP/GTP exchange.

7.5.4.3 Structure of the Heterotrimer In the known structures of heterotrimeric G proteins, contacts of the bc-complex to Ga are mediated only via the b-subunit, which binds in the region of the switch regions I and II and in the region of the N terminus of the a-subunit. The binding to switches I and II of the a-subunit partially masks interaction sites for downstream effector proteins, and prevents the transmission of a signal in the ground state. The c-subunit is located at the side of the b-subunit and does not itself interact with the a-subunit.

7.5.4.4 Conformational Changes Upon Activation by GTP ﬁ In all, the active and inactive forms of Ga,t have a very similar structure. Signi cant conformational changes on transition between the two functional states were found for three structural elements, known as switches I, II, and III, that include only 14% of the amino acids of transducin. The c-phosphate interacts with three amino acids that move switch I upwards and thus cause a coupled movement of switches I and II (Figure 7.22).

& Critical regions of Ga: — Switches I, II, and III change upon activation.

Overall, the switch function can be described in terms of conserved conformational changes of the switch I and switch II regions triggered by a universal “loaded spring” mechanism (Figure 7.23). Depending on the identity of the G protein, the extent of the conformational changes can vary from small to large changes, and the size of the switch regions must be deﬁned for each protein. One major consequence of GTP binding is dissociation of the bc-complex and the exposure of binding sites for downstream effectors. The bc-complex binds to the switch regions I and II of the a-subunit and thereby masks major interaction 340 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.23 “Loaded spring” mechanism of interactions with the c-phosphate of GTP. regulatory GTPases. A schematic diagram of Release of the c-phosphate after GTP the universal switch mechanism involving the hydrolysis allows the switch I and II regions to switch I and switch II domains of regulatory relax into the GDP-specific conformation. The GTPases. Invariant Thr35 and Gly60 residues extent of the conformational change is (numbering for Ras; see Chapter 11) are fixed different for different proteins, and may involve in a strained conformation by H-bond extra elements for some proteins.

sites with the downstream effector proteins; thus, binding of the effector is only possible if the bc-complex has dissociated. The conformational changes in switch II that are triggered by GTP binding lead to a dissociation of the bc-complex, exposing the binding sites for downstream effectors. For example, the binding site for the effector molecule adenylyl cyclase includes the switch II, and a conformational change in switch II makes a major contribution to binding and activation of adenylyl cyclase.

bc-complex binds to switches I and II Effectors bind to switch II.

7.5.4.5 Mechanism of GTP Hydrolysis Hydrolysis of the bound GTP terminates signaling by the Ga-subunit. The rate of GTP hydrolysis, and thus the lifetime of the activated Ga-GTP state, is a major determinant of the intensity of signaling by G proteins. Consequently, the mechanism of GTP hydrolysis has undergone intensive examination. It is generally assumed that hydrolysis of the c-phosphate bond proceeds via an

SN2 mechanism, as shown in Figure 7.24a, whereby the leaving phosphate is stabilized by two critical residues, namely Gln204 and Arg178 (numbering of

transducin, Gat). Whereas, Gln204 is part of the G-domain, Arg178 is located on the helical domain of the a-subunit. Both residues play an essential role in catalysis and are found in many Ga-subunits at equivalent positions. 7.5 The Heterotrimeric G Proteins 341

Figure 7.24 (a) “In-line” attack and hydrolysis. This dissociative mechanism of dissociative mechanism of GTP hydrolysis. GTP hydrolysis is now widely assumed to be Hydrolysis of GTP takes place via an “in-line” used by regulatory GTPases; (b) Binding of attack of a water molecule at the c-phosphate. GDPAlF4 at the active site of Gia. The The reaction passes through a transition state representation is based on the structure of the c in which the -phosphate adopts a Gia GDP AlF4 complex. The coordination metaphosphate-like, planar configuration. The sphere and the distances from AlF4 (in A)to þ c-phosphate, the water molecule and the Arg178, Gln204, the Mg2 ion and other leaving group (GDP) are oriented in form of a residues of the GTPase center are shown. The trigonal bipyramide, with an asymmetric catalytically important residues Gln204 and charge distribution. A surplus of negative Thr181 fix a water molecule that is located “in charge is located on the leaving group (GDP), line” to an oxygen atom of the c-phosphate of and stabilizing this charge by positive residues GDP. of the protein will enhance the rate of GTP 342 7 G Protein-Coupled Signal Transmission Pathways

& Residues important for GTP hydrolysis by Ga,t: — Gln204 — Arg178.

Information on the residues of Ga involved in stabilization of the transition state ﬁ has been obtained rst from structures of Ga-proteins in complex with GDP AlF4 , and a comparison of this structure with that of GaGTPcS, the activated a-subunit.

& GDP AlF4 : Transition state analog, fixes activated state

In the presence of AlF4 , a permanent activation of the G protein is observed: ﬁ Ga GDP is xed by the binding of AlF4 in a conformation that permits activation of the effector molecule. GDP AlF4 functions as a transition-state analog, in which c the AlF4 adopts the position of the -phosphate in the supposed transition state of GTP hydrolysis (Figure 7.24b).

7.5.4.6 Acceleration of GTP Hydrolysis by GTPase-Activating Proteins In all, the a-subunits of the G proteins possess a slow GTPase activity. A reduction of the lifetime of the activated GaGTP state, and thus a weakening of the signal transmission, can be achieved by the binding of speciﬁc GTPase-activating proteins such as the RGS proteins (Section 7.5.7) to GaGTP. The RGS proteins stimulate the GTPase activity of different a-subunits by up to three orders of magnitude.

& RGS proteins: — Stabilize the transition state — Accelerate GTPase activity up to 100-fold.

Mechanistically, the GTPase-activating activity of the RGS proteins is explained, in particular, by a stabilization of the transition state. It is assumed that the RGS proteins ﬁx the catalytic residues of the GTPase center and bring it into a position that is favorable for hydrolysis. GTPase stimulation of the Ras protein by the corresponding GAP proteins proceeds, in contrast, by another mechanism (see Section 11.5.3).

7.5.5 Structure and Function of the Gbc-Complex

The Gbc complex functions as a single entity that performs distinct functions, both in pathways associated with activated GPCRs and in pathways that are independent of GPCRs. During these functions, Gbc interacts with many different effectors, 7.5 The Heterotrimeric G Proteins 343 some that are also regulated by Ga-subunits and others that are not part of classical G protein signaling pathways.

7.5.5.1 Structure of the Gbc-Complex Structures of the Gbc-complex could be obtained in the free state, in the GaGDP- bound state and in complex with effectors such as phosducin and GRK [18]. A characteristic structural feature of all Gb-subunits is the presence of seven distinct b-sheet motifs arranged like the blades of a propeller. Each b-sheet motif is composed of four antiparallel b-sheets, and each contains a WD repeat, a tryptophan-aspartic acid sequence, at a spacing of 40 amino acids.

& b-Subunit: — Seven WD repeats, — b-Propeller structure c-Subunit: — Carries a prenyl anchor

The WD repeat motif is found in a large number of proteins, and is considered to be a stable platform that can reversibly form complexes with other proteins. There is no great structural difference between the free and Ga-bound forms of the bc-complex, and therefore activation of the latter for interaction with the corresponding effector molecule (see below) appears to be based only on its release from the inactive GaGDPbc complex. The Ga-subunit has the function of a negative regulator here, which inactivates the bc-complex by masking the interaction region for signal proteins next in the sequence.

7.5.5.2 Specificity of Gbc Signaling It is still not well understood how the downstream effector proteins recognize Gbc, and there is no readily apparent consensus sequence or structure that mediates the binding of these proteins to Gbc. In most known crystal structures of Gbc with effectors, the overall structure of Gbc is unperturbed. However, the results of nuclear magnetic resonance studies of Gbc have suggested that Gbc contains an intrinsically flexible region that may accommodate multiple modes of binding, allowing interaction with multiple targets. Another critical question asks for the specificity of Gbc signaling and for the ability of different bc combinations to interact with distinct GPCRs and other effectors. There are five b-subunits and 12 c-subunits, which would allow for a large number of different Gbc dimers. The sequences of the subunits are highly conserved among mammalian species and the expression patterns are highly tissue-specific, which points to unique or specialized roles of distinct dimer combinations in signal transduction pathways. 344 7 G Protein-Coupled Signal Transmission Pathways

7.5.5.3 Signaling by the bc-Complex The bc-complex, in addition to binding to the Ga-subunit, interacts speciﬁcally with its own effector molecules, taking part itself in the propagation and termination of signal transmission by GPCRs.

& bc-Complex has specific signaling functions

The intrinsic signaling function of the bc-complex is based on its interaction with the effector proteins listed in Table 7.3. The large list of identified effector proteins of the bc-complex indicates that bc-signaling can regulate and influence major signaling pathways in the cell [19]. For example, the bc complex takes part in the organization of larger GPCR–G protein signaling complexes activated by ligand binding to the GPCR. During this task, the bc-complex interacts with the same type of effector proteins that are also targeted by the Ga-subunit. In addition, the bc-complex performs many functions that are not directly coupled to GPCR signaling. Noteworthy here is the direct interaction of bc-complexes with ion channels, allowing the regulation of ion fluxes. Furthermore, the bc-complexes have been shown to interact with scaffolding proteins such as the RACK proteins, which indicates a coupling of bc-signaling to larger signaling complexes. The RACK proteins are scaffolding proteins that bind PKC and serve as adaptors for other signaling pathways (see Section 9.4.4). Interaction of the bc-complex with GRK1 (bARK; see Section 8.3.3.3) appears to be of special regulatory importance. The function of the bc-complex in this system is shown in Figure 7.25. The bc-complex binds specifically to the bARK and translocates this to the cell membrane. The translocation of bARK is necessary to switch off and modulate signal transmission via adrenaline. In addition to the classical signaling associated with GPCRs and G proteins, the bc-complex has been shown to interact with a variety of intracellular signaling molecules, independent of GPCRs (this aspect will be discussed in Section 7.6).

Table 7.3 Major effectors of bc-complexes.

Effector protein of bc-complexes Function

Adenylyl cyclase cAMP signaling þ Phospholipase C-b Ca2 , diacylglycerol signaling þ þ Ca2 -channels; potassium channels Ca2 -signals electrical signals G protein-coupled receptor kinases (GRK) Attenuation of GPCR signaling Phosducin Regulation, dampening of rhodopsin Regulators of G protein signaling (RGS) Negative regulation of Ga PI3-kinase type c Akt kinase pathways RACK proteins Protein kinase C 7.5 The Heterotrimeric G Proteins 345

Figure 7.25 Desensitization of GPCRs via substrate proteins. One of the substrates is cAMP-dependent protein kinases. Starting also the receptor that is phosphorylated in the from an activated receptor, the signal is region of the cytoplasmic domain by the transmitted via the Ga-subunit of the G protein activated protein kinase A. The ligand-bound to adenylyl cyclase. The latter is activated and receptor is preferentially phosphorylated. As a forms cAMP; this activates a protein kinase of consequence of phosphorylation, activation of type A that passes the signal in the form of a further G proteins by the receptor is Ser/Thr-specific protein phosphorylation to suppressed.

7.5.6 Membrane Association of the G Proteins

Signal transmission via G proteins is inseparably linked with their membrane association. The preceding reaction partners are transmembrane proteins, while the subsequent effector molecules, such as adenylyl cyclase, are either also transmembrane proteins or are associated with the membrane.

& Membrane anchors of G proteins: — Myristoyl anchor on Gi — Palmitoyl anchor near N terminus of Ga — Prenyl anchor on c.

The membrane association of the G proteins is mediated by membrane anchors (see Section 2.9) that are introduced during the course of a PTM at the N terminus of the a-subunit and at the C terminus of the c-subunit. a The -subunits of Gi and Go subtypes possess a lipid anchor in the form of a myristoylation at the N-terminal glycine residue. All Ga-subunits, except the ﬁ photoreception-speci cGa,t, contain a palmitate anchor at a cysteine residue near the N terminus. Myristoylation and/or palmitoylation of the Ga-subunits affects 346 7 G Protein-Coupled Signal Transmission Pathways

targeting to specific cell membrane regions and regulates interactions with other proteins such as adenylyl cyclase, GPCRs, and the bc-complex. The c-subunits have a membrane anchor in the form of prenyl residues, in a similar way to Ras protein. In addition, the terminal carboxyl group is esterified with a methyl group, which further increases the hydrophobicity of the C terminus. The length of the appended c isoprenoid grouping is variable. While the -subunit of the Gt,c protein has a fi farnesyl chain encompassing 15 C atoms, a modi cation with a C20 geranyl-geranyl c subunit is to be found in -subunits of Go-proteins in the brain.

7.5.7 Regulators of G Proteins: Phosducin and RGS Proteins

Signal transmission via GPCRs and the corresponding heterotrimeric G proteins is subject to tissue- and cell-speciﬁc regulation at different levels. The regulation is mostly of a negative, suppressing character and serves two purposes in particular. First, the cell must try to weaken the cytoplasmic answer under conditions of persistent activation of the receptor. Secondly, the cell needs mechanisms to rapidly terminate the signal. The most important regulatory attack points at the level of the G proteins and their receptors are (Figure 7.26):

Desensitization: phosphorylation of the receptor on the cytoplasmic side and uncoupling of the receptor from the G protein (Section 7.3.3.1) as a reaction to persistent stimulation. Downregulation of the number of receptor molecules: regulation at the levels of expression, stability, and internalization of the receptor. Inactivation of the bc-complex: binding of phosducin to the bc-complex. Reduction of the lifetime of the GaGTP complex: activation of the GTPase of GaGTP by RGS proteins.

Figure 7.26 Regulation of GPCRs and of G-proteins. For details, see the text. 7.5 The Heterotrimeric G Proteins 347

At the level of the G proteins, negative regulation by phosducin or RGS proteins stands out in particular.

7.5.7.1 Phosducin Phosducin is an abundant protein in photoreceptor cells of the retina that is widely assumed to regulate light sensitivity through interaction with the bc-subunits of the visual G protein transducin [20]. Related proteins, the phosducin-like proteins, are found in other tissues, such as the brain and the pineal gland. Phosducin and phosducin-like proteins regulate G protein-mediated signaling by binding to the bc-subunit and removing the dimer from cell membranes. & Phosducins: — Negative regulators of G proteins — Bind to bc-complex.

The main function of the phosducins appears to lie in a negative regulation of signaling by G proteins. Through binding to the bc-complex, the phosducins sequester this complex and prevent formation of the functional transducin Gatbc complex. Interestingly, the phosducin function is subject to regulation by phosphorylation through various protein kinases which provides an “on”–“off” switch for þ phosducin’s function on bc-modulation. Both, PKA and the Ca2 /calmodulin- dependent kinase II (CaM kinase II) have been found to speciﬁcally phosphorylate multiple Ser residues of phosducins. Phosphorylation decreases the afﬁnity of phosducin for transducin Gbc; this allows the re-formation of transducin’s heterotrimeric complex and a further round of activation.

7.5.7.2 RGS Proteins To a large part, the intensity of signaling by G proteins is regulated by proteins that function as GAPs. Two types of protein form part of this control: the regulators of G protein signaling (RGS proteins); and various effector proteins. The RGS proteins comprise a family of proteins which play crucial roles in the physiological control of G protein signaling [21]. At least 37 different RGS genes have been found in the human genome, and the encoded proteins mainly act as negative regulators of G protein signaling by accelerating the intrinsic GTPase activity of the Ga-subunit and reducing the lifetime of the activated state of Ga. The hallmark of the RGS proteins family is the presence of a speciﬁc domain, termed the RGS domain, which is conserved among the members of the RGS family and mediates contacts to the a-subunits. & RGS proteins: — Activate GTPase — Terminate signaling — Contain RGS box — Multiple domains — Many interacting proteins. 348 7 G Protein-Coupled Signal Transmission Pathways

By activating the GTPase rate of Ga up to 2000-fold, the RGS proteins attenuate and dampen heterotrimer-linked signaling. X-ray analysis indicates that the stimulatory action of the RGS proteins on the GTPase activity of the a-subunits can be described in terms of a stabilization of the transition state of GTP hydrolysis. A speciﬁc assignment of RGS proteins to particular a-subunits is to be assumed, whereby most of the known RGS proteins act as GAPs towards members

of the Gi subfamily. In addition to their GTPase-activating function, the RGS proteins serve many other regulatory functions and form part of a complex G protein signaling network. As illustrated by the presence of a variety of signaling modules in addition to the RGS box, RGS proteins participate in a broad spectrum of regulatory processes (Figure 7.27). Signaling modules of RGS proteins include PDZ domains, PH domains, phosphotyrosine-binding (PTB) domains, and Ras-binding domains, among others.

& RGS domains bind to Ga and are found on many proteins: — RGS proteins — GRK — AKAPs — p115 Rho-GEF — Axin.

Figure 7.27 Multidomain structure of RGS family members RGS 6 and RGS 12. DEP, Disheveled/ Egl-10/Pleckstrin homology domain; GGl, Gc-like domain; RBD, Ras-binding domain (see Chapter 10; GoLoco, Gai/o-Loco interacting motif; for details of PDZ and PTB, see Section 10.2.1). 7.5 The Heterotrimeric G Proteins 349

In addition to the “classical” GTPase-activating function directed towards Ga- GTP, other regulatory functions of RGS proteins in G protein signaling are known. Effectors of activated Ga subunits also have been identiﬁed as direct targets of RGS proteins. For example, RGS2 and RGS3 can directly bind to and inhibit adenylyl cyclase members, and PLC-b has been suggested to be regulated by RGS members independent of their GAP activity. Examples include the GPCR kinase GRK (Section 7.3.3.3), AKAP scaffold proteins (Section 9.3.3), the protein axin which forms part of the Wnt signaling pathway (Section 16.9), and the Rho exchange factor p115-Rho-GEF. The latter signaling protein provides a direct link of G protein signaling to the Rho family of small G proteins (see Chapter 11) which regulate a multitude of transcriptional and cytoskeletal processes.

7.5.7.3 Non-G Protein Signaling by RGS Proteins The RGS box is found in many signaling proteins that are not included in the RGS family. The presence of a RGS box enables these proteins to interact directly with the Ga-subunit and to modulate or regulate G protein functions. For example, the protein kinase GRK2 contains a RGS domain that mediates binding to the bc-complex and allows for the downregulation of Gbc signaling. In addition, RGS- box containing proteins are involved in multiple signaling events that are not directly linked to G protein signaling. These signaling functions include:

Interaction with central signaling proteins: RGS proteins have been shown to directly interact with the p85 subunit of PI3 kinase (Section 9.4) and with components of the TGFb signaling pathway (Section 14.3). Furthermore, RGS proteins can directly bind to the eukaryotic initiation factor eIF-2B (Section 5.6.2). RGS proteins as signal transducers: There are several examples of RGS box- containing proteins that are effectors in central signaling paths and function as signal transducers. An examples is the protein axin, which forms part of the Wnt signaling pathway (Section 16.9). Axin has an atypical RGS box but does not exhibit GAP activity towards Ga. Another example is p115RhoGEF, which

functions both as a GAP and an effector of Ga,12 and stimulates via a Dbl homology domain the GTP/GDP exchange on the small GTPase Rho (Chap- ter 11). Another interesting example of RGS protein signaling is RGS12 that functions as an adapter in MAP kinase signaling. For details, see Ref. [21].

A new, “non-classical” function of some RGS proteins is suggested from the discovery of the GoLoco motif in some members of the RGS superfamily, as for example RGS7. GoLoco motif-containing proteins generally bind to GDP-bound Ga-subunits and act as guanine nucleotide dissociation inhibitors (GDIs), slowing the spontaneous dissociation of GDP and inhibiting the association with bc-subunits. 350 7 G Protein-Coupled Signal Transmission Pathways

7.6 Receptor-independent Functions of Heterotrimeric G Proteins

Summary Novel functions of G proteins have emerged that do not involve activation by GPCRs. G proteins have been shown to interact directly with cytoskeletal elements and to play unique roles in cell division. Furthermore, G protein- mediated signaling is observed at distinct subcellular locations such as the mitochondrion and the nucleus.

In conventional G protein signaling, GPCR-activated G proteins regulate well- known signaling pathways at the cell membrane. However, further functions of G proteins have been discovered which involve direct interactions with cytoskeletal elements, unique roles in cell division, and signaling at distinct subcellular locations other than the cell membrane [22]. The following nonconventional signaling functions of heterotrimeric G proteins have been identiﬁed:

Interaction with cytoskeletal elements: Both, Ga- and Gbc-subunits have been shown to bind directly to components of the cytoskeleton and to regulate the dynamics of microtubule formation. Furthermore, functional interactions of

Ga,12/13 subunits with cell adhesion proteins such as cadherin, integrins and b-catenins have been demonstrated. G proteins at organelles: G protein subunits are speciﬁcally targeted to the mitochondrion, the Golgi, the endoplasmic reticulum and endosomes, where they bind to the protein components of these compartments and regulate signaling at these locations. Details of these regulations remain to be revealed. G proteins in the nucleus: Gbc-subunits have been shown to bind to and modulate the activity of transcriptional regulators such as the glucocorticoid receptor.

7.6.1 Novel G Protein Cycle

A novel G protein cycle that is independent of a transmembrane receptor has been discovered in Caenorhabditis elegans and in Drosophila. This nonclassical approach to G protein signaling seems to be evolutionarily conserved also in vertebrates, and is required for positioning the mitotic spindle and the regulation of microtubule pulling forces in cell division. There are two novel elements in this noncanonical pathway:

Ga cycles between GDP- and GTP-bound states without the involvement of GPCRs. 7.6 Receptor-independent Functions of Heterotrimeric G Proteins 351

The GDP-bound form of Ga, rather than the GTP-bound form, is the active form in force generation.

In C. elegans, the transition between GDP- and GTP-bound states requires the actions of a cytosolic GDP exchange factor, Ric-8, and the GTPase-activating protein RGS-7. Another key element of the GTPase cycle is the GDP-dissociation inhibitor GPR-1/2 that binds to Ga via a GoLoco motif. In one model (Figure 7.28), the GEF and GAP function in concert to transition the G protein from an inactive heterotrimer to an active Ga-GDP-state. Plasma membrane-localized Ga-GDP is thought to localize GPR1/2 to the microtubules, and GPR1/2 is assumed to be the effector of Ga-GDP. The link between Ga-GDP and microtubule regulation appears to be provided by the ability of GPR1/2 to bind to the coiled-coil protein Lin-5, which is involved – together with the motor protein dynein – in microtubule

Figure 7.28 A noncanonical heterotrimeric G exchange factor Ric-8 and a GTPase-activating protein cycle in cell division. During G protein RGS protein function in concert to transition function in cell division, the “active” element is the G protein from an inactive heterotrimer to GDP-bound Ga. The cycle appears to function an active Ga-GDP. The details of this transition in the absence of GPCR regulation. According await further clarification. After Ref. [22]. to one model, the guanine-nucleotide 352 7 G Protein-Coupled Signal Transmission Pathways

force generation. At present, the function of the Gbc complex in this novel cycle remains open.

7.7 Effector Molecules of G Proteins

Summary Activated G proteins pass the signal on to subsequent effector molecules that have enzyme activity or function as ion channels (see Figure 7.18). Important effector molecules are adenylyl cyclase, phospholipases, ion channels, and cGMP-specific phosphodiesterases. The activation of these enzymes leads to concentration changes of diffusible signal molecules such as cAMP, cGMP, þ diacylglycerol, inositol triphosphate and Ca2 , which trigger further specific reactions (Chapter 8). The G protein-mediated opening of ion channels may lead to changes in membrane potential, and also to changes in the ion environment, þ where variations in Ca2 concentration are of particular importance. Another effector molecule which provides a link to the signaling pathway of small GTPases of the Rho-family, is a G-nucleotide exchange factor (GEF) specific for

the Rho GTPases. The Rho-specific GEF p115 is activated by G12-subunits and thereby allows a crosstalk between the G protein and Rho signaling pathways.

7.7.1 Adenylyl Cyclase and cAMP as “Second Messenger”

Summary Adenylyl cyclases (ACs) transmit G protein signals further by producing the 0 0 second messenger 3 ,5 -cyclic AMP (cAMP) in response to stimulation by Ga,s- or Gbc-subunits. At least nine different types of ACs with transmembrane structure and one cytosolic AC are known in mammals. The transmembrane ACs are constructed from two transmembrane sections, each composed of six transmembrane helices, and two cytosolic domains that interact with the G protein subunits and form the active site for synthesis of cAMP from ATP. The transmembrane AC subtypes are subject to distinct regulation by multiple cellular signaling molecules. The activation of AC subtypes can be mediated by 2þ Ga,s- and Gbc-subunits, by Ca /calmodulin, and by PKC. Furthermore, some 2þ subtypes are inhibited by Ga,i,Ca and phosphorylation by PKA.

The adenylyl cyclases catalyze the formation of 30,50-cyclic AMP (cAMP) from ATP (Figure 7.29). 7.7 Effector Molecules of G Proteins 353

Figure 7.29 Formation and degradation of cAMP.

& Adenylyl cyclases: — Form cAMP from ATP — Contain 2 6 TM helices — Catalytic site on cytoplasmic domains C1 and C2. 354 7 G Protein-Coupled Signal Transmission Pathways

cAMP is a widespread signal molecule that functions primarily via the activation of protein kinases. The synthesis of cAMP by adenylyl cyclase is opposed by degradation and inactivation by phosphodiesterases (PDEs) (Section 8.2).

7.7.1.1 Structure of Adenylyl Cyclase In mammals, at least nine different types of ACs have been described which are transmembrane proteins; known as types I–IX, these ACs show a high degree of sequence homology (ca. 50%) [23]. In addition, a cytosolic AC is known which is þ regulated by Ca2 and bicarbonate and plays an essential role in sperm motility. The ACs of types I–IX are large transmembrane proteins with a complex transmembrane topology. The assumed topology (Figure 7.30) shows a short cytoplasmic N-terminal section, followed by a transmembrane domain M1 with six transmembrane sections and a large cytoplasmic domain C1. The structural motif is repeated so that a second transmembrane domain M2 and a second cytoplasmic domain C2 can be differentiated. Information on the structure–function relationship of ACs is available, in particular for the cytoplasmic domain. According to this, the important functions of AC – namely interactions with the G protein and the synthesis of cAMP – are localized on the cytoplasmic C1 and C2 domains. The latter are homologous to a high degree between the different subtypes, whereas the transmembrane domain, in contrast, is little conserved. Structural determinations of the complex of – Ga,s GTP and a C1 C2 dimer have indicated that the active center is at the cleft of the C1–C2 dimer. The ATP binding site and a binding site for the activator

Figure 7.30 Topology of adenylyl cyclase. In larger cytoplasmic portion (C1 and C2). mammals, adenylyl cyclase is a Sequence analysis predicts six transmembrane transmembrane protein that is composed of helices for each of the domains (numbering two homologous domains, each with a from 1---12). The active site is formed by transmembrane domain (M1 and M2) and a residues from C1 and C2. 7.7 Effector Molecules of G Proteins 355 forskolin are also located there. Similar to DNA polymerases, ACs appear to employ a two-metal-ion mechanism for catalysis.

The activator Gs,a binds to the C2 domain and triggers a rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation.

7.7.1.2 Regulation of Adenylyl Cyclase The ACs are master regulators of major physiological processes such as learning and memory formation, pain perception, aging, circadian rhythm, pattern formation in the cortex, and many others. The AC isoforms are expressed in a tissue-speciﬁc manner and allow for both tissue- and cell-speciﬁc responsiveness to regulators’ signals [24]. A multitude of regulatory inputs regulate the biological functions of this enzyme class. Typically, ACs can integrate multiple positive and negative signals that act directly through the stimulation of GPCRs or indirectly via intracellular signaling pathways. Besides classical regulatory mechanisms mediated by GaGTP, Gbc, ACs can also be modulated by direct protein–protein interactions, which highlights the importance of binding partners in the selectivity of AC members.

& Regulation of ACs: — Differential regulation of isoforms Main regulatory inputs:

— Ga-subunits — bc-subunits — PKC þ — Ca2 — CamKinase

Stimulation of AC may take place by

Ga,s GTP þ Ca2 /calmodulin Protein kinase C bc-subunits of G proteins

Inhibition of AC is possible by

Ga,i GTP þ Ca2 Protein kinase A

The stimulatory and inhibitory inﬂuences that take effect on important groups of ACs are summarized in Figure 7.31. 356 7 G Protein-Coupled Signal Transmission Pathways 7.7 Effector Molecules of G Proteins 357

þ The regulation of subtypes I, III and VIII by Ca2 /calmodulin is especially þ prominent, with all three subtypes being stimulated by Ca2 , albeit over different þ concentration regions. Ca2 is a central intracellular messenger substance (see Chapter 8), and an increase in its concentration is observed when the different signal transduction processes are activated. þ The Ca2 /calmodulin regulation of AC is particularly important in the brain, where the enzyme is concentrated in the vicinity of receptors for N-methyl-D- þ aspartate (NMDA) that provide effectively regulated entry points for Ca2 . As the þ entry point for Ca2 and AC are in close proximity, a rapid response of the enzyme þ to changes in Ca2 concentration is ensured. Based on the results of knockout studies in mice, AC isoforms play an important role in the nervous system by, for example, inﬂuencing the processes of learning, memory, and behavior. Overall, the ACs represent a “meeting point” at which different regulatory signals arrive and are weighed up against each other. In many aspects, the ACs resemble a coincidence detector that is only activated when several signals become effective in þ simultaneous fashion. Ca2 /calmodulin-dependent ACs are seen as important elements in learning processes and in memory formation, and a coincidence mechanism has been postulated for both cases.

7.7.2 Phospholipase C

Summary Phospholipase C enzymes catalyze the formation of the two second messengers

diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) from phosphatidyl-inositol-4,5 bisphosphate (PtdIns(4,5,)P2). To date, four major PLC subtypes have been identified: PLC-b, PLC-c, PLC-d, and PLC-e. These enzymes are multidomain proteins that are regulated by diverse cellular stimuli to 2þ b produce DAG and Ca signals. PLC- is stimulated specifically by Gq- and Gbc- subunits. PLC-c forms part of the receptor-tyrosine kinase pathways, while PLC-d þ is regulated by Ca2 and forms part of the Ras signaling pathways.

J Figure 7.31 Multiple modes of regulation of ACVII. Note that Gbc regulation of ACII is adenylyl cyclase isoforms. (a) The pattern of dependent on Gas coactivation and does not regulation of ACI as illustrated is activate AC by itself. PKC can use AC as a representative also for ACIII and ACVIII. R1 substrate, resulting in an elevation of basal represents a GPCR, such as the glucagon or activity and an inhibition of the Gbc b 2-adrenergic receptor, that couples to the superactivation; (c) The pattern of regulation stimulatory G protein Gas. R2 represents a of ACV is representative also of ACVI. PKA, GPCR, such as the muscarinic M2 or a1- protein kinase A; PKC, protein kinase C; CaM, adrenergic receptor, that couples to the calmodulin; CaMK, calmodulin-dependent inhibitory G protein Gai; (b) The pattern of kinase; NO, nitric oxide; VDCC, voltage- þ regulation of ACII as illustrated is dependent Ca2 channel. representative of the regulation of ACIV and 358 7 G Protein-Coupled Signal Transmission Pathways

Figure 7.32 Classification of the importance for signal transduction, phospholipases and the reaction of phospholipase C (PL-C) catalyzes the cleavage phospholipase C. (a) Cleavage specificity of of phosphatidylinositol-4,5-bisphosphate phospholipases A1, A2, C, and D; (b) Cleavage (PtdIns(4,5)P2) into the messenger substances of inositol-containing phospholipids by diacylglycerol and inositol-1,4,5-triphosphate phospholipase C. In a reaction of particular (Ins(1,4,5)P3).

Another large class of effector molecules activated by G proteins is the b-subfamily of the phospholipases of type C (PLC-b). Phospholipases are enzymes that cleave phospholipids, and phospholipases of type A1, A2, C and D can be differentiated according to the speciﬁcity of their attack point on the phospholipid. The bonds cleaved by these phospholipases are shown in Figure 7.32a.

& Phospholipases A1, A2, C, and D are classified by cleavage specificity

The cleavage of inositol-containing phospholipids by PLC is of particular regulatory importance, as this reaction generates two second messengers. PLC catalyzes the release of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins 7.7 Effector Molecules of G Proteins 359

(1,4,5)P3) from phosphatidyl inositol-4,5-diphosphate (PtdIns(4,5)P2, a phospholipid that occurs at low concentrations in the membrane (Figure 7.32b). Thus, PLC has a key function in formation of the intracellular messenger substances DAG, 2þ Ins(1,4,5)P3, and Ca (Chapter 8). The stimulation of PLC isoenzymes by extracellular stimuli such as neurotransmitters, hormones, inﬂammatory mediators and odorants represents one of the major mechanisms employed by cell-surface receptors to trigger downstream signaling events. To date, at least 13 isoforms have been identiﬁed in the mammalian PLC family, of which the PLC-b, PLC-c, PLC-d, PLC-e and PLC-f enzymes [25] are the best- characterized.

& Major PLC types: — PLC-b — PLC-c — PLC-d — PLC-e.

The domain structures of the PLC families shown in Figure 7.33 indicate the presence of diverse signaling and scaffolding modules in these enzymes, and points to multiple regulation mechanism and functions.

Figure 7.33 Domain architecture of the major homology domain (Section 2.4.4); CDC25, G- þ phospholipase C families. EF, Ca2 -binding nucleotide exchange module (Section 11.3); module; X, Y, catalytic domains, C2 (Section RA, Ras-association module. 2.5); CT, C-terminal domain; PH, pleckstrin 360 7 G Protein-Coupled Signal Transmission Pathways

Common to all PLCs, with the exception of PLC-f, is the occurrence of pleckstrin homology (PH) domains. The PH domains serve as interaction modules that mediate protein–membrane interactions and protein–protein interactions (Section 2.4.4). Two distinct functions have been assigned to the PH domains of PLC-b enzymes, namely the speciﬁc binding of phosphatidyl inositol phosphates and binding to Ga-GTP and Gbc-subunits.

& Important domains of PLCs: — PH domain — Catalytic domains EF, X, and Y.

It is generally assumed that the PH domain has the function of associating the

phospholipase with the membrane-localized substrate, the PtdIns(4,5)P2, and of ensuring an effective conversion of the substrate. In addition, PLC contains þ domains known as EF-hands which mediate the low-afﬁnity binding of Ca2 . The catalytic core is formed by the EF, X, Y, and C2 domains. The PLC enzymes are activated mainly via signaling paths originating from the activation of GPCRs and receptor tyrosine kinases (see Chapter 10) in an isozyme-speciﬁc fashion. The links of these receptors to the various PLC isozymes are shown in Figure 7.34. The activation of PLC isozymes via the major membrane-associated receptor types points to a central role for PLC subtypes in signal transduction. The products of PLC activation – the second – ’ messengers DAG and Ins(1,4,5)P3 represent the cell s central tools for þ regulating protein phosphorylation via PKC and generating Ca2 -signals, respectively (see Chapters 8 and 9).

7.7.2.1 Phospholipase C-b Phospholipases of type C-b function as effector enzymes in signal transmission by various GPCRs. The initiating external signals are diverse (see Figure 7.18) and include hormones, neurohormones and sensory signals such as odorous agents and light (in nonvertebrates).

& PLC-b is activated by: — Gq — bc

The effector function of PLC-b enzymes in G protein signaling is based on, and mediated by, the following functions and interactions [26]:

a Activation by the Gq subfamily of pertussis toxin-insensitive -subunits Activation by bc-subunits GTPase-activating function toward Gq-subunits Interaction with PDZ domain-containing proteins. 7.7 Effector Molecules of G Proteins 361

Figure 7.34 Activation of various PLC (PDGF), epidermal growth factor (EGF) and isozymes. Various extracellular signals nerve growth factor (NGF) can activate PLC-c stimulate the hydrolysis of isozymes. Upon growth factor stimulation, c phosphatidylinositol-4,5-bisphosphate (PIP2) PLC- is recruited to activated growth factor by PLC to two important second messengers, receptors via SH2 domain-phosphotyrosine DAG and inositol-1,4,5-trisphosphate (IP3). interaction and then subjected to PLC-b subtypes are activated by GPCR through phosphorylation by receptor tyrosine kinases several mechanisms. Pertusis toxin (PTX)- (Chapter 10). PLC-e can be activated by both insensitive heterotrimeric Gq family proteins GPCR and RTK activation with a distinct (e.g., Gq,G11) activate PLC-b subtypes via GTP- activation mechanism involving small loaded Ga subunits. PLC-b subtypes are also regulatory GTPases Rap, Rho, or Ras (Chapter activated by Gbc subunits liberated from PTX- 11). PLC-d is activated by Ga subunits and 2þ sensitive Gi family proteins. Various growth Ca -signals. factors such as platelet-derived growth factor 362 7 G Protein-Coupled Signal Transmission Pathways

The main effector function of PLC-b enzymes is based on their stimulation by bc b Ga,q and the -complex, whereby each of the various PLC- isoforms has a bc different sensitivity to Gaq- and -subunits. b Other activities of PLC- enzymes include a GAP activity towards Gq-subunits which is thought to improve signal quality by decreasing agonist-independent noise. Furthermore, PLC-b enzymes contain regions that mediate interaction with PDZ domains of scaffolding proteins. PDZ-containing proteins are involved in the clustering and structural organization of receptors and their downstream signaling proteins, and thereby regulate agonist-dependent signal transduction in, for example, neuronal cells. It is speculated that this interaction contributes signiﬁcantly to the strong association PLC-b with membrane fractions.

7.7.2.2 Phospholipase C-c As phospholipases of type C-c are activated by receptor tyrosine kinases (see Chapter 10), PLC-c is involved in growth factor-controlled signal transduction pathways. The receptor tyrosine kinases phosphorylate the enzyme at speciﬁc tyrosine residues and initiate an activation of the enzyme that mobilizes internal calcium stores and engages multiple protein kinase pathways.

& PLC-c: — Contains SH2, SH3 domains — Activated by: receptor tyrosine kinases nonreceptor tyrosine kinases.

A characteristic of the structure of PLC-c is the occurrence of SH2 and SH3 domains. These represent protein modules that serve to attach upstream and downstream partner proteins. The SH2 domains mediate binding to Tyr- phosphates of the activated, autophosphorylated receptor tyrosine kinase. During this process, the PLC-c enzymes are phosphorylated on Tyr-residues and are thereby activated.

7.7.2.3 Phospholipases C-d The subfamily of PLC-d comprises three isoenzymes that appear to be regulated by þ Ca2 levels. The most abundant isoform, PLC-d1, contains a PH domain that ﬁ shows a high af nity for PtdIns(4,5)P2. This property is thought to be mainly responsible for the tethering of PLC-d1 to the cell membrane. Modulation of the

levels of PtdIns(4,5)P2 by external stimuli leads to the dissociation from the membrane. All PLC-d isoforms contain nuclear localization signals and show a coordinated intracellular translocation during the cell cycle.

7.7.2.4 Phospholipase C-e PLC-e appears to be a multifunctional enzyme, and contains a Ras-association domain and a CDC25 domain which is a nucleotide exchange motif (see Section 11.3). 7.8 GPCR Signaling via Arrestin 363

Upstream regulators of PLC-e have been reported to be members of the Ras and Rho e subfamily of small GTPases. Furthermore, PLC- has been shown to be activated by Gs, e Gi/o and G12/13 subunits, revealing that PLC- is another PLC enzyme regulated by G proteins.

& PLC-e is activated by: — Ras and Rho proteins — Gs,Gi/o and G12/13.

7.8 GPCR Signaling via Arrestin

Summary In addition to G proteins, GPCRs interact with another downstream effector, the arrestins, to transmit extracellular signals across the cell membrane. By binding to the cytoplasmic parts of GPCRs, arrestins are major players in the downregulation of GPCR signaling. In addition, these proteins function as scaffolds that direct the recruitment and activation of distinct signaling complexes in response to GPCR activation and other stimuli. Many arrestin- binding partners have been identified including protein kinases, protein phosphatases, phosphodiesterases, a guanine nucleotide exchange factor, and actin assembly proteins. An important scaffolding function of arrestins is based on the recruitment and activation of the MAPK cascade in response to GPCR signals which may serve to transmit GPCR signals down to the level of transcription.

Arrestin has long been known as a modulator of GPCR signaling. Indeed, the function of arrestin in GPCR signaling has long been interpreted in terms of the downregulation and desensitization of GPCRs: receptor phosphorylation by GRKS or second-messenger-activated protein kinases leads to the binding of arrestins and uncouples the heterotrimeric G protein from the activated receptor. During recent years, novel functions of arrestins in GPCR signaling have been discovered that are independent of heterotrimeric G proteins, and it is now well established that GPCRs can use arrestins as an own effector that couples GPCR signaling to distinct downstream signaling events. Currently, two arrestin members, arrestin-1 and arrestin-2, are considered as versatile scaffolds that direct the recruitment, activation and scaffolding of distinct cytoplasmic signaling complexes. This mechanism regulates various aspects of cell motility, chemotaxis, and apoptosis [12]. A large number of arrestin-binding partners has been identiﬁed, including protein MAP kinases, Src kinase, PI3 kinase, Akt kinase, Protein phosphatase 2A, phosphodiesterases, the GEF Epac, and actin assembly proteins. 364 7 G Protein-Coupled Signal Transmission Pathways

& Arrestins: — Bind to phosphorylated GPCRs — Trigger the internalization of GPCRs — Activate tyrosine kinases — Activate the MAPK cascade

The following signaling functions of arrestins are predominant:

Scaffold for protein kinases: Nonreceptor tyrosine kinases such as the Scr kinase can be activated and used to transmit the signal further. This is an example of where the arrestins have a positive role in signal transduction. Furthermore, arrestins are involved in the activation of Akt kinase (Section 9.4) and of the transcription factor NFkB. Activation of MAPK cascade (Figure 7.35): This is observed mainly for Class B GPCRs. A well-studied example of an arrestin-dependent signaling system leads to the activation the MAP kinase Erk. Here, arrestin bound to GPCRs serves as a scaffolding protein that organizes the protein kinases of the MAPK pathway (see Chapter 12) into functional complexes. The recruitment of MAPKs can occur in association with internalization events, but it can also occur independently. Which MAPK path will be activated appears to depend on the type and phosphorylation pattern of the receptor. Overall, there is a highly complicated relationship between arrestins and MAPKs that is not well understood.

Figure 7.35 Multiplicity of arrestin signaling (see text for details). Questions 365

Scaffold for actin reorganization: arrestins bind to a number of actin assembly proteins and, as a consequence, arrestins can mediate cytoskeletal reorganization and cell migration during for example, chemotaxis.

How the arrestins are differentially recruited to distinct GPCRs and how the many downstream effectors are selected is not well understood. It has been postulated that different binding modes of arrestins on GPCRs exist, namely a more transient binding and a stable binding. Transient binding may be directed by the cytoplasmic regions of the receptor in dependence of agonist binding, and both the sequence of the receptor and the chemical nature of the agonist are thought to be relevant to this type of binding. Phosphorylation of the receptor is postulated to promote stable arrestin binding, and this may function synergistically with agonist binding. Structural determinations suggest that arrestin binding to the receptor leads to distinct conformational changes of arrestin, depending on the signaling pathway activated. Movements of the N- and C-termini of arrestin towards and away from each other have been observed, depending on the nature of the agonist that is bound to the GPCR. The multiple conformations that arrestins can adopt will present distinct interaction surfaces for binding of the downstream effectors, which may explain the multitude of signaling events that arrestins can trigger.

Questions 7.1. What are the major structural parts of transmembrane receptors? Explain the functions of these regions. Which types of transmembrane regions can you name, and how are these structures organized? Give examples. 7.2. Which major mechanisms are used for the regulation of signaling via transmembrane receptors? Give at least two examples of transmembrane receptor regulation. 7.3. Give examples of transmembrane receptors with different transmembrane topology. Describe the functions of the receptors. 7.4. Which types of extracellular signals can activate GPCRs? Name the GPCR families and at least one of the cognate ligands. Give the chemical structure of at least ﬁve different signaling molecules that can activate GPCRs. 7.5. Describe the main structural features of GPCRs and their involvement in the activation of the downstream effector. 7.6. Which types of GPCR ligand analogs can you name, and what is the basis of their classiﬁcation? How may a particular analog contribute to the selection of a downstream effector? 7.7. What are the principal mechanisms for the regulation of GPCRs? Which types of intracellular proteins may interact with and regulate GPCRs? 366 7 G Protein-Coupled Signal Transmission Pathways

7.8. Which major mechanisms are used for the downregulation of GPCRs? What is the function of phosphorylation in downregulation, and which protein kinases are involved? How are these kinases targeted to the GPCR? 7.9. Explain the phenomenon of “signal switching” by GPCRs. Which mechanisms may be involved? 7.10. Describe the role of arrestin in GPCR signaling. 7.11. Describe the switch function of regulatory GTPases. Which types of proteins control the passage through the GTPase cycle, and what is the function of these proteins? 7.12. Which compounds can be used to interfere with the GTPase cycle?

7.13. Which classes of Ga-subunits can you name? Give the functions and a downstream effector for each class. 7.14. How do cholera toxin and pertussis toxin interfere with the GTPase cycle of G-proteins? Which reactions are involved?

7.15. Describe the structural basis of the switch function of Ga-subunits. 7.16. Which lipid modiﬁcations are found on components of GPCR/G-protein signaling? 7.17. What is the function of the bc-complex in G-protein signaling? Give examples of downstream effectors of the bc-complex. 7.18. What are the major regulators of G-proteins? Explain their function(s). 7.19. Name the major downstream effectors of G-proteins and describe the signaling function of these proteins. 7.20. Describe the structural properties of adenylyl cyclase. Which signals are used for the regulation of this enzyme class? 7.21. Name the enzyme classes that hydrolyze phospholipids. Which signaling molecules may be derived from phospholipids? What is the function of these signaling molecules? 7.22. Which classes of PLC can you name, and how are these enzymes regulated? Which signaling pathways are involved in these regulations?

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8 Intracellular Messenger Substances: “Second Messengers”

Extracellular signals are registered by membrane receptors and conducted into the cell via cascades of coupled reactions. The ﬁrst steps of signal transmission often take place in close association with the membrane, before the signal is conducted into the cell interior. The cell uses mainly two mechanisms for transmission of signals at the cytosolic side of the membrane and in the cell interior. First, signal transmission may be mediated by a protein–protein interaction, with the proteins involved being receptors, proteins with adapter function alone, or enzymes. Second, signals may be transmitted with the help of low-molecular-weight messenger substances, termed “second messengers.” The intracellular messenger substances are formed or released by speciﬁc enzyme reactions during the process of signal transduction and serve as effectors, by which the activity of proteins further along the sequence is regulated (Figure 8.1).

8.1 General Properties of Intracellular Messenger Substances

The intracellular messengers are diffusible signal molecules and reach their target proteins mostly by diffusion. Two types of intracellular messenger substance can be differentiated (Figure 8.1):

Messenger substances with a hydrophobic character, such as diacylglycerol or the phosphatidylinositol derivatives, are membrane-localized. A main function of the hydrophobic messengers is the promotion of membrane localization of the target proteins. Hydrophilic messengers with good aqueous solubility are localized in the cytosol and reach their target proteins by diffusion. In many cases, the target proteins are activated by an allosteric mechanism upon binding of the second messenger. The major effector molecules of the hydrophilic messengers are protein kinases. Other important targets are G-nucleotide exchange factors (see Chapters 5 and 9) and ion channels.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 370 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.1 Function and formation of these for signal transmission further. intracellular messenger substances in Hydrophobic messenger substances, in signaling pathways. Starting from the activated contrast, remain in the cell membrane and receptor, effector proteins next in sequence are diffuse at the level of the cell membrane to activated that create an intracellular signal in membrane-localized target proteins. PK, the form of diffusible messenger substances. protein kinase; S, substrate of the protein The hydrophilic messenger substances diffuse kinase. to target proteins in the cytosol and activate

& Cytosolic second messengers: — cAMP, cGMP — Inositol phosphates þ — Ca2 . Membrane-associated second messengers: — Diacylglycerol — Phosphatidyl inositides.

The following properties make the intracellular “second messengers” particularly suitable as elements of signal transduction:

Intracellular messenger substances can be formed and degraded again in speciﬁc enzyme reactions. Via enzymatic pathways, large amounts of messenger substances can be repeatedly and rapidly created and inactivated. þ Messenger substances, such as Ca2 , may be stored in special storage organelles, from which they can be rapidly released by a signal. 8.2 Cyclic AMP 371

Messenger substances may be produced in a location-speciﬁc manner, and they may also be removed or inactivated according to their location. It is therefore possible for the cell to create signals that are spatially and temporally limited.

8.2 Cyclic AMP

Summary The second messenger 30,50-cyclic AMP (cAMP) is produced from ATP by the action of adenylyl cyclases, and is degraded by the action of phosphodiesterases. The signaling function of cAMP is based primarily on binding to protein kinase A (PKA), ion channels, and the transcription factor Epac. To a large part, signaling by cAMP is restricted to discrete locations at the cell membrane and intracellular membrane compartments where cAMP production, binding to its target proteins and cAMP degradation each occur within defined supramolecular complexes organized by specific anchoring proteins such as the A-kinase anchoring proteins (AKAPs).

30,50-cyclic AMP (cAMP), which is produced from ATP by the action of adenylyl cyclases (ACs; see Section 7.7.1 and Figure 7.30), inﬂuences many cellular functions including gluconeogenesis, glycolysis, lipogenesis, muscle contraction, membrane secretion, learning processes, ion transport, differentiation, growth control, and apoptosis. The concentration of cAMP is controlled primarily by two means, namely via new synthesis by ACs and via degradation by phosphodiesterases. Both enzymatic activities cooperate in forming cAMP gradients in the cell with speciﬁc temporal and local characteristics. An important feature of cAMP signaling is the colocalization of the enzymes of cAMP metabolism and the targets of cAMP. For example, ACs, phosphodiesterases and protein kinase A have been found to colocalize at the same subcellular sites, allowing for a precise control of cAMP formation, degradation, and target selectivity.

& cAMP: — Formed by adenylyl cyclases — Degraded by phosphodiesterases.

8.2.1 Formation and Degradation of cAMP

8.2.1.1 Formation by ACs As outlined in Chapter 7, the ACs comprise a family of enzymes each of which exhibits a distinct pattern of regulation. Two types of ACs are responsible for cAMP 372 8 Intracellular Messenger Substances: “Second Messengers”

formation, namely transmembrane ACs and soluble ACs. Whereas, the transmem- 2þ brane ACs are mainly regulated by Ga- and Gbc-subunits and Ca - ions þ (Section 7.7.1), the soluble ACs are uniquely regulated by bicarbonate and Ca2 , and is insensitive to heterotrimeric G protein regulation [1]. The soluble ACs are widely expressed and is not strictly a soluble protein; rather, it is present at discrete subcellular localizations in a wide variety of cells.

8.2.1.2 Phosphodiesterases and cAMP Breakdown Pivotal in shaping and controlling intracellular cAMP gradients in cells are the cyclic nucleotide specific phosphodiesterases (PDEs), [2], which provide the sole means of degrading cAMP. In total, 11 families of PDEs derived from 21 genes have been identified, with additional diversity resulting from alternative mRNA splicing. The isoforms differ in their regulatory and kinetic properties as well as their specificity. According to substrate specificity, PDEs can be divided into cAMP- specific, cGMP-specific and dual-specificity PDEs that cleave both cAMP and cGMP. The PDEs are subject to a variety of regulatory influences, including regulation by þ Ca2 /calmodulin and by protein phosphorylation. Due to their central role in regulating the levels of cyclic nucleotides, the PDEs represent a popular target for the development of specific inhibitors for pharmaceutical use.

& Classes of PDEs: — cAMP-specific — cGMP-specific — Dual specificity.

8.2.2 Targets of cAMP

cAMP functions as an activator of downstream signaling proteins which possess speciﬁc cAMP binding sites and are regulated by cAMP via allosteric mechanisms. The proteins involved in cAMP signal conduction perform their function, without exception, in association with the cell membrane. Cyclic AMP binds to and activates the following signaling proteins (Figure 8.2):

cAMP-gated ion channels: An important function of cAMP is the regulation of ion passage through cAMP-gated ion channels. cAMP binds to cytoplasmic structural elements of these ion channels and regulates their open state. An þ example is the cAMP-regulated Ca2 passage through cation channels. cAMP also performs this function during the perception of smell in mammals. Protein kinase A (PKA): The majority of the biological effects of cAMP are mediated by the activation of protein kinases, classiﬁed as PKA. cAMP binding to PKA relieves autoinhibition of the enzyme and allows the phosphorylation of substrate proteins. The mechanism of activation of protein kinases of type A by cAMP is shown schematically in Figure 8.3. An increase in cAMP concentration, 8.2 Cyclic AMP 373

Figure 8.2 Main signaling functions of cAMP AKAP, A-kinase anchoring protein (Section and cGMP. For details, see the text. For Rap1, 9.3.3); PKG, cGMP-dependent protein kinase, B-raf and MAPK, see Chapters 11 and 12. PDE, protein kinase G (Section 9.3.2.). phosphodiesterase; PKA, protein kinase A;

Figure 8.3 Regulation of protein kinase A dissociation of the tetrameric enzyme into the (PKA) via cAMP. PKA is a tetrameric enzyme R2 form with bound cAMP and free C subunits. composed of two catalytic subunits (C) and In the free form, C is active and catalyzes the two regulatory subunits (R). In the R2C2 form, phosphorylation of substrate proteins (S) at PKA is inactive. Binding of cAMP to R leads to Ser/Thr residues. 374 8 Intracellular Messenger Substances: “Second Messengers”

triggered by the activation of AC and/or the inhibition of phosphodiesterase, leads to the cooperative binding of two molecules of cAMP to two sites on the regulatory subunit. Upon binding of four molecules of cAMP, the enzyme dissociates into an R subunit dimer with four molecules of cAMP bound and two free C subunits which are now released from inhibition by the regulatory subunits and can thus phosphorylate Ser/Thr residues on specific substrate proteins. The nature of the substrate proteins of protein kinase of type A is very diverse; the substrates may be other regulatory proteins or enzymes of intermediary metabolism. The specificity of signaling by PKA and the selection of its substrates is mainly determined by specific anchor proteins, the AKAP proteins (Section 9.3.3). Epac, a Guanine Nucleotide Exchange Factor (GEF): A further second-messenger function of cAMP involves activation of the protein Epac, which is a GEF for the small GTPase Rap1 (see Section 11.7). The binding of cAMP to the multidomain Epac proteins causes a conformational change leading to a release of autoinhibition, and an increased exchange activity towards Rap1 and Rap1 activation. Activation of Rap by Epac links cAMP signaling to activation of the protein kinase B-Raf and the mitogen-activated signaling (MAPK) pathway (see Chapter 12). Epac and protein kinase A may act independently, but are often associated with the same biological process, in which they fulfill synergistic or opposite effects. Like PKA, Epac proteins are targeted to distinct cellular sites by scaffold proteins. With the help of cAMP analogs that selectively activate Epac, the involvement of Epac proteins in the control of key cellular processes has been shown. For example, roles for Epac in diverse cellular processes ranging from insulin secretion to cardiac contraction have been demonstrated [3].

& Targets of cAMP: — Protein kinase A — Ion channels — Epac, a transcription factor.

8.2.3 Compartmentalization of cAMP Signaling

To a large part, signaling by cAMP is restricted to discrete locations at the cell membrane and intracellular membrane compartments. cAMP gradients that are timely and locally variable have been demonstrated, and these gradients have been shown to arise from a sequestration of cAMP signaling components at the cell membrane within deﬁned supramolecular complexes. The major components of these complexes are speciﬁc anchoring proteins, the AKAPs (Section 9.3.3), which 8.3 cGMP and Guanylyl Cyclases 375 form a scaffold that binds and organizes PKA, Epacs and PDEs within larger supramolecular complexes at spatially discrete sites. By this mechanism, the formation and degradation of cAMP and the activation of PKA occurs at spatially restricted sites, allowing for the initiation of a localized reaction. The inclusion of PDEs in these complexes is critical to shaping and interpreting cAMP gradients because of the high turnover number of these enzymes [4]. This aspect of signal transduction, known as “targeting,” is described in greater detail in Section 9.3.3.

8.3 cGMP and Guanylyl Cyclases

Summary The second messenger 30,50-cyclic GMP (cGMP) is produced from GTP by the action of guanylyl cyclases. cGMP binds to and activates cGMP-dependent protein kinases, ion channels and some phosphodiesterases. Guanylyl cyclases are grouped into two types: receptor guanylyl cyclases and soluble guanylyl cyclases. The receptor guanylyl cyclases are transmembrane proteins that are activated by extracellular binding of peptide ligands to form cGMP by the cytoplasmic catalytic domain. The cytoplasmic localized guanylyl cyclases are regulated by the second messenger nitric oxide (NO).

As with cAMP, 30,50-cyclic GMP (cGMP) is a intracellular messenger that is present in life forms ranging from bacteria to yeast to human. Many targets of cGMP contain a cyclic nucleotide recognition module termed GAF (cyclic GMP, adenylyl cyclase, FhlA) domain. Originally, the GAF domain was believed to bind only cGMP, but it has now been found also to bind cAMP. Examples of proteins with GAF domains are non-membrane adenylyl cyclases, guanylyl cyclases, and phosphodiesterases [5].

8.3.1 Guanylyl Cyclases

Analogous to cAMP, cGMP is formed by catalysis via guanylyl cyclase from GTP. Although the guanylyl cyclases catalyze a similar reaction to the ACs, the two enzyme classes differ considerably in their structure and mechanism of activation. Two groups of guanylyl cyclases are found in vertebrates: one group comprises cytoplasmically localized enzymes that contain a heme group and are referred to as soluble guanylyl cyclases; a second group contains one transmembrane segment. The members of the second group are directly regulated by extracellular ligands and are referred to as guanylyl cyclase receptors. 376 8 Intracellular Messenger Substances: “Second Messengers”

& Guanylyl cyclase receptors: — Transmembrane proteins with a single transmembrane helix — Activated by extracellular peptide ligands — Synthesize cGMP in an ATP-dependent manner.

8.3.1.1 Guanylyl Cyclase Receptors The guanylyl cyclase receptors constitute a unique class of transmembrane guanylyl cyclases that transmit an extracellular signal directly into the formation of an intracellular second messenger substance [6]. cGMP formed in this way regulates important physiological processes such as the relaxation of blood vessels. Mammals express seven single-membrane-spanning forms known as guanylyl cyclases A to F (GC-A to GC-F) that exist as homodimers (Figure 8.4). Soluble guanylyl cyclases (see below) and transmembrane guanylyl cyclases such as natriuretic peptide receptor (NPR)-C that lack the large intracellular domains have also been identiﬁed. The transmembrane guanylyl cyclase contains an extracellular ligand-binding domain, a single transmembrane helix and various intracellular domains that are required for the ligand-regulated activation of the enzyme. Peptides with vasodilatory properties, such as atrial natriuretic peptide (ANP), have been identiﬁed as ligands for the guanylyl cyclase receptors. Guanylyl cyclases activated

Figure 8.4 Schematic of mammalian intermolecular disulfide bond. Abbreviations: guanylyl cyclases and their ligands. Seven ANP, atrial natriuretic peptide; BNP, B-type transmembrane and four soluble guanylyl natriuretic peptide; CNP, C-type natriuretic cyclases are expressed in mammals. The peptide; CO, carbon monoxide; CO2/HCO3 , similarity of the extracellular domain color carbon dioxide/bicarbonate; GCAPs, guanylyl represents primary amino acid sequence cyclase activator proteins; Gn, guanylin; Hb, similarity between receptors. The red circles hemoglobin; NO, nitric oxide; NPR-C, indicate known phosphorylation sites. The natriuretic peptide receptor C; Sta, heat-stable black bar between NPR-C subunits indicates an enterotoxin; Uro, uroguanylin. After Ref. [6]). 8.3 cGMP and Guanylyl Cyclases 377 by ANP belong to the GC-A subfamily and are also termed NPRs. Peptide binding to the extracellular ligand binding domain activates cGMP synthesis by the intracellular guanylyl cyclase domain. Structural studies have suggested that ligand binding to the extracellular domain of GC-A induces a twisting of the homodimer that is ﬁnally transmitted into activation of the cyclase domain. The regulation of GC-A is complex. Phosphorylation of an intracellular domain termed the kinase- homology domain is also required for the activation of GC-A. Furthermore, ATP increases the activity of GC-A by a mechanism that remains to be clariﬁed.

8.3.1.2 Soluble Guanylyl Cyclases The soluble guanylyl cyclases [7] exist as heterodimers and are regulated by the second messenger, NO (Section 8.10.3). A heme group that confers NO-sensitivity is bound at the N terminus of these enzymes; the binding of NO to this heme group results in an activation of guanylyl cyclase activity.

& Cytoplasmic guanylyl cyclases: — Contain a heme group — Are activated by NO.

8.3.2 Targets of cGMP

The second messenger functions of cGMP are mainly directed towards three targets (Figure 8.2):

cGMP-dependent protein kinases: The cGMP-dependent protein kinases (cGKs; also known as protein kinase G, PKG) are activated by cGMP binding [2] and have structural elements similar to those of PKA. In contrast to PKA, the regulatory and catalytic functions in cGMP-dependent protein kinases are localized on one protein chain. cGKs exist as homodimers, and the binding of cGMP to the regulatory domain relieves autoinhibition by the N terminus and allows phosphorylation of substrate proteins. cGKs modulate many physiological functions, such as smooth muscle relaxation (e.g., the vasculature, gastrointestinal tract, bladder and penile), platelet aggregation, renin release, intestinal secretion, learning, and memory. The most important in vivo substrates in þ smooth muscle cells are Ca2 -channels and a myosin-specific protein phosphatase. Phosphorylation of these two substrates by a cGMP-specific protein kinase þ modulates Ca2 -levels and thereby provides control of smooth muscle tone. Cation channels: Cation channels are known which are gated by cGMP and possess cGMP-binding sites on their intracellular side. The binding of cGMP to the cation channel induces an opening of the channel and an influx of cations. In þ the vision process, the role of cGMP is to regulate Ca2 influx via cGMP-gated cation channels. cAMP-specific phosphodiesterases: Some types of cAMP-specific phosphodiesterases are regulated by cGMP. 378 8 Intracellular Messenger Substances: “Second Messengers”

& Targets of cGMP: — cGK — cGMP-gated cation channels — cAMP-dependent phosphodiesterases.

8.4 Metabolism of Inositol Phospholipids and Inositol Phosphates

Summary Inositol-containing phospholipids of the plasma membrane are the starting compounds for the enzymatic formation of multiple low-molecular-weight inositol messengers in response to intracellular and extracellular signals. The activation of PLC leads to the formation of the central second messengers

diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG activates PKC, 2þ whilst IP3 mediates the release of Ca from internal storage organelles. Furthermore, multiple membrane-bound phosphatidylinositol phosphates, collectively termed phosphoinositides, have been identified that perform distinct signaling functions at the cell membrane

Inositol-containing phospholipids of the plasma membrane are the starting compounds for the formation of multiple low-molecular-weight inositol messengers in response to intracellular and extracellular signals. These messengers include the central second messengers diacylglycerol (DAG) and inositol trispho-

sphate (ITP3), as well as membrane-bound phosphatidylinositol phosphates, collectively termed phosphoinositides. Theplasmamembranecontainsthephospholipid phosphatidylinositol (PtdIns), in which the phosphate group is esteriﬁed with a cyclic alcohol, myo- D-inositol (Figure 8.5). Starting from PtdIns, a series of enzymatic transformations leads to the generation of a diverse number of second messengers. These transformations include the phosphorylation of speciﬁc hydroxyl groups of inositol as well as hydrolysis of the bond between the glycerol moiety and the phosphorylated inositol.

& PtdIns-derived messengers: — Diacylglycerol — InsP3 — InsPx — PtdIns(3,4,5)P3 — PtdInsPx. 8.4 Metabolism of Inositol Phospholipids and Inositol Phosphates 379

Figure 8.5 Formation of diacylglycerol, Ins(1,4,5)P3 and PtdIns(3,4,5)P3. PLC, phospholipase of type C; PI3-kinase, phosphatidyl inositol-30-kinase. 380 8 Intracellular Messenger Substances: “Second Messengers”

The most important PtdIns-derived messengers are:

PtdIns(3,4,5)P3: This compound binds to PH domains and is formed in a reaction catalyzed by the enzyme phosphatidylinositol-3-kinase (PI3K) (Sec- tion 8.4). Diacylglycerol and inositol-1,4,5-triphosphate (Ins(1,4,5)P3): Hydrolysis of phos-

phatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2) by PLC produces two second messengers, namely Ins(1,4,5)P3 and DAG. Ins(1,4,5)P3 activates the release of þ Ca2 , whilst DAG acts primarily by stimulation of PKC.

PtdIns is ﬁrst phosphorylated by speciﬁc kinases at the 40 and 50 positions of

the inositol residue, leading to the formation of PtdIns(4,5)P2. This compound and other inositol-containing glycerophospholipids are collectively known as phosphoinositides.

& Key enzymes of PtdIns metabolism: — Phospholipase C type b and c — PI3 kinase — Phosphoinositide phosphatases.

From PtdIns(4,5)P2, two pathways lead to physiologically important messenger substances. PtdIns(4,5)P2 may be further phosphorylated by PI3K to yield PtdIns (3,4,5)P3, which functions as a membrane-localized messenger (Section 8.6). In a “ further reaction, PtdIns(4,5)P2 may be cleaved by PLC, forming the second ” messengers Ins(1,4,5)P3 and DAG.

8.4.1 Other Inositol Messengers

Further transformations of inositol phosphates and phosphoinositides are known which lead to the formation of almost 30 inositol-containing compounds with potential messenger function. These messengers perform multiple messenger functions at the cell membrane, in the cytosol, and in other subcellular compartments as for example, the nucleus [8]. These reactions include the

phosphorylation of Ins(1,4,5)P3 to inositol polyphosphates (e.g., by the enzyme inositol polyphosphate kinase, IPK) as well as speciﬁc dephosphorylation by inositol phosphatases (e.g., by inositol polyphosphate-5-phosphatase). Another inositol phosphate with second messenger function is inositol-1,4,5,6-tetrakispho-

sphate (Ins(1,4,5,6)P4), which acts in a similar way as InsP3 to activate the InsP3 receptor. The signaling functions of these other inositol messengers are just 8.4 Metabolism of Inositol Phospholipids and Inositol Phosphates 381

Figure 8.6 Formation and major functions of Akt kinase (see Section 9.4.2); PDK1, inositol second messengers. Abbreviations: phosphoinositide-dependent protein kinase 1 PI3K: phosphatidyl inositide 3-kinase; PLC: (see Section 8.4.2); for PH, ENTH, and ANTH Phospholipase C; PKC: protein kinase C; Akt: domains, see Section 8.6.2.

beginning to be clariﬁed and a discussion of their messenger function is beyond the scope of this book. The routes of formation and the assumed physiological roles of a selected number of inositol-containing compounds are summarized in Figure 8.6.

8.4.2 Activation of PLC and Inositol Phosphate Formation

Phospholipase C, which occurs in different subtypes in the cell (Section 7.2), is a key enzyme of phosphatide inositol metabolism (for cleavage speciﬁcity, see Figure 7.33). Two central signaling pathways regulate the PLC activity of the cell in a positive fashion (Figure 8.7), triggering inositol phosphate and þ Ca2 signals. Phospholipases of type C-b (PLC-b) are activated by G proteins and are thus linked into signal pathways starting from G protein-coupled receptors (GPCRs). Phospholipases of type c (PLC-c), in contrast, are activated by transmembrane receptors with intrinsic or associated tyrosine kinase activity (see Chapters 10 and 13). The extracellular stimuli activated by the two major reaction pathways are very diverseinnature,whichiswhythePLC activity of the cell is subject to multiple regulation. 382 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.7 Formation and function of activates protein kinase C (Section 9.5), which diacylglycerol (DAG) and Ins(1,4,5)P3. has a regulatory effect on cell proliferation, via Formation of DAG and Ins(1,4,5)P3 is subject phosphorylation of substrate proteins. Ins to regulation by two central signaling (1,4,5)P3 binds to the corresponding receptors 2þ pathways, which start from transmembrane (InsP3R) and induces release of Ca from receptors with intrinsic or associated tyrosine internal stores. The membrane association of

kinase activity (see Chapters 10 and 13) or DAG, PtdIns(3,4)P2 and PL-C is not shown from G protein-coupled receptors. DAG here, for clarity. 8.5 Storage and Release of Ca2þ 383

8.5 þ Storage and Release of Ca2

Summary þ Ca2 is a ubiquitous signaling molecule, the signaling function of which is þ activated by its release from intracellular stores or through Ca2 -entry þ channels from the extracellular side. The free Ca2 concentration is subject þ to strict regulation, and the targeted increase of Ca2 represents a universal means of controlling a vast array of metabolic and physiological reactions. For release from the intracellular stores, three types of messenger are

predominant, namely Ins(1,4,5)P3, cADP ribose, and nicotinic acid adenine þ dinucleotide phosphate (NAADP). In addition, Ca2 itself is used as a þ trigger for Ca2 release. 2þ The major Ca -entry channels are the IP3 receptor that is regulated by IP3 and þ þ Ca2 , and the ryanodine receptor that is activated primarily by Ca2 . The signal- þ þ þ directed release of Ca2 is opposed by Ca2 transport systems, which carry Ca2 back into the extracellular region or into the storage organelles. The coordinated þ release and removal of Ca2 leads to the creation of temporally and spatially þ distinct signals which are observed as Ca2 spikes, waves or oscillations. The þ information encoded in transient Ca2 signals is deciphered by various þ intracellular Ca2 -regulated proteins that convert the signal into a wide variety of þ biochemical changes. Many signaling enzymes are directly activated by Ca2 . þ Furthermore, Ca2 -sensor proteins are known that translate the physiological þ changes in Ca2 concentration into specific cellular reactions by binding to and activating signaling enzymes, often protein kinases. The most prominent of these sensor proteins is calmodulin.

2þ The primary signal function of Ins(1,4,5)P3 is the mobilization of Ca from storage organelles. þ The concentration of free Ca2 in the cytosol of “resting” cells is very low, at þ about 10 7 M. One reason that the cell attempts to maintain a low free Ca2 concentration is the ability of these ions to form poorly soluble complexes with þ inorganic phosphate. The low concentration of free cytosolic Ca2 is opposed by a þ large storage capacity for Ca2 in speciﬁc cytosolic compartments, and by a high þ concentration in the extracellular region where Ca2 is present at millimolar þ concentrations. In the cytosol, Ca2 is stored not only in the mitochondria but also in special storage compartments of the endoplasmic reticulum (ER). In the ER- þ þ associated storage, Ca2 exists in complex with various Ca2 storage proteins such as calreticulin, calsequestrin, Grp94, and BIP [9]. In the protein-bound and þ compartmentalized form, Ca2 is not freely available but may be released in the process of signal transduction. 384 8 Intracellular Messenger Substances: “Second Messengers”

þ & Ca2 distribution: — Extracellular: about 1 mM þ — Intracellular: free Ca2 : 10 7 M. þ Ca2 stores: — Endoplasmic reticulum — Sarcoplasmic reticulum — Mitochondria. þ Ca2 storage proteins: — Calreticulin — BIP — Grp94.

þ In muscle cells, Ca2 is stored in the sarcoplasmic reticulum, notably by binding þ to the storage protein calsequestrin. The release of Ca2 from storage by a neural stimulus (Section 8.5.1) initiates muscle contraction. The cell has available a multitude of tools for creating speciﬁc concentration þ changes of free Ca2 [10], and these tools (see Figure 8.11) allow the cell to shape þ Ca2 signals in the dimensions of space, time, and amplitude. An overview of the main pathways leading to an increase or decrease of intracellular calcium is provided in Figure 8.8.

8.5.1 þ þ Release of Ca2 from Ca2 Storage

þ þ The mobilization of Ca2 from Ca2 stores is induced by binding of the second 2þ messengers Ins(1,4,5)P3, cADP ribose and NAADP to ligand-gated Ca channels in which the receptor and ion channel form a structural unit. (Figure 8.9).

& 2þ Opening of the intracellular ligand-gated Ca channels InsP3-R and ryanodine receptor is regulated by: — InsP3 — cADP ribose — NAADP þ — Ca2 .

8.5.1.1 The InsP3 Receptor 2þ The InsP3 receptor is a ligand-gated Ca channel that is subject to regulation by 2þ InsP3, ATP, and Ca itself [11]. The binding of InsP3 to the InsP3 receptor leads to þ an opening of the receptor channel, so that Ca2 , which is present in the ER stores ﬂ at high concentration, can ow into the cytosol. The InsP3 receptor is a 8.5 Storage and Release of Ca2þ 385

2þ Figure 8.8 Pathways for the increase and The entering Ca binds to InsP3 receptors on þ þ reduction of cytosolic Ca2 concentration. the membrane of Ca2 storage organelles and 2þ Influx of Ca from the extracellular space induces, together with InsP3, their opening. þ þ takes place via Ca2 channels; the open state Ca2 flows out of the storage organelle into the of these is controlled by binding of ligand (L) cytosol via the ion channel of the InsP3 þ or by a change in the membrane potential receptor. Transport of Ca2 back into the (DV). According to the type of ion channel, the storage organelles takes place with assistance þ ligand may bind from the cytosolic or the from ATP-dependent Ca2 transporters. extracellular side to the ion channel protein. homotetrameric channel with six transmembrane helices on the C terminus of each subunit. The cytosolic domain comprises about 75% of the total protein, and harbors binding sites for InsP3 and calcium ions. There is an inhibitory domain near the N terminus, and a clam-like InsP3-binding region that appears to trigger conformational changes and relieve autoinhibition upon InsP3 binding. Opening 2þ of the InsP3 receptor is subject to multiple regulation involving Ca , InsP3, ATP, phosphorylation and dephosphorylation. Multiple regulatory protein interactions 386 8 Intracellular Messenger Substances: “Second Messengers”

þ Figure 8.9 Tetrameric Ca2 channels and potential change DV induces the opening of þ þ þ control of Ca2 release. (a) A change in the voltage-gated Ca2 channels. Ca2 passes membrane potential (DV) induces a through, which serves as a trigger for the þ þ conformational change in the dihydropyridine release of Ca2 from Ca2 storage organelles receptor of skeletal muscle; this is transmitted by binding to ryanodine receptors on the as a signal to the structurally coupled surface of the storage organelles; þ ryanodine receptor. Opening of the Ca2 (c) Membrane-associated signaling pathways þ channel takes place and efflux of Ca2 from the are activated by ligands and lead, via activated sarcoplasmic reticulum into the cytosol occurs; receptor and phospholipase C (PL-C) to the 2þ 2þ (b) In cardiac muscle, the release of Ca takes formation of InsP3 and the release of Ca þ place by a Ca2 -induced mechanism. A from storage organelles.

fi and modi cations have been reported for the InsP3 receptor, including the binding of AKAP proteins and phosphorylation by PKA and PKB (see Chapter 9) [12]. fi 2þ 2þ Binding of InsP3 to the receptor increases its af nity for Ca , and only after Ca þ þ has been bound can trafficking of Ca2 into the cytosol take place. For Ca2 2þ concentrations of up to 500 nM, Ca functions synergistically with InsP3 to 2þ activate the IP3 receptors, but at higher concentrations the cytosolic Ca inhibits InsP3 receptor opening, inducing its closure.

& InsP3 receptor: — Homotetramer þ — Releases Ca2 from internal stores — 2þ Opening activated by InsP3, low Ca þ — Closure induced by high Ca2 . 8.5 Storage and Release of Ca2þ 387

þ By this mechanism, Ca2 has a biphasic action on the receptor, with a þ þ stimulatory effect at low Ca2 and an inhibitory effect at high Ca2 . The latter is thought to be a crucial mechanism for terminating channel activity, for shaping þ þ Ca2 waves, and for preventing pathological increases in Ca2 .

8.5.1.2 The Ryanodine Receptor The ryanodine receptor takes its name from its stimulation by the plant alkaloid ryanodine [13]. Overall, it has a similar composition to the InsP3 receptor and is þ involved in Ca2 signal conduction in many excitatory cells (cells of banded and smooth musculature, neurons, etc.). þ The open state of the ryanodine receptor is controlled in part by Ca2 ,whichbindsto þ the receptor and induces its opening. The Ca2 -induced opening of the ryanodine receptor provides the cell with a cooperative, self-amplifying mechanism that can trigger a þ þ rapid increase in Ca2 concentration. An initial increase in Ca2 concentration, induced þ þ by Ca2 inﬂux from the extracellular space due to the opening of voltage-gated Ca2 þ channels, will in turn initiate opening of the ryanodine receptors. The additional Ca2 emerging from the membrane compartments will then open even more ryanodine 2þ receptors,leadingtoasteepincreaseinCa concentration. As with the InsP3 receptors, þ þ high Ca2 concentrations can inhibit Ca2 ﬂux through the channel.

& Ryanodine receptor: — Structurally related to InsP3 receptor þ — Opening induced by low Ca2 , by cADP ribose þ — Closure induced by high Ca2 .

þ A specialized coupling between extracellular Ca2 inﬂux and the ryanodine þ receptor exists in muscle cells, where a voltage-dependent Ca2 channel in the cell membrane – termed the L-type voltage-gate calcium channel – can couple directly to the cytoplasmic domain of the ryanodine receptor localized in the membrane of the sarcoplasmic reticulum (Figure 8.9). Depolarization of the cell membrane is transmitted in this system via an electromechanical coupling directly to the gating state of the ryanodine receptor.

8.5.1.3 cADP-Ribose and NAADP In some cell types (including cardiac muscle cells and pancreatic cells), an alternative second messenger – cyclic ADP-ribose (cADP-ribose) – is involved in opening the ryanodine receptors [14]. cADP-ribose is formed from NADP via an enzymatic pathway with the help of an ADP-ribosyl cyclase (Figure 8.10). This þ messenger initiates Ca2 release from the ryanodine receptors and sensitizes the þ receptors to further activation by Ca2 . This feed-forward mechanism, which is þ þ þ known as Ca2 -induced Ca2 release, can lead to prolonged Ca2 signals. A further second messenger, NAADP, can be generated by the action of ADP- ribosyl cyclase (Figure 6.10) and has been identiﬁed in brain and other tissues as þ being the most potent natural Ca2 -mobilizing agent. The nature of NAADP’s 388 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.10 Reactions of ADP ribosyl cyclase. replacement of the nicotinamide group of Structures of NADP, nicotinic acid adenine NADP (yellow) with nicotinic acid to generate dinucleotide phosphate (NAADP) and cyclic NAADP. ADP-ribosyl cyclase can also catalyze ADP-ribose phosphate (cADPRP). ADP-ribosyl cyclization of NADP to cADPRP. cyclase, in base exchange mode, can catalyze

target calcium release channel is controversial, however, as is the subcellular þ localization of its receptor. NAADP is known to release Ca2 from the intracellular

stores independently of InsP3 and cyclic ADP ribose signals [15].

þ 8.5.1.4 Ca2 Channels and Apoptosis The results of several studies have suggested that an inappropriate regulation of

InsP3 receptors contributes towards apoptosis. Proteins with central functions in 2þ ﬂ apoptosis have been found to bind to and regulate Ca - ux through the InsP3 receptor. The antiapoptotic protein Bcl-2 (see Chapter 17) is known to bind directly

to the InsP3 receptor, protecting the cell from death. However, the binding of cytochrome c, released from the mitochondria during apoptosis, causes an 2þ inhibition of InsP3 receptor closure and leads to elevated Ca levels and the promotion of cell death. 8.5 Storage and Release of Ca2þ 389

þ 8.5.1.5 Tool Kit for Ca2 Release þ Overall, multiple pathways can be used for mobilizing Ca2 from the internal þ stores. A Ca2 signaling “toolkit” is available from which cells can select speciﬁc þ components to activate the internal Ca2 stores and to generate a variety of þ different Ca2 signals that suit their physiology. In summary, the following þ pathways can induce Ca2 release from internal stores (Figure 8.11):

þ þ þ Ca2 -induced Ca2 release from ryanodine receptors caused by inﬂux of Ca2 þ through voltage-operated Ca2 channels on the plasma membrane. þ Cyclic ADP-ribose-evoked Ca2 release. þ NAADP-evoked Ca2 release. þ InsP3-evoked Ca2 release. 2þ Ca release by interaction of InsP3 receptors with calcium-binding proteins. 2þ Ca release triggered by sphingolipids or leukotriene B4. þ Ca2 release from mitochondria.

2þ 2þ Figure 8.11 Tools for Ca release. The figure PLC/InsP3-evoked release of Ca from InsP3 illustrates the major pathways for mobilizing receptors or RyRs. 3, cyclic ADP-ribose þ þ þ Ca2 from internal stores. 1, Ca2 -induced (cADPR)-evoked Ca2 release. 4, Nicotinic acid þ Ca2 release from ryanodine receptors (RyRs) adenine dinucleotide phosphate (NAADP)- þ þ þ caused by the influx of Ca 2 through voltage- evoked Ca2 release. 5, Ca2 release evoked þ or ligand-gated channels on the outer cell by sphingosine. 6, Ca2 release from membrane. This release may be also triggered mitochondria. by direct interaction of the channel with RYR. 2, 390 8 Intracellular Messenger Substances: “Second Messengers”

8.5.2 þ Influx of Ca2 from the Extracellular Region

þ In the extracellular region, the Ca2 concentration is over 10 3 M, and very high in þ comparison to the free cytosolic Ca2 concentration. The cell membrane contains a þ þ variety of different Ca2 channel types that enable Ca2 inﬂux to take place from þ the extracellular region into the cytosol. One of the primary functions of Ca2 entry þ is to charge up the internal stores, which can then release an internal Ca2 signal. þ The main Ca2 inﬂux channels are

Voltage-gated channels are opened by a depolarization or change in membrane potential. Ligand-gated channels are activated by the binding of an agonist to the extracellular domain of the channel. Examples are provided by the acetylcholine receptor and the N-methyl-D-aspartate receptor. Mechanically activated channels are present on many cell types and respond to mechanical stress. þ þ þ Store-operated Ca2 channels named CRAC (Ca2 release-activated Ca2 ) chan- þ nels are activated in response to depletion of the intracellular Ca2 stores.

2þ In addition, Ca channels are known that are controlled by Ga proteins and þ Ca2 channels that are gated by sphingolipids.

þ & Properties of ion channels involved in Ca2 influx: — Ligand-gated — Voltage-gated — Mechanically operated — Store-operated.

8.5.3 þ Removal and Storage of Ca2

þ Generally, the cytosolic Ca2 concentration is increased only temporarily and locally þ during the stimulation of cells. The cell possesses efﬁcient Ca2 transport systems, þ which can rapidly transport Ca2 back into the extracellular region or into the þ storage organelles. Ca2 uptake into the mitochondria is another important way of þ þ removing cytosolic Ca2 ions: by sequestering Ca2 ions, mitochondria can þ modulate the kinetics and spatial dimensions of cellular Ca2 signals.

þ & Ca2 removal occurs via: þ — Ca2 -ATPases þ þ — Na /Ca2 exchange proteins.

þ Two types of channel proteins are responsible for removing Ca2 from the þ þ cytosol: (i) Ca2 -ATPases are pumps that carry out an active transport of Ca2 8.5 Storage and Release of Ca2þ 391 against its concentration gradient, using the hydrolysis of ATP as an energy source; þ þ and (ii) Na /Ca2 exchange (NCX) proteins, which are bidirectional exchange þ þ proteins that can exchange Na for Ca2 . These exchange proteins are located especially in muscle cells and in neurons. þ þ Most important for Ca2 removal are the Ca2 -pumps of which two major types are known:

2þ þ Plasma membrane Ca (PMCA) pumps are Ca2 -ATPases involved in draining þ the Ca2 from the cytosol back into the extracellular region. 2þ Sarco(endo)plasmic reticulum Ca (SERCA) pumps are responsible for trans- þ porting Ca2 back into the intracellular store of the sarcoplasmic and endoplasmic reticula.

þ þ The opening of Ca2 -channels leads to a local increase in the cytosolic Ca2 þ concentration from 10 7 Mto106 M. In this concentration region, the Ca2 transport systems mentioned above operate very efﬁciently; however, if an increase þ in Ca2 concentration over 10 5 M takes place – for example, due to cell damage – a þ þ Ca2 level that is critical for the cell will be reached. In this case, Ca2 must be þ pumped into the mitochondria, aided by Ca2 transport systems localized in the mitochondrial inner membrane.

8.5.4 þ Temporal and Spatial Changes in Ca2 Concentration

þ Ca2 is a versatile signaling molecule which is used by the cell for the creation of þ temporally and spatially distinct signals. Most Ca2 -signaling components are þ organized into macromolecular complexes in which Ca2 -signaling functions are þ carried out within highly localized environments. The Ca2 -signals produced are of þ variable shape and may appear as “elementary” Ca2 signals or as global signals in the form of spikes or waves. þ Generally, the formation of cell-specific and highly variable global Ca2 signals is þ based on the differential use of the various mechanisms that produce the Ca2 “on” þ and “off” states. From the large Ca2 signaling tool kit (Figure 8.11), each cell employs a specific set of channels and pumps to create signals that are highly variable in space and in time. When the membrane channels and the intracellular release channels are þ activated, only brief pulses of Ca2 are produced, as these channels have short þ open times. These “elementary” Ca2 signals are localized around the channels and provide local control of many physiological reactions such as the activation þ of other ion channels and nucleus-specificCa2 signals. The coordinated þ recruitment of many elementary Ca2 release and entry channels allows the þ formation of global Ca2 signals that persist over a longer time and have a larger þ spatial distribution. Commonly, these global Ca2 signals are of a pulsatile nature and appear as waves or spikes. The mechanisms by which waves and spikes are generated are diverse and are used in a cell-specific fashion. The 392 8 Intracellular Messenger Substances: “Second Messengers”

differential sensitivity of the InsP3 receptor and the ryanodine receptor to low þ and high Ca2 concentrations is one mechanism that is assumed to contribute þ þ to Ca2 wave and spike formation. A small increase in Ca2 concentration due to þ an elementary Ca2 signal will activate some release channels and allow the þ þ influx of more Ca2 in a cooperative manner. The Ca2 concentration increases þ until a threshold value is reached that is sufficient to inhibit Ca2 influx through þ the channel, such that the Ca2 concentration falls again. In a further reaction, þ the released Ca2 activates PLC enzymes, leading to an increased formation

of InsP3 that diffuses to the InsP3 receptors and brings about the release of þ more Ca2 .

8.5.4.1 Calcium Oscillations þ Various feedback mechanisms exist that ensure a decrease in Ca2 concentration and concomitant peak and wave formation under conditions of constant exposure to stimulatory signals. A coupling of inﬂux and efﬂux systems can lead to periodic þ changes in Ca2 concentration that can last for a longer period, even after the initial þ stimulus has faded away. Such Ca2 oscillations are observed for example, in excitable cells and are driven by changes in the plasma membrane potential that þ activate voltage-dependent Ca2 channels. The major means of evoking oscillations, however, is through the activation of cell-surface receptors (GPCRs, receptor

tyrosine kinases) that activate PLC, which form the messengers DAG and IP3, þ leading to the Ca2 spike formation described above. When the stores become þ depleted of Ca2 , the store-operated CRAC channels open and allow the influx of þ Ca2 from the extracellular side such that the stores can be refilled. Such a þ recycling of Ca2 across the internal stores can last for up to minutes. The ensuing þ þ Ca2 oscillations are detected by downstream Ca2 sensors that translate the oscillation into distinct cellular responses. Much like electronic or optical signals in þ control engineering, the information content of Ca2 signals may be determined by þ the location, frequency, period and amplitude of the Ca2 peak. Yet, how the þ frequency of an oscillating Ca2 signal is decoded or integrated and incorporated into specific biochemical reactions is not well understood. Two sensors that have þ þ been implicated in decoding Ca2 -oscillations, are Ca2 -sensitive protein kinase C þ subtypes and the Ca2 /calmodulin-dependent protein kinase II, CaM kinase II. þ The properties of CaM kinase II and its ability to decode Ca2 oscillations will be discussed in Section 9.6.2.

8.6 Functions of Phosphoinositides

Phosphorylation at the 30 position of the inositol part of PtdIns derivatives leads to further second messengers of central regulatory importance. The reaction is catalyzed by a class of enzymes known as phosphatidylinositide-3-kinases (PI3- kinases; see Figure 8.6 and Section 9.4.1). The PI3-kinases phosphorylate various phosphatidyl inositol compounds at the 30 position. 8.6 Functions of Phosphoinositides 393

8.6.1

The Messenger Function of PtdIns(3,4,5)P3

A major substrate of PI3-kinase is PtdIns(4,5)P2, which is converted into PtdIns (3,4,5)P3. This compound is a membrane-localized second messenger that exerts most of its cellular functions by binding to the pleckstrin homology (PH) domains of signal proteins. PH domains are found as independent protein modules in many signal proteins (Section 2.4.4) that mediate protein–lipid and possibly also protein– protein interactions.

PtdIns(3,4,5)P3 formed by PI3-kinase serves to recruit PH domain-containing proteins to the membrane and to involve them in signal conduction. In addition to membrane targeting, PtdIns(3,4,5)P3 binding to PH domains can also bring about an allosteric activation of effector proteins. Many protein kinases contain PH domains and are therefore subject to regulation by PtdIns(3,4,5)P3. ﬁ The speci c binding of PtdIns(3,4,5)P3 has also been reported for other protein modules found in signaling proteins, namely SH2 domains, PTB domains, the C2 domain of protein kinase C, and the FYVE ring ﬁnger domain of several membrane proteins.

& Function of PtdIns(3,4,5)P3: — Binding to PH domains of effector proteins such as PI3 kinase, PDK1 — Activation of effector proteins by: Allosteric processes Membrane targeting.

8.6.2

Functions of PtIns(4,5)P2 and Other Phosphoinositides

By varying the site of attachment of the phosphate group on the inositol moiety of phosphatidylinositol, a collection of phosphoinositides is created in the cell with multiple functions in cellular signaling, in membrane trafficking, and in microfilament formation and degradation. The DAG moiety of the various phosphoinositides serves for membrane association, whereas the exposed headgroups bind to effector proteins realizing their specific signaling function. These effector proteins bind the various phosphoinositides via a diverse range of interaction domains with considerable specificity, allowing for a high selectivity of an effector for a particular phosphoinositide species. In addition to the well-known PH domain, other specific phosphoinositide-binding domains have been identified, such as the ENTH domain, the ANTH domain, the FYVE domain, as well as patches of basic amino acids used by some of the effector proteins for phosphoinositide binding.

PtdIns(4,5)P2 represents a focal point in phosphoinositide signaling. Not only is this compound the substrate of PLC to yield the second messengers InsP3 and DAG, rather it also binds to a number of effector proteins through which it is a critical regulator of actin polymerization and anchorage to cell membranes, regulated secretion, endocytic 394 8 Intracellular Messenger Substances: “Second Messengers”

vesicle formation and other membrane-associated trafﬁcking functions [16]. As interaction modules, PH domains, ANTH and ENTH domains have been identiﬁed.

The phosphoinositides PtdIns(4)P, PtdIns(3)P and PtdIns(3,5)P2 are associated predominantly with intracellular membranes (Golgi, endosomes) where they are involved in membrane trafﬁcking and vesicle turnover. Four principal interaction domains have been implicated in targeting functional proteins to these compartments, named the PH, ANTH, FYVE, and PX domains.

& The phosphoinositides

— PtdIns(4,5)P2 — PtdIns(3,5)P2 — PtdIns(4)P, — PtdIns(3)P. are involved in: — Actin polymerization — Secretion — Vesicle trafficking — Target domains: PH, ENTH, ANTH, FYVE.

8.7 þ Ca2 as a Signal Molecule

þ Ca2 is a central signal molecule of the cell. Following hormonal or electrical þ stimulation, an increase in cytosolic Ca2 occurs, leading to the initiation of other reactions in the cell. As outlined above, this increase is limited in time and in þ space, and allows the formation of a variety of differently shaped Ca2 signals. þ Examples of Ca2 -dependent reactions are numerous and affect many important þ processes of the organism, including Ca2 signals in the form of temporally and þ spatially variable changes in Ca2 concentration that serve as elements of intracellular signal conduction in many signaling pathways.

þ & Ca2 is involved in: — Muscle contraction — Vision process — Cell proliferation — Secretion — Cell motility — Formation of cytoskeleton — Gene expression — Reactions of intermediary metabolism.

þ Three main pathways for increases in Ca2 concentration predominate (Table 8.1; see also Figures 8.7 and 8.8), and there are two principal mechanisms þ by which Ca2 can perform a regulatory function: 8.7 Ca2þ as a Signal Molecule 395

þ Table 8.1 Receptors of the plasma membrane that mediate increase of intracellular Ca2 .

Mediated via PLCb Mediated via PLCc Direct a1-Adrenergic receptor Epidermal growth factor Nicotinic acetylcholine receptor receptor Muscarinic acetylcholine Platelet-derived growth factor Glutamate receptors receptors receptor Glucagon receptor Fibroblast growth factor receptor Serotonin receptor T cell receptor Vasopressin receptor Ocytocin receptor Angiotensin II receptor Thrombin receptor Bombesin receptor Bradykinin receptor Tachykinin receptor Thromboxane receptor

þ Direct activation of proteins: Many proteins have a speciﬁc binding site for Ca2 , þ þ and their activity is directly dependent on Ca2 binding. The available Ca2 concentration thus directly controls the activity of these proteins (Table 8.2). þ There are many enzymes that have a speciﬁcCa2 -binding site in the active þ center and for which Ca2 has an essential role in catalysis. An example of a þ Ca2 -dependent enzyme is PLA2, which catalyzes the hydrolysis of fatty acid

þ Table 8.2 Ca2 -binding proteins.

Protein Function

Troponin C modulator of muscle contraction Caldesmon modulator of muscle contraction a-Actinin bundling of actin Villin organization of actin ﬁlaments Calmodulin modulator of protein kinases and other enzymes Calcineurin B protein phosphatase Calpain protease PLA2 release of arachidonic acid PKC ubiquitous protein kinase þ þ Ca2 -activated K channel effector of hyperpolarization 2þ InsP3 receptor intracellular Ca release þ Ryanodin receptor intracellular Ca2 release þ þ þ þ Na /Ca2 transporter exchange of Na and Ca2 via the cell membrane þ þ Ca2 ATPase transport of Ca2 through cell membrane Recoverin regulation of guanylyl cyclase þ Parvalbin Ca2 storage þ Calreticullin Ca2 storage þ Calbindin Ca2 storage þ Calsequestrin Ca2 storage 396 8 Intracellular Messenger Substances: “Second Messengers”

þ esters at the 20 position of phospholipids (see Figure 7.33), where Ca2 plays an þ essential role. This enzyme has two Ca2 ions bound tightly at the active center; þ one of the two Ca2 ions is directly involved in catalysis and binds the substrate in the ground state and also helps to neutralize charge in the transition state þ of ester hydrolysis. The second Ca2 ion is assigned a role in the stabilization of the transition state, in addition to a structural function. þ Other examples of Ca2 -regulated enzymes are PKC (Section 9.5) and PLC-c (Section 7.7.2). Many proteins are also known that are without enzyme þ activity but which have Ca2 -regulated functions. Proteins involved in the complex process of polymerization and depolymerization of the cytoskeleton þ are also often regulated by Ca2 binding. These include the annexins, fi mbrin, gelsolin and villin (the latter two are also regulated via PtInsP2). 2þ Both, Ca and PtInsP2 also have antagonistic effects on the polymerization þ state of microfilaments. Ca2 promotes the depolymerization of microfila-

ments, while PtInsP2 promotes their polymerization. 2þ Binding to Ca sensors: Another central mechanism of signal transduction via þ þ þ Ca2 is its binding to Ca2 -binding proteins, also known as Ca2 sensors.In þ response to physiological changes in Ca2 concentration, the sensor proteins undergo a conformational change that exposes a binding site recognized by downstream effectors. Temporal and spatial changes in calcium concentration in þ þ the range of 0.1 to 10 mM lead to specific binding of Ca2 to Ca2 -binding sites on the sensor and concomitant conformational changes that modulate the interaction with downstream target proteins. Accordingly, the calcium affinity of the various calcium sensors is fine-tuned in this concentration range.

þ & Signaling function of Ca2 is based on: — Direct activation of enzymes þ — Binding to Ca2 -sensors such as: Calmodulin Troponin C Recoverin.

8.7.1 þ The EF Hand: A Ca2 -Binding Module

The basic structural unit of most calcium sensors is a calcium-binding motif called EF-hand, and proteins harboring this motif are also called EF-hand proteins. The EF-hand has a characteristic helix–loop–helix structure and is found pairwise in a stable four-helix bundle (Figure 8.12a), called the EF domain. The pairing of EF- þ hands enables cooperativity in the binding of Ca2 ions, which is essential for þ generating a clean response to the relatively modest change in Ca2 concentration þ during cellular signaling. Binding afﬁnities for Ca2 vary widely for different EF- þ hands, with dissociation constants of Ca2 binding between 10 5 M and 10 9 M. 8.7 Ca2þ as a Signal Molecule 397

Figure 8.12 Calmodulin structure. (a) Basic with the calmodulin-binding domain of the þ structural features of EF-hand Ca2 -binding protein kinases is mediated by short helices, þ proteins. Shown are the structures of: isolated shown in green and blue. CaM-CaMKII, Ca2 / EF-hand motif, EF-hand domain from calmodulin-dependent protein kinase II; CaM- þ þ calmodulin (CaM), intact CaM, and the Ca2 - CaMKK, Ca2 /calmodulin-dependent protein þ sensor S100; (b) Comparison of different kinase kinase; CaM-MLCK, Ca2 /calmodulin- þ Ca2 /calmodulin structures. The figure dependent myosin light chain kinase; CaM-EF, þ illustrates the different conformations of Ca2 /calmodulin-dependent edema factor, an calmodulin when bound to target protein adenylyl cyclase, from Bacillus anthracis. From kinases. Calmodulin is shown in yellow and Ref. [18]). calcium ions are depicted in blue. Interaction 398 8 Intracellular Messenger Substances: “Second Messengers”

8.7.2 þ Calmodulin as a Ca2 Sensor

þ The most widespread family of Ca2 sensors are the calmodulins, which are small EF-hand proteins of about 150 amino acids [17]. Calmodulin comprises four EF-hands organized in two globular EF-domains that þ are separated by a flexible a-helical linker (Figure 8.12a). It binds four Ca2 ions fi ¼ 7 6 fi 2þ with an af nity (KD 5 10 Mto5 10 M) that ts into the intracellular Ca þ concentrations exhibited by most cells. The degree of Ca2 binding is therefore þ þ well suited to mirror changes in Ca2 during Ca2 signaling. Importantly, calcium affinities of the N- and C-terminal EF-domains may be very different, allowing the occupation of one domain at low calcium concentration whereas the other domain is occupied only at high concentrations.

& Calmodulin: þ — Widespread Ca2 -sensor þ — Binds four Ca2 ions via four EF-hands — Organized in two EF domains — Flexible structure þ — Affinity for Ca2 in micromolar range — Binding to target protein in different conformations possible.

þ Significant structural changes are induced in calmodulin upon Ca2 binding, leading to the exposure of a hydrophobic surface in each of the two domains that mediate binding of the target proteins. The conformational change is akin þ to a Ca2 -controlled unfolding of calmodulin, and it is assumed that þ interactions with the target proteins of Ca2 /calmodulin are mediated by the newly exposed hydrophobic residues. Furthermore, the flexible linker between the two domains enables a great deal of variability in the relative orientations of the two domains, which is a critical factor in the ability of calmodulin to interact with a large number of targets. Calmodulin can bind in very different modes to target proteins. The canonical and best-characterized binding mode is the wrapping mode, where the two domains bind to a single region (Figure 8.12b). When bound in this þ mode, Ca2 /calmodulin has a collapsed structure in which the two globular þ domains are much closer together than in free Ca2 /calmodulin, and it wraps around and sequesters the helical target peptide. Calmodulin can also bind in an extended mode such that the two domains interact with very different regions of the target. In another binding mode, calmodulin induces dimerization of the target, as exemplified by the complex with glutamate decarbox- ylase. The mechanisms by which calmodulin regulates its target proteins are diverse and can be categorized into several classes, the most important of which are: 8.7 Ca2þ as a Signal Molecule 399

þ Irreversible binding of calmodulin to the target protein irrespective of Ca2 .An example is phosphorylase kinase, an enzyme that contains calmodulin as a þ ﬁrmly bound subunit and is activated in the presence of Ca2 . þ Formation of inactive, low-afﬁnity complexes with calmodulin at low Ca2 þ concentrations and transition to an active complex in the presence of high Ca2 . This class includes the protein phosphatase calcineurin. þ Activation by Ca2 /calmodulin. Target proteins exhibiting this more conven- þ tional behavior include the Ca2 /calmodulin-dependent protein kinases (Sec- tion 9.6). þ Inhibition by Ca2 /calmodulin. This class includes members of the GPCR

kinases and subtypes of the InsP3 receptor.

þ From the structures of the substrates and their complexes with Ca2 /calmodulin [18], three main mechanisms of substrate activation have emerged (Figure 8.13). By one mechanism, an autoinhibitory element is displaced from the active site of the target enzyme, so as to relieve autoinhibition. Another protein activation mechanism þ of Ca2 /calmodulin employs a remodeling of the active site of the target protein.

8.7.3 þ Target Proteins of Ca2 /Calmodulin

þ The Ca2 /calmodulin complex is a signal molecule that mediates a myriad of cellular processes such as cell division and differentiation, gene transcription, neuronal signal transduction, membrane fusion, muscle contraction, and glucose metabolism. Different calmodulin subtypes are known which regulate a plethora of target proteins. The most well-known target proteins are the calmodulin-dependent ACs, phosphodiesterases, the protein phosphatase calcineurin (Section 9.7.5), protein kinases such as the CaM kinases (Section 9.6), and the myosin light chain kinase (MCLK), involved in the contraction of smooth musculature. A scanning of the human proteome for calmodulin targets has revealed the number of calmodulin-binding proteins to exceed 100, illustrating the huge range of functions þ that are potentially controlled by Ca2 /calmodulin [19].

8.7.4 þ Other Ca2 Sensors

þ The cell contains other Ca2 sensors, some of which are related to calmodulin, which occur in specialized tissue and perform speciﬁc functions there:

þ S100: A major family of Ca2 -sensor proteins are the S100 proteins which are found as dimers comprised of two EF-hand domains. The S100 proteins are associated with multiple target proteins that promote cell growth, cell-cycle regulation, transcription and cell-surface receptor signaling. Troponin C: This protein is structurally closely related to calmodulin. It is a component of the contraction apparatus of muscle and harbors two EF domains, 400 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.13 Mechanisms of activation inducing an active conformation (anthrax þ þ of target proteins by Ca2 /calmodulin. adenylyl cyclase); (c) Ca2 /calmodulin-induced þ þ (a) Binding of Ca2 /calmodulin relieves dimerization of K -channels. AID, autoinhibition (CaM-kinases, calcineurin); autoinhibition domain. From Ref. [18]. þ (b) Ca2 /calmodulin remodels the active site,

þ þ þ one of which binds Ca2 with such a high Ca2 afﬁnity that it is loaded with Ca2 þ even in the resting state. The other domain has a lower Ca2 afﬁnity and serves þ as the Ca2 -directed regulator of the interaction with the proteins involved in muscle contraction. þ Recoverin and the myristoyl switch: Another regulatory Ca2 receptor is recoverin, which performs an important control function in the signal transduction cascade of the vision process, by inhibiting the activity of rhodopsin kinase þ (Section 7.3.3). Recoverin is a Ca2 receptor with four EF structures and two þ Ca2 binding sites; it can exist in the cytosol or associated with the membrane and has an N-terminal myristoyl residue as a lipid anchor. The distribution 8.9 Other Lipid Messengers: Ceramide, Sphingosine, and Lysophosphatidic Acid 401

þ between free and membrane-associated forms is regulated by Ca2 . Binding of þ Ca2 to recoverin leads to its translocation from the cytosol to the membrane of þ þ the rod cells. Structural determination of recoverin in the Ca2 -bound and Ca2 - free forms indicates that membrane association of recoverin is regulated by a þ Ca2 -myristoyl switch (Section 2.9.6).

þ & The Ca2 sensor recoverin: — Is involved in the vision process þ — Binds two Ca2 -ions þ — Ca2 -binding leads to membrane localization þ — Is regulated by a Ca2 -myristoyl switch.

8.8 Diacylglycerol as a Signal Molecule

& Diacylglycerol: — Is formed from PtdInsP2 by PLC — Is a source of arachidonic acid — Activates PKC.

During the cleavage of PtInsP2 by PLC, two signal molecules are formed, namely InsP3 and DAG. Whilst InsP3 acts as a diffusible signal molecule in the cytosol after cleavage, the hydrophobic DAG remains in the membrane. DAG can be produced by different pathways, and has at least two functions (Figure 8.14). First, it is an important source for the release of arachidonic acid, from which prostaglandins are biosynthesized. The glycerine portion of the inositol phosphatide is often esteriﬁed in the 20 position with arachidonic acid, which is subsequently cleaved off by the action of type A2 phospholipase. Second, DAG performs an important regulatory function by stimulating PKC (Section 9.5).

& Functions of DAG: — Source of arachidonic acid — Activation of PKC.

8.9 Other Lipid Messengers: Ceramide, Sphingosine, and Lysophosphatidic Acid

Summary In addition to the membrane-associated messenger substances DAG and PtdIns

(3,4,5)P3, other lipophilic compounds such as ceramide, sphingosine and lysophosphatidic acid are known that are formed specifically during the process of signal transduction and which function as messenger substances. 402 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.14 Formation and function of acid (Ptd), and the action of phosphatases diacylglycerol. The figure shows, schematically, results in DAG. Arachidonic acid, the starting the two main pathways for the formation of point for the biosynthesis of prostaglandins diacylglycerol (DAG). DAG can be formed and other intracellular and extracellular from PtdInsP2 by the action of phospholipase messenger substances, can be cleaved from C (PL-C). Another pathway starts from DAG. PKC, protein kinase C; PtdIns, phosphatidyl choline. Phospholipase D (PL-D) phosphatidylinositol. converts phosphatidyl choline to phosphatidic

8.9.1 Ceramide

Ceramide is a lipophilic messenger that regulates diverse signaling pathways involving apoptosis, stress response, cell senescence, and differentiation. For the most part, ceramide’s effects are antagonistic to both cell growth and survival [20]. Cera- mide can be created in two ways, namely de novo synthesis and from sphingomyelin via the action of the enzyme sphingomyelinase (Figure 8.15). Sphingomyelinase has a similar cleavage speciﬁcity to PLC, in that it cleaves an alcohol–phosphate bond. The activation of sphingomyelinase is observed in response to diverse stress challenges that include irradiation, exposure to DNA-damaging agents, or treatment with pro-apoptotic ligands such as tumor necrosis factor a (TNFa; see Chapters 14 and 17). Because of these properties, ceramide is a potent apoptogenic agent.

& Ceramide: — Formed from sphingomyelin by sphingomyelinase or by de novo synthesis — Activates protein kinases and protein phosphatases — Is pro-apoptotic. 8.9 Other Lipid Messengers: Ceramide, Sphingosine, and Lysophosphatidic Acid 403

Figure 8.15 Formation and function of Sphingomyelinase is activated via a pathway ceramide. The starting point for the synthesis starting from tumor necrosis factor a (TNFa) of ceramide is sphingomyelin, which is and its receptor. Ceramide serves as an converted to phosphocholine and ceramide activator of protein kinases and protein by the action of a sphingomyelinase. phosphatases. R1, fatty acid side chain.

Ceramide (and also ceramide-1-phosphate), produced by the action of sphingomyelinase, is a membrane-located messenger substance that binds to and activates various downstream targets including stress-activated protein kinases such as the c-Jun-terminal protein kinase (JNK) (Section 12.4.2) and the protein phosphatases 1 and 2 (Section 9.7). Based on its location and synthesis, ceramide may be compared to DAG but the two compounds have opposite effects on cell growth; whereas, DAG stimulates cell growth via PKC, ceramide is a potent inhibitor of cell proliferation.

8.9.2 Sphingosine

This signaling molecule is formed from ceramide and can be phosphorylated to yield sphingosine-1-phosphate (S-1P). The latter has signaling functions both inside and outside of the cell; inside the cell it binds to intracellular targets and has a proliferation-promoting effect, whereas outside the cell it signals through binding to GPCRs, the S-1P receptors. 404 8 Intracellular Messenger Substances: “Second Messengers”

8.9.3 Lysophosphatidic Acid (LPA)

Messenger substances derived from phospholipids can also function as hormones and serve for communication purposes between cells. One important extracellular messenger substance formed from phospholipids is lysophosphatidic acid (LPA; 1-acyl-sn-glycerin-3-phosphate). LPA is released by platelets and other cells, and reaches its target cells via the blood circulation.

& Lysophosphatidic acid: — Extracellular messenger — Binds to GPCR — Signals via Gq-, Gi-orG12-proteins.

As a product of the blood-clotting process, LPA is an abundant constituent of serum, where it is found in an albumin-bound form. LPA binds and activates speciﬁc GPCRs found in many cells [21]. The LPA receptor can transmit the signal 2þ to Gq-, Gi-orG12-proteins. If Gq is involved, an InsP3 and Ca signal is produced ﬂ in the cell, whereas signal conduction via Gi-orG12-proteins ows into the Ras pathway or activates the Rho proteins, respectively (see Chapter 11).

8.10 The NO Signaling Molecule

Summary NO is a short-lived radical that functions as a universal messenger substance. Signal transduction via NO involves redox reactions and takes place by covalent modification of the target protein, primarily by reaction with cysteine-SH groups þ or metal ion centers to form S-nitrosyls (SNO) or metal nitrosyls (MeX –NO). Well-known targets of NO signaling are the soluble guanylyl cyclase and hemoglobin. NO is formed enzymatically from arginine with help from NO synthases (NOS). These enzymes are regulated either at the level of gene þ expression or via activation by Ca2 signals, which allows for NO-formation in a signal-directed fashion. Two major routes exist for transferring NO bioactivity to protein targets. In one route, the NO produced by NOS directly modifies a reactive cysteine thiol of a target protein. In a second route, which operates by transnitrosylation, the proteins become S-nitrosylated by accepting the NO group from a S-nitrosylated donor. S-nitrosylation is now a well-established regulatory modification that serves to regulate the activity of a large number of proteins. When NO is formed in excess amounts and in a deregulated fashion, nonspecific reactions are observed with various cell constituents including proteins, lipids and DNA, and NO acts as a toxic compound. 8.10 The NO Signaling Molecule 405

Nitric oxide (NO) is a universal messenger substance that is found in almost all living cells. NO takes part in intercellular and intracellular communication processes in higher and lower eukaryotes, and it is also found in bacteria and in plant cells. Although NO is a short-lived radical, several criteria qualify it as an intracellular and intercellular messenger:

NO is formed with the assistance of speciﬁc enzyme systems activated by extracellular and intracellular signals. NO is synthesized intracellularly and reaches its targets either directly or via a distinct transport form.

NO potentially acts via multiple downstream signaling mechanisms, depending on the concentration:

Low concentrations mediate physiological signaling (e.g., neurotransmission or vasodilatation). Higher concentrations mediate immune/inﬂammatory actions and are, for example, neurotoxic.

Therefore, NO produced in a dysregulated fashion is a toxic substance that is involved in the pathogenesis of many disorders such as Alzheimer’s disease and stroke. In contrast to the classical extracellular messengers such as the steroid hormones, signal transduction via NO involves redox reactions and occurs via a covalent modification of the target protein, leading to a change in the latter’s biological activity. The modification of the target protein is, for the most part, reversible and transient, and the modified protein can transmit the signal to other effector proteins.

8.10.1 Reactivity of NO

The NO radical is water-soluble and can cross membranes fairly freely by diffusion. However, because of its radical nature NO has a lifetime in aqueous solution of only 1–5 s. The reactions of NO are thought to proceed in a complicated manner via þ its radical form and via the oxidized NO and the reduced NO forms (Figure 8.16) which ultimately leads to the nitrosylation (addition of NO), nitrosation (addition þ of NO ) and nitration (addition of NO2) of biomolecules. The main targets are the sulfhydryl groups of proteins, glutathione and free cysteine, the N-containing side chains of amino acids, and the metal ion centers of proteins. The reaction products, þ namely S-nitrosyls (SNO), N-nitrosyls (NNO) and metal nitrosyls (MeX – NO) represent the bioactive forms of NO within which NO can be exchanged and transported, whereas NO itself is barely detectable in free form. 406 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.16 Reactions of NO in biological products of this reaction, NO2/N2O3 and systems. NO reacts in biological systems with peroxynitrite (OONO), react further by SH groups of glutathione (GSH), with SH- nitrosylation of nucleophilic centers to yield groups of target proteins, and with transition S-nitroso (SNO), N-nitroso, or nitrated metals (Me). Furthermore, NO reacts with O2 compounds. and with the superoxide anion O2 . The

& Nitric oxide (NO): — Short-lived radical — Formed from arginine by NO synthase (NOS) — – Signals via covalent adducts with metal ions, SH groups, NH2-groups of target proteins þ — Reacts with Me2 in metal ion centers — Reacts with O2 and O2 to yield NOx products that form covalent adducts with biomolecules.

NO formed by NO synthase (NOS) does not react readily with the side chains of proteins; rather, it is thought to react primarily with metal ion centers in proteins or with free oxygen O2 and the superoxide radical O2 .NO2 and N2O3 have been ﬁ identi ed as the main reaction products with oxygen O2, and these compounds are þ assumed to produce the nitrosium ion NO required for the S- or N-nitrosylation of the target proteins. Reaction of NO with the superoxide radical O2 yields the strong oxidant peroxynitrite, ONOO which can lead to nitration of for example, tyrosine residues in target proteins. The nature and amount of the oxidized nitrogen compounds is strongly dependent on the amount of oxygen and superoxide radical present in the tissue, and therefore the redox status of the tissue is a critical determinant of the extent of nitrosylation and nitration of the target proteins. 8.10 The NO Signaling Molecule 407

Figure 8.17 Biosynthesis of NO. The starting point of NO synthesis is arginine. Arginine is converted by NO synthase, together with O2 and NADP, to NO and citrulline. Arginine can be regenerated from citrulline via reactions of the urea cycle.

8.10.2 Synthesis of NO

NO is formed enzymatically from arginine with assistance from NOS, producing citrulline (Figure 8.17). Both, citrulline and arginine are intermediates of the urea cycle, and arginine can be regenerated from citrulline by the action of urea cycle enzymes. The NOS are enzymes of complex composition (molecular weight ca. 300 kDa) that are active as dimers but can also exist as inactive monomers.

& Cofactors and substrates of NOS: — FAD, FMN — L-Arginine — Tetrahydrobiopterin — Heme — NADPH — O2.

There are three major forms of NOS [22], each with a characteristic pattern of tissue-speciﬁc expression:

Constitutive neuronal NO synthase (nNOS or NOS I) Constitutive endothelial NO synthase (eNOS or NOS III) Inducible NO synthase (iNOS or NOS II). 408 8 Intracellular Messenger Substances: “Second Messengers”

Figure 8.18 Main pathways for activation of NO synthases. For details, see text.

þ The constitutive enzymes nNOS and eNOS are primarily regulated by Ca2 / calmodulin. These enzymes are found at specific intracellular sites and can produce NO in a highly compartmentalized and localized manner. Very small amounts (picomolar concentrations) of NO are formed by the constitutive enzymes, and this serves mostly for a highly specific and transient regulatory þ modification of target proteins. Due to their Ca2 -dependence, the NOS respond to þ a large number of extracellular and intracellular stimuli that release Ca2 , offering a wide array of regulatory options. Other regulatory modifications of the constitutive NOS include phosphorylation and nitrosylation by exogenous NO. Some important pathways for activation of the constitutive NO synthases are summarized in Figure 8.18.

8.10.2.1 nNOS nNOS is localized predominantly to neuronal regions, and plays a major role in synaptic plasticity by its localization to postsynaptic densities. It contains a PDZ þ domain that mediates association with glutamate- and voltage-dependent Ca2 channels, the NMDA receptors, at the postsynaptic density. The colocalization of nNOS with NMDA receptors allows for NO production that is triggered by electrical signals and the neurotransmitter glutamate.

8.10.2.2 eNOS eNOs is the primary source of endothelial NO which acts as a potent vasodilator with a critical role for example in cardiovascular physiology. In addition to binding þ of the Ca2 /calmodulin complex, eNOS requires dimerization and cofactor binding 8.10 The NO Signaling Molecule 409 for activity, and additional myristoylation and phosphorylation events for full activation. A major target of NO produced by eNOS is the soluble guanylate cyclase that is activated by NO to produce cGMP, a second messenger with multiple functions (see below).

8.10.2.3 iNOS þ The inducible NO synthases are Ca2 -independent and regulated mostly at the level of gene expression. iNOS can be induced by proinﬂammatory cytokines, such as TNFa, interferon (IFN)-c and interleukin (IL)-1b (see Chapter 12). Furthermore, the iNOS family can be induced under stress conditions and by bacterial lipopolysaccharides, leading to the formation of large quantities (nanomolar concentrations) of NO several hours after exposure and which may continue in a sustained manner. A large part of the transcriptional regulation of iNOS is mediated by the transcription factor NFkB.

& Constitutive NO synthases: — nNOS (NOS I) — eNOS (NOS III) þ þ — Activated by Ca2 and Ca2 /calmodulin. Inducible NO synthase: — iNOS (NOS II) — Induced for example, by IFN-c, TNFa, IL-1 — Produces large amounts of NO — Important target: NFkB.

8.10.3 Physiological Functions of Nitrosylation

The physiological importance of nitrosylation is based on a regulatory, toxic, and defense function. NO produced in a regulated manner serves to control a multitude of cellular reactions; however, an excessive production of NO can have toxic effects and hence has also been employed in mammals for antimicrobial purposes.

8.10.4 Nitrosylation of Metal Centers

There are two well-studied examples of the regulatory function of NO by binding to metal ion centers, namely NO-sensitive guanylyl cyclase and the nitrosylation of hemoglobin.

8.10.4.1 NO-Sensitive Guanylyl Cyclase The ﬁrst cellular target of NO to be identiﬁed was the cytoplasmic guanylyl cyclase (Section 9.3). Cytoplasmic guanylyl cyclase is a heme-containing, cGMP-generating, 410 8 Intracellular Messenger Substances: “Second Messengers”

heterodimeric NO receptor. Binding of NO to the heme group activates the enzyme about 200-fold, and the associated increase in cGMP levels has multiple consequences. The cGMP can stimulate cGMP-dependent protein kinases which have many proteins as substrates, including PI3 kinase and phosphatases. Another target of cGMP includes the cGMP-gated ion channels which regulate the efﬂux of þ þ þ K from the cell and the inﬂux of Ca2 and Na into the cell, and thereby play an essential role in vasodilation and during phototransduction and neurotransmission in the retina. The binding of cGMP opens the channels and, as a consequence, a þ Ca2 signal is produced and a broad palette of biochemical reactions in the cell is activated.

8.10.4.2 Nitrosylation of Hemoglobin Hemoglobin (Hb) is regulated by NO, both by NO-binding to its metal ion centers and by S-nitrosylation (Figure 8.19). The Hb molecule is tetrameric, composed of two a-chains and two b-chains. In humans, each chain has a heme system and the b-chains have a reactive cysteine group (Cys93). Hb is able to bind NO at two sites: ﬁrst, NO can bind to the Fe(II) of the heme grouping; second, NO can accumulate at Cys93 of the b-chain by forming an S-nitrosyl, Hb-SNO.

& Metal ion centers as targets of NO: — Guanylyl cyclase: Produces cGMP from GTP Activated by NO-binding to heme group cGMP activates protein kinases and opens ion channels. — Hemoglobin: NO binds to heme group and/or Cys93 of b-chain NO binding is linked to O2 tension O2 regulates delivery of NO for blood vessel relaxation.

The nitrosylation of Hb has been shown to perform a dynamic vehicle function

where gradients of O2 are linked to the delivery of NO to blood vessels [23]. At high oxygen levels, when Hb is in the oxygenated, relaxed R-state, the heme groups are

occupied by O2 and NO-binding to Cys93 is favored. However, at low oxygen, when Hb is in the tense (T-state) conformation, Hb-SNO formation is disfavored and the NO group is transferred to thiol groups in the red blood cell membrane and then in the plasma. The major acceptor for NO disembarked from Hb-SNO is the anion exchanger protein (AE1) that is present in abundant amounts in the cell membrane of red blood cells. The AE1 protein can accept the NO group directly from Hb-SNO, a step that is essential for the efﬂux of NO’s biological activity from the red blood cells. By employing this S-transnitrosylation mechanism, the NO vasodilator activity is delivered to oxygen-depleted tissues, resulting in blood vessel relaxation and an increased blood ﬂow. 8.10 The NO Signaling Molecule 411

Figure 8.19 Generation of NO bioreactivity at T-state which can occur in a complex with the the membrane of red blood cells. In the transmembrane protein AE1. Concomitantly, T-state, NO can bind to the heme group NO is transferred from Cys93 to a cysteine of hemoglobin as a Fe---NO complex. thiol within AE1. This can transfer NO activity Oxygenation in the lungs promotes the out of the red blood cell and into the vessel transition from T to R state and the transfer of wall. Both, extracellular and intracellular heme-liganded NO to Cys93 on the b-subunit. glutathione-SNO (GSNO) can provide a In the vascular periphery, deoxygenation is source of NO groups in equilibrium with associated with the transition from R- to Hb-SNO.

8.10.5 Regulatory Functions of Protein S-Nitrosylation

8.10.5.1 Selectivity of Protein S-Nitrosylation SNO formation is a redox-based signal with exquisite speciﬁcity based on the selective modiﬁcation of single cysteine residues in target proteins. The selectivity 412 8 Intracellular Messenger Substances: “Second Messengers”

of S-nitrosylation has been shown to be modulated by the sequence context of the cysteine and the colocalization of target protein and NOS [24]. The study of the sequence dependence of S-nitrosylation has revealed two motifs that direct speciﬁc S-nitrosylation. In one motif, the target cysteine is located between an acidic and a basic amino acid, as revealed in either the primary sequence or the tertiary structure. In the other motif, the cysteine is contained in a hydrophobic region.

& S-nitrosylation, SNO formation: — Redox-based signal — Arises from reaction of NOx with –SH of proteins, GSH and Cys — Formed in a site-specific manner — Dynamic modification — Multiple targets as for example, IkB and NMDA receptor.

The colocalization of target proteins with NOS is another mechanism that contributes to selective S-nitrosylation. High local concentrations of NO can be formed by NOS and the NO can react directly with thiols of proteins in close neighborhood to NOS. In this case, local hydrophobicity of the target cysteine is of importance. Examples of the colocalization of NOS and target protein include the NMDA receptor and the ryanodine receptor.

8.10.5.2 Transnitrosylation Cysteine residues that do not appear to be candidates for direct NO modiﬁcation based on their reactivity can be S-nitrosylated by transnitrosylation. This process includes the transfer of NO from a donor SNO-compound to the target protein. For many target proteins, S-nitroso-glutathione (GSNO), serves as the NO group donor. However, direct thiol–thiol transfer can occur between an S-nitrosylated donor protein and an acceptor protein. Important examples of the latter mechanism include the transfer of the NO-group from the b-chain Cys93 of SNO-Hb to the anion-exchange protein AE-1. Similarly, SNO-caspase-3 (see Chapter 17) can nitrosylate the X-linked inhibitor of apoptosis (XIAP), and thereby inhibit its E-3 ubiquitin ligase activity. In these examples, the donor of the NO-group functions as a nitrosylase, by analogy to protein kinases.

8.10.5.3 Denitrosylation The level of SNO in cells of higher organisms is determined not only by the rate of SNO formation but also by its degradation. A major tool for SNO removal is the enzymatic degradation of GSNO by GSNO reductase (GSNOR), an enzyme which has GSNO as a speciﬁc substrate. It is thought that SNOs from S-nitrosylated proteins are transferred by transnitrosylation to glutathione to yield GSNO, which is then reduced to glutathione (GSH) by GSNOR:

Protein1 SNO þ GSH , GSNO þ protein1 þ þ GSNO þ NADH þ H , GSSG þ NH4 GSNOR; GSH 8.10 The NO Signaling Molecule 413

Table 8.3 Regulatory attack points of NO. Proteins are included for which a direct regulation by NO has been reported.

Binding Subcellular localization site Membrane Cytosol (incl.. compart- Nucleus Extracellular ments)

Thiol NMDA receptor Aldolase AP-1 Glutathione NADPH oxidase GAPDH OMDM trans- Albumin ferase Protein kinase C Plasminogen activator Adenylyl cyclase Aldehyde dehydrogen- (type I) ase NFkB, IkB kinase Metal Guanylyl cyclase Hemoglobin Aconitase/IREP1 Cyclooxygenase Cyt P450

Abbreviation: NMDA: N-methyl-D-aspartate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IREP1: iron-responsive element-binding protein; OMDM transferase: O6-methylguanine-DNA methyltransferase.

By this mechanism, GSH can collect protein-SNOs to form GSNO allowing for the modulation or full termination of SNO signaling.

8.10.5.4 Target Proteins for S-Nitrosylation The ubiquitous influence of NO on cellular functions is exerted to a large part by the S-nitrosylation of cysteine residues within a broad functional range of proteins. More than 1000 proteins have been identified that are modified by S-nitrosylation [24], and protein S-nitrosylation is now a well-established regulatory modification that is involved in the regulation of a wide range of cellular processes including vascular homeostasis, vectorial membrane trafficking, neurotransmission, cellular immune response, apoptosis, and gene transcription. Furthermore, aberrant SNO- signaling has been implicated in the pathogenesis of many disorders such as arthritis, diabetes, septic shock, stroke, and asthma. A selection of central cellular proteins that are regulated by S-nitrosylation is presented in Table 8.3.

8.10.6 Toxic Action of NO and Nitrosative Stress

The bioactivity of NO depends heavily on its intracellular concentration. For the regulatory purposes of S-nitrosylation of specific targets, normally low amounts of NO, produced in a dynamic way, are employed. However, when NO is formed in excess amounts and in a less than regulated fashion by, for example, the induction of iNOS during inflammatory conditions or bacterial infections, then nonspecific reactions with various cell constituents including proteins, lipids and DNA are 414 8 Intracellular Messenger Substances: “Second Messengers”

observed. This situation has been termed nitrosative stress, in analogy to oxidative stress caused by the generation of reactive oxygen species (ROS), such as the superoxide anion, O2 . NO reacts readily with the superoxide anion to produce the strong oxidant peroxynitrite, which can then nitrate cellular targets and lead to, for example, the formation of 3-nitrotyrosine residues in proteins. A defense against nitrosative stress and the excessive formation of SNO is provided by antioxidants and primarily by GSH. The latter can protect proteins against hazardous levels of S- nitrosylation by scavenging the SNO and by the reduction of GSNO by GSNOR (see also above). Nitrosative stress can also be used in a beneficial fashion by the cell, however, and it is being increasingly recognized as an efficient tool for combating microbial infections. During infections, increased NO production is observed in humans and experimental animals, specifically in those cells engaged in antimicrobial defense, such as phagocytes. Although the microbial targets of NO responsible for the antimicrobial effects are poorly characterized, microbial proteins containing metal ion centers and reactive cysteine residues (e.g., cysteine proteases) are likely attack points.

Questions 8.1. Which types of adenylyl cyclases do you know, and how are these enzymes regulated? 8.2. Which features of cAMP production and degradation may be responsible for producing locally acting signals? Name the major cellular targets of cAMP. 8.3. Describe the main structural features of guanylyl cyclases and their regulation. Name the major cellular targets of cGMP. 8.4. Give the structure of the major second messengers derived from phosphoinositides. What is the major function of these compounds? þ 8.5. Which types of ion channels are involved in the formation of Ca2 signals? Which signals are used for regulation of these channels? þ 8.6. Describe the cellular distribution of Ca2 . What tools do cells have available þ for the mobilization of Ca2 ? þ 8.7. How can cells produce Ca2 signals that are variable in time and in space? 8.8. Which interaction modules show binding speciﬁcity for phosphoinositides, and what is the signaling function of ligand binding to these modules? Give at least two examples of signaling proteins that are regulated by phosphoinositide binding. þ 8.9. Which features qualify Ca2 as signaling molecule? Which proteins are the þ major targets of Ca2 signaling? Describe the signaling function of at least þ three proteins that are regulated by Ca2 . 8.10. Describe the major signaling pathways that can trigger formation of the second messenger DAG. References 415

8.11. Which types of second messengers can be formed from membrane lipids? What are the primary targets of these messengers? 8.12. How is NO produced? Which enzyme subtypes are responsible for formation of NO, and how are these enzymes regulated? þ 8.13. How may NO signals lead to an influx of Ca2 ? 8.14. Name the major reaction products of protein modification by NO. Give examples of proteins modified this way. 8.15. A major target of NO is hemoglobin. Give an overview of how NO may regulate oxygen transport by hemoglobin.

References

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9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Summary Reversible phosphorylation of proteins on Ser/Thr and Tyr residues is a regulatory signal that functions as a switch in signaling pathways. The phosphate esters formed on proteins by the action of protein kinases are stable modifications that cause profound changes in the activity of cellular proteins. Because of the stability of the phosphate esters, protein phosphatases are required for their removal. The concerted and highly regulated action of both protein kinases and protein phosphatases is used by the cell to create a temporally and spatially restricted signal influencing the activity state of proteins in a highly specific fashion.

The reversible phosphorylation of amino acid side chains is a widely used principle for regulating the activity of enzymes and signaling proteins (see Section 2.5). Via this function, protein kinases and protein phosphatases play pivotal roles in regulating various aspects of metabolism, gene expression, cell growth, cell division and cell differentiation. Almost all intracellular signaling pathways use protein phosphorylation to create signals and conduct them further. The protein kinases are certainly one of the largest protein families in the cell, as is illustrated by the identification of about 500 protein kinase genes in the human genome [1]. Of the various protein kinases, the Ser/Thr-specific and Tyr-specific enzymes are the best-characterized (details of Tyr-specific protein kinases are provided in Chapters 11 and 13). At this point, before proceeding to the protein family of Ser/Thr-specific protein kinases, an approximate classification of the protein kinases will be presented.

9.1 Classification, Structure, and Characteristics of Protein Kinases

The first protein kinase to be obtained in a purified form was the Ser/Thr-specific phosphorylase kinase of muscle, in 1959 [2]. With the discovery of the Tyr-specific

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 418 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

protein kinases [3], the Ser/Thr-speciﬁc protein kinases were joined by another extensive class of protein kinases of regulatory importance, to which a central function in growth and differentiation processes was soon attributed. In addition, protein kinases are known that phosphorylate other amino acids. However, these protein kinases are of minor importance in eukaryotic signal transduction and will not be discussed in detail here. Based on the nature of the acceptor amino acids, four classes of protein kinases can be distinguished (Figure 9.1):

Ser/Thr-speciﬁc protein kinases esterify a phosphate residue with the alcohol group of Ser and Thr residues.

Figure 9.1 Amino acid specificity of protein kinases. 9.1 Classification, Structure, and Characteristics of Protein Kinases 419

Tyr-specific protein kinases create a phosphate ester with the phenolic OH group of Tyr residues. Histidine-specific protein kinases form a phosphorous amide with the 1 or 3 position of His. The members of this enzyme family also phosphorylate Lys and Arg residues. Aspartate- or glutamate-specific protein kinases create a mixed phosphate- carboxylate anhydride.

Examples of cellular activities regulated by protein kinases are diverse, affecting practically all of the cell’s performance.

& Protein phosphorylation is found in: — Enzymes (Chapters 2, 7, 8, 10, and 13) — Adapter proteins (Chapter 8) — Signal proteins (Chapter 5, 7, and 8) — Transcription factors (Chapter 1) — Ion channels — Transmembrane receptors (Chapters 5, 8, 11, and 12) — Ribosomal proteins (Chapter 6) — Structural proteins — Transport proteins.

The switch function of protein phosphorylation is based on different mechanisms that may operate either alone or in combination. Phosphorylation by protein kinases inﬂuences the function, activity and subcellular location of the protein substrate, especially in the following ways:

Induction of conformational changes by allosteric mechanisms (Section 2.5.2) Direct interference with binding of substrate or other binding partners, for example, isocitrate dehydrogenase (Section 2.5.2) Creation of binding sites for effector molecules in the sequence: examples of this are binding of Tyr-P to SH2 and PTB domains, and binding of Ser-P to 14-3-3 proteins (Chapter 10).

On the basis of sequence and structure, the Ser/Thr and Tyr-speciﬁc protein kinases form a closely related superfamily that is distinct from the histidine kinases and other phosphotransfer enzymes. The current classiﬁcation of protein kinases splits the protein kinase “kinome” into two broad groups: conventional protein kinases (ePKs) and atypical protein kinases (aPks). The aPKs are a small set of protein kinases that do not share clear sequence similarity with ePKs, but have been shown experimentally to have protein kinase activity. There are 478 ePKs members and 20 aPK members in the human genome [1]. The ePKs are divided into eight subfamilies (as listed in Table 9.1), with selected subfamily members. 420 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Table 9.1 The protein kinase complement of the human genome [1].

Conventional protein Examples of ePK members No. of kinase genes in kinase (ePK) family human genome

AGC PKA, PKB (Akt kinase), PKC, 84 GRK, Ribosomal protein S6 kinase CAMK (calmodulin- CamKI, CamKII, CamKIV, 98 regulated kinase) Myosin-light chain kinase CK1 (casein kinase) CK1 12 CMGC Cyclin-dependent kinases 70 (CDKs) Mitogen-activated kinases Glycogen synthase kinase (GSK) RGC Receptor guanylate 5 cyclase kinase STE “sterile” 61 TK Tyrosine kinases Receptor tyrosine kinases, e.g., 93 EGFR, PDGFR Src kinase TKL (tyrosine kinase-like 55 kinase) 478 Atypical PKs (aPKs): 4 20 families

9.2 Structure and Regulation of Protein Kinases

Summary Ser/Thr- and Tyr-specific protein kinases are constructed from a small and a large lobe, with the active site forming a cleft between the two lobes. The two lobes are connected by a hinge region. The small lobe harbors the binding site for ATP, while the large lobe provides catalytic residues and a docking surface for peptide/protein substrates. The structural elements that are most important for catalysis and for protein kinase control comprise the P-loop, the C-helix, the catalytic loop, and the activation loop. In many kinases, the activation loop is the site of regulatory phosphorylations or interaction with activity modulators. The Ser, Thr or Tyr residues of the activation loop may be phosphorylated in response to activating signals, and this phosphorylation promotes an active conformation of the kinases. Protein kinases perform a switch function by toggling between an “active” and “inactive” state in response to diverse signals. Whereas, the active state adopts a similar structure in most protein kinases, the 9.2 Structure and Regulation of Protein Kinases 421

structures of the inactive states are highly variable. Multiple mechanisms have been identified that control the activity of protein kinases, including the binding of protein inhibitors and protein activators, the binding of second messengers and phosphorylation-regulated subcellular distribution, among others.

The Ser/Thr- and Tyr-speciﬁc protein kinases share many common features. The catalytic mechanism, structural properties and mechanisms of kinase control are very similar for the two kinase classes. In the following, the main properties of both classes will therefore be presented together.

9.2.1 The Protein Kinase Reaction

The common catalytic function of Ser/Thr- and Tyr-speciﬁc protein kinases is the covalent phosphorylation of substrate proteins via transfer of the c-phosphate of ATP to the OH group of serine, threonine, or tyrosine residues. This catalytic function is carried out by a catalytic domain of about 270 amino acids, the structure and catalytic residues of which are highly conserved among the two protein kinase families. The conserved structure of the kinase domain shows two lobes that are linked by a ﬂexible hinge, and the catalytic center is formed by residues from both lobes. The proposed mechanism of phosphate transfer by protein kinases is shown in Figure 9.2 [4]. The key players of the reaction are

Acidic amino acids required for:

stabilization of the transition state activation of the OH group of the acceptor amino acid for nucleophilic attack on the c-phosphate. One or two metal ions that coordinate the c-phosphate of ATP, help to ﬁx the ATP, and stabilize negative charges in the transition state Basic amino acids that serve to stabilize negative charges in the transition state and in the leaving group, ADP.

& Conserved amino acids of protein kinases: — D166: in catalytic loop, substrate-OH activation — N171: H-bond to D166 þ — D184: part of DFG motif in activation loop, Mg2 -binding — K72: orients a- and b-P of ATP, ion pair with E91 — E91: in C-helix, ion pair with K72.

Sequence comparisons and the results of mutation experiments and biochemical studies have indicated an essential function in the catalysis of phosphate transfer 422 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Figure 9.2 Schematic representation of key OH-group of the acceptor serine for a interactions at the catalytic site and the nucleophilic attack on the c-phosphate of ATP. activation loop of protein kinase A. A possible The catalytic mechanism is not definitively mechanism is shown in which the carboxylate established. of Asp166 functions as a base and activates the

for the conserved amino acids Lys72, Glu91, Asp166, Asn 171, and Asp184 (numbering of PKA). It is generally assumed that Asp166, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl, and that the reaction takes place by an “in-line” attack of the Ser-OH at the c-phosphate. Overall, the mechanism of phosphate transfer by protein kinases is related to nucleotide transfer by nucleic acid-polymerizing enzymes that also use metal ions and acidic residues as key elements in catalysis.

9.2.2 Main Structural Elements of Protein Kinases

The core of all eukaryotic Ser/Thr- and Tyr-speciﬁc protein kinases adopts a common fold, as illustrated in Figure 9.3 for the tyrosine kinase domain of the 9.2 Structure and Regulation of Protein Kinases 423

Figure 9.3 Structure of the tyrosine kinase indicated. The P-loop is shown in yellow, the domain of the insulin receptor with bound ATP activation loop in green, and the catalytic loop and peptide substrate. The a-helices are in orange. The amino and carboxy termini are shown in red, the b-strands in blue. The denoted by N and C. functional important structural elements are insulin receptor. The structure comprises a small and large lobe with the active site forming a cleft between the two lobes. The two lobes are connected by a hinge region. The small N-terminal lobe contains ﬁve b-structures and one a-helix, named the C-helix. In contrast, the larger C-terminal lobe is mostly b-helical and comprises a four-helix bundle, additional a-helices, and two short b-strands. The small lobe provides the binding site for ATP, while the large lobe provides catalytic residues and a docking surface for peptide/protein substrates. Opening and closing of the active site cleft is an essential part of catalysis. The following structural elements have been found to be critical for catalysis and for protein kinase control (amino acid numbering of PKA):

& Structural elements of protein kinases: — P-loop — C-helix — Catalytic loop — Activation segment with activation loop — Autoinhibitory elements.

C-helix of the N-terminal lobe: In most active protein kinase conformations, residue E91 of the C-helix forms a salt bridge to an invariant Lys-residue (K72) 424 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

within the N-terminal lobe, allowing optimal positioning of the ATP phosphates. Regulatory mechanisms often modulate kinase activity by altering the conformation of the C-helix, thereby affecting the integrity of these interactions. Glycine-rich loop in the N-terminal lobe: In all canonical protein kinases, a glycine-rich loop (also termed ATP phosphate-binding P-loop or G-loop) with the conserved amino acid consensus motif GxGxxG forms a flexible clamp that covers and anchors the nontransferable ATP b/c-phosphates, leaving the c-phosphate solvent exposed. By binding and positioning the ATP appropriately for c-phosphate transfer to the substrate, the P-loop controls nucleotide affinity/ specificity and c-phosphoryl transfer rate. The P-loop is found in similar form in the Ga-proteins (Section 7.4.3) Catalytic loop: The catalytic loop is located at the base of the active site and contains a conserved Asp-residue (D166), presumed to be the catalytic residue. Furthermore, a conserved Asn-residue (N171) is found on this loop. Activation segment and activation loop: The primary sequence of the activation segment is defined as the region between and including two conserved tripeptide motifs (DFG . . . APE) within the large lobe, and comprises 20–35 amino acids. Moving from N to C terminus, the secondary structural elements within the þ activation segment are the Mg2 -binding loop, comprising the DFG motif, the activation loop (also called T-loop for the cyclin-dependent protein kinases; see Chapter 15) and the P þ 1 loop, involved in peptide substrate binding. Residue D184 of the conserved DFG motif is involved in metal-ATP binding. The activation loop shows considerable structural diversity and conformational plasticity, and is one of the most important control elements of protein kinase activity. In many kinases, the activation loop is the site of regulatory phosphorylations or interaction with activity modulators. Ser, Thr or Tyr residues of the activation loop may be phosphorylated in response to activating signals, and this phosphorylation promotes an active conformation of the kinases. In many inactive states of protein kinases, the activation loop collapses into the active site and blocks the binding of both nucleotide and peptide substrate. Upon phosphorylation, it moves away from the active site and adopts an open conformation allowing for substrate binding and catalysis (Figure 9.4). Critical for this conformational change is the electrostatic interaction between one of the phosphate residues of the loop and a basic pocket containing an Arg-Asp motif which is conserved among kinases regulated by phosphorylation. The placement and the number of phosphorylation sites varies from kinase to kinase. Overall, the presence of the phosphate residues creates a network of interactions that properly orient the C-helix and the catalytic residues and promote lobe closure. For some protein kinases, as for example the MAP kinases (Figure 9.4), phosphorylation of the activation loop facilitates homodimerization of the kinase that is required for the nuclear localization of the enzyme. Autoinhibitory sequence elements: Many protein kinases contain autoinhibitory elements that help to fix an inactive conformation by intramolecular binding to the substrate-binding site. These elements may fold into the active site, blocking the binding of both the nucleotide and peptide substrate. As they lack 9.2 Structure and Regulation of Protein Kinases 425

Figure 9.4 Activation of MAP kinase by (shown in cyan) and promotes lobe closure phosphorylation of the activation segment. such that the kinase becomes active. The MAP kinase (Chapter 12) undergoes Furthermore, the pThr organizes the C- phosphorylation at a threonine (Thr183) and a terminal extension (shown in yellow) into a tyrosine residue within the activation loop functionally important homodimerization which induces a movement of the activation interface. Dimerization via this interface is loop (red). This creates a network of required for the nuclear localization of the interactions that properly orient the aC helix enzyme.

phosphorylatable residues, these elements are also termed pseudosubstrates. The autoinhibitory elements may be localized on the same polypeptide as the kinase domain, or they may reside on a separate subunit of an oligomeric kinase. An N-terminal myristoic acid anchor may also participate in autoinhibition, as shown for the Abl tyrosine kinase (Section 10.3.3).

In inactive kinase structures, two or more of the above-mentioned structural elements are aligned in a way that prevents substrate binding and/or catalysis. The spatial relationship of the critical protein kinase activity modulation sites is shown in Figure 9.5.

9.2.3 Substrate Binding and Recognition

Taking into account the many Ser, Thr and Tyr residues in proteins, the question arises of which parameters define the phosphorylation site of a substrate protein. With the help of targeted exchange of amino acids in substrate proteins, sequence comparisons of phosphorylation sites, and the use of defined peptides as substrates it has been possible to show clearly that the sequence in the neighborhood of a Ser/ Thr or Tyr residue is an important determinant of specificity. The different Ser/ Thr- and Tyr-specific protein kinases show different requirements with respect to the neighboring sequence of the residues to be phosphorylated, so that each subfamily has its own consensus sequence for phosphorylation. It should be pointed out that several Ser/Thr residues are found in many phosphorylation 426 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Figure 9.5 Alignment of the activity salt bridge to K72 which helps to coordinate modulation sites of protein kinases. Close-up the a- and b-phosphates of ATP correctly. This of the critical elements of protein kinase helix corresponds to the PSTAIRE helix of control as found in active kinase structures, CDK2 (see Chapter 15), and is the focus of represented here by the structure of active several regulatory mechanisms among protein protein kinase A. The phosphothreonine acts kinases. In inactive kinase structures, two or as an organizing center that forms contacts to more of the critical activity modulation sites Arg165, which is neighbored to the catalytic are misaligned or blocked by intra- or D166 and to the C-helix. The C-helix forms a extramolecular interaction.

sequences, so that multiple and cooperative phosphorylation is possible in a sequence segment. Phosphorylation of the large subunit of the RNA polymerase II (see Section 4.2.7) is particularly marked. At the C terminus, this contains 52 copies of the heptamer sequence YSPTSPS as potential phosphorylation sites.

& Substrate specificity of protein kinases is determined by: — Sequence context of the phosphorylation site — Interaction modules distinct from the phosphorylation site — Colocalization of kinase and substrate.

In addition to the sequence context of the phosphorylation sites, other structural parts of the substrate protein have also been shown to contribute to substrate binding. The coupling of a protein kinase and its protein substrate can be achieved, for example, via structural parts that are located far away from the phosphorylation consensus site on the substrate and the substrate binding site on the kinase. An example is provided by the docking of substrate proteins on receptor tyrosine kinases via SH2-phosphotyrosine interactions (see Chapters 10 and 13). This 9.2 Structure and Regulation of Protein Kinases 427 interaction helps to clamp the substrate onto the kinase, thereby ensuring a high efficiency of phosphate transfer. Another major determinant of protein kinase specificity is the targeting of protein kinases to the neighborhood of selected substrates. The colocalization of protein kinases and their substrates at distinct subcellular sites greatly enhances the specificity of the kinase reaction. Only those substrates that have been translocated to the specific subcellular site will be phosphorylated by the protein kinase. The mechanisms of colocalization are diverse and are discussed separately in Section 9.2.4. The peptide substrate-binding site is located on the C-terminal lobe of the protein kinases. Structural information available to date shows that the peptide substrate is contacted via multiple interactions, both N- and C-terminal, to the residue to be phosphorylated. There is a marked complementarity between the binding pocket on the kinase and the peptide substrate with regards to shape, hydropathy and electrostatic potential. For several protein kinases, such as the insulin receptor tyrosine kinase (Section 10.1.3) and CDK2 (Section 15.2.1), an intact peptide- binding site is not present in inactive forms of the kinase. Rather, a substrate recognition site is created in these kinases only after activation loop phosphorylation and a subsequent conformational change. Most information on substrate recognition by protein kinases has been obtained by using peptide substrates bound to the catalytic domain. The structure of a complete protein substrate–kinase complex, namely eIF-2a bound to the catalytic domain of PKR (RNA-dependent protein kinase; Section 4.2.6) shows extensive contacts between eIF-2a and an a-helix (aG) of the C-lobe [5]. The N-lobe is not involved in substrate binding; rather, it mediates the dimerization of PKR.

9.2.4 Control of Protein Kinase Activity

Protein kinases can exist in active and inactive forms, which is why they are able to perform the function of a switch in signaling pathways. For most of the time, protein kinases are found in the “off” state that has minimal activity, but upon specific signals being received they are converted into the “on” state that is maximally active. As illustrated by the multifaceted character of activating and inactivating signals, the cell possesses a broad palette of tools to induce a transition between the two activity states. As all protein kinases catalyze the same reaction, they all adopt catalytically active “on”-conformations that are structurally very similar. However, the structures of the off-states are variable and the protein kinases have evolved in different ways by which the adoption of the on-state conformation is impeded. Overall, the protein kinase structures contain several flexible elements that can be fixed in either an active or an inactive conformation. The flexible elements comprise the hinge between the two lobes, the activation loop, the P-loop and the C-helix, and they move in a highly coordinated and cooperative fashion upon transition between the “on” and “off” state. 428 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

The activated state of protein kinases is characterized by the following features:

The two lobes are closely packed together. The ATP is buried between the two lobes and its phosphates contact the P-loop. The C-helix is positioned for salt bridge formation to a b-strand of the N-terminal lobe. The binding sites for the peptide substrate and ATP are fully accessible. The phosphorylated activation loop has moved away from the active site and adopts an extended conformation. The loop helps to organize the catalytic residues for optimal phosphate transfer, and it forms part of the substrate binding site. Movement of the activation loop is often coupled to a movement of the C-helix.

Whereas, fully active protein kinases adopt a similar active conformation, the inactive states of protein kinases are very diverse. A high plasticity of the kinase domains is observed that allows the adoption of distinct inactive conformations in response to phosphorylation or interactions with specific regulatory domains on the same kinase molecule or on other proteins. Overall, the inactive states are characterized by a more open conformation of the two lobes, precluding an optimal orientation of the residues involved in substrate binding and catalysis. The mechanisms for fixation of the inactive protein kinase states are diverse and may be used either singly or in combination. Mechanisms for the fixation of inactive protein kinase states include (Figure 9.6):

Binding of protein inhibitors. Protein kinase inhibitors ﬁx inactive states by, for example, deforming the N-terminal lobe and destroying the ATP-binding site; see inhibitor p21Kip (Section 15.2.3). Inhibitory phosphorylations (e.g., Thr14, Tyr15 on CDKs; see Chapter 15; phosphorylation of Src-kinase; Section 10.3.2). Binding of regulatory subunits (PKA; Section 9.3). Autoinhibition. An inhibitory structural element that is itself part of the protein kinase or part of a kinase subunit is often used to ﬁx an autoinhibited, inactive state of the kinase. The autoinhibitory elements are mobile and can adopt conformations that induce misalignment of the catalytic residues and blockage of the substrate-binding sites.

A multitude of mechanisms may be used either singly or in combination for activation of the kinase. The transition from the inactive to the active state is induced by:

Binding of activating subunits (cyclins; Section 15.2.2). Binding of chemical messengers (cAMP; Section 9.3.2) with concomitant release of inhibitory subunits. þ Binding of cofactors such as Ca2 /calmodulin, diacylglycerol (DAG), phospholipids (Section 9.5.3). Phosphorylation of the activation loop. 9.2 Structure and Regulation of Protein Kinases 429

Figure 9.6 Mechanisms of activation and inactivation of protein kinases.

Dephosphorylation of inhibitory phosphorylated sites. Changes in the oligomerization state of the kinase as a consequence of ligand binding to the extracellular domain of the transmembrane protein kinase (Section 10.1.2). Binding of a phosphorylated substrate. The polo-like kinase 1 (PLK1) is kept in an inactive state by intramolecular binding of a repressive domain, the polo box domain 1 (PBD1), to the active site. The binding of a phosphorylated substrate by PBD1 relieves the inhibition.

The control of kinase activity operates at the following regulatory levels:

Expression of kinase and of regulatory subunits (inhibitory or activating subunits as, for example, the cyclins; see Chapter 15). Signal-induced destruction of kinases or regulatory subunits via the ubiquitin- proteasome pathway (Chapter 15). Activation of upstream protein kinases that phosphorylate regulatory sites as, for example, the activation loop. Protein kinase cascades may be formed in this way (Chapter 12). 430 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Activation of upstream protein phosphatases that dephosphorylate regulatory sites. Binding of extracellular ligands to transmembrane protein kinases (e.g., receptor tyrosine kinases; Chapter 10). Signal-induced formation of second messengers that activate protein kinases þ (Ca2 , DAG; Chapter 8). Binding of metabolites that activate the kinase. One such example is the AMP- activated protein kinase that senses the cellular AMP/ATP ratio. Colocalization of kinase and protein substrate (Section 9.2.4). The targeted recruitment of protein kinases to speciﬁc subcellular compartments as, for example, the cell membrane is major method of protein kinase control.

9.2.5 Regulation of Protein Phosphorylation by Subcellular Localization and Specific Targeting Subunits

The targeted localization of protein kinases and protein phosphatases represents a major mechanism by which the selectivity of protein phosphorylation is enhanced and a tight control of phosphorylation, dephosphorylation, and interaction with cofactors is made possible. The principle of targeted localization is shown in Figure 9.7.

Figure 9.7 Main mechanisms of targeted phosphatidyl inositol-3,4,5 trisphosphate; PH, localization of protein kinases. Targeting of pleckstrin homology domain; SH2, sarc protein kinases (or protein phosphatases) by homology domain 2; PTB, phosphotyrosine- the mechanisms shown provides for access to binding domain. membrane-associated substrates. PIP3, 9.3 Protein Kinase A 431

& Targeting of kinases and phosphatases to subcellular sites is mediated by: — Specific localization or regulatory subunits — Lipid anchors — Protein–protein interaction modules of the kinase.

Many substrates of protein kinases occur either as membrane-associated or particle-associated forms. In order for protein kinases or protein phosphatases to perform their physiological function in a signal transduction process, they must in many cases be transported to the location of their substrate. During the course of activating signal transduction pathways, a redistribution of protein kinases and protein phosphatases to new subcellular locations is often observed. By restricting the action of the two enzyme classes to a distinct subcellular site, the persistence, amplitude and signal-to-noise ratio of phosphorylation signals are improved. Furthermore, signals from other effectors can be integrated more efficiently in these multiprotein signaling units. The subcellular targeting of protein kinases and protein phosphatases is often mediated by the regulatory subunits of these enzymes, which can bind specifically to scaffolding, adapter, or anchoring proteins located at distinct subcellular sites. These anchoring proteins may be multivalent and allow the assembly of several signaling proteins. The mechanisms by which the anchoring proteins assemble at a distinct subcellular site are diverse, but structural membrane proteins, transmembrane receptors, ion channels or cytoskeletal proteins can each serve as the anchoring target. Other anchoring proteins interact with the membrane via lipid anchors or with distinct membrane-anchoring sequences. Generally, the nature of the regulatory subunit of the protein kinase or protein phosphatase determines in which compartment of the cell and at which membrane section the protein phosphorylation signal will become active. The subunit functions as a localization moiety that determines at which place in the cell the protein kinase or phosphatase will gain access to its substrates. Another important aspect of protein kinase targeting is the colocalization of sequentially acting protein kinases by means of scaffolding proteins (see Chapter 12). By assembling protein kinases with the upstream and downstream signaling partners – either a protein kinase or other signaling proteins – a high local concentration of signaling proteins is achieved allowing that permits the creation of localized and efficient signal events at specific subcellular sites.

9.3 Protein Kinase A

Summary Protein kinase A (PKA) is primarily regulated by cAMP, and in the absence of cAMP PKA exists in an autoinhibited tetrameric state. The PKA tetramer is 432 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

composed of two regulatory R subunits and two catalytic C subunits. Binding of cAMP to the R subunits triggers a relief of autoinhibition via dissociation of the tetramer into R2 and free C subunits. Signaling by PKA occurs in a timely and locally controlled manner by the association of PKA with A kinase-anchoring proteins (AKAPs). The AKAPs are scaffolding proteins that are multivalent due to the presence of multiple interaction domains and also help to assemble multiprotein complexes that may contain, in addition to PKA, other protein kinases and phosphatases.

The cAMP-dependent protein kinase (protein kinase A; PKA) has been classiﬁed as a member of the superfamily of AGC kinases that includes, among others, the following protein kinases:

PKA PKB, also named Akt kinase PKC PKG, cGMP-dependent kinase.

The AGC kinases have a very similar structure of the catalytic domain and are regulated in a similar fashion by phosphorylation in the activation loop. Of the protein kinases, PKA is the best investigated and characterized to date. The substrates and functions of PKA are diverse, and the enzyme is involved in the regulation of metabolism of glycogen, lipids, and sugars. In addition, cAMP/ protein kinase A plays an important role in controlling ion channels and in long- term modiﬁcations at nerve synapses. Furthermore, PKA is involved in the cAMP- stimulated transcription of genes that have a cAMP-responsive element in their control region. An increase in cAMP concentration leads to an activation of PKA, which in turn activates the transcription factor CREB by phosphorylation at Ser133 [6]. Phosphorylated CREB then recruits the transcriptional coactivator CBP, a histone acetylase, to the cAMP-response element, and this leads to a transcription stimulation of the target genes (Section 4.4.3.1).

9.3.1 Structure and Substrate Specificity of PKA

& PKA structure and regulation: — Inactive PKA: autoinhibited R2C2 tetramer — þ Activation: cAMP binding: R2(cAMP)4 2C — Isoforms: RIa,RIb, RIIa,RIIb,Ca,Cb,Cc.

The activity of PKA is controlled primarily by cAMP. In the absence of cAMP, PKA exists as a tetramer composed of two regulatory R subunits, each containing two cAMP-binding sites, and two catalytic C subunits (Figure 9.8). The catalytic activity 9.3 Protein Kinase A 433

Figure 9.8 Control of protein kinase A. PKA is inhibitor proteins. A negative feedback can targeted to distinct subcellular sites via form via PKA-mediated phosphorylation of a association of the R-subunits with AKAP phosphodiesterase. The phosphodiesterase is proteins. On activation of PKA, the catalytic C- activated by the phosphorylation and subunits are released and phosphorylate hydrolyzes cAMP to AMP, whereby the signal substrates that are located close to the AKAP transduction via protein kinase A is reduced or anchoring site. Substrate phosphorylation can terminated. be downregulated by the binding of PKA is masked in the holoenzyme C2R2, as the R subunit blocks the entrance to the active site due to the presence of an inhibitory motif on R. Two classes of PKA holoenzymes, types I and II, have been identiﬁed that differ in their R subunits (RI and RII). Both R subunits (RI and RII) and the C subunit exist as multiple isoforms (RIa,RIb, RIIa, RIIb,Ca,Cb,Cc). The existence of multiple R and C subunits harboring different biochemical features allows for the formation of a number of holoenzymes with different biological characteristics, which in turn contributes to the speciﬁcity and variability of PKA signaling observed in the cell. cAMP binds cooperatively to two sites termed A and B on each R subunit. In the inactive holoenzyme, only the B site is exposed and available for cAMP binding; however, when the B site is occupied this enhances the binding of cAMP to the A site in a cooperative fashion. The binding of cAMP to the R subunit leads to a large conformational change in R, as revealed by crystal structures of the C subunit in complex with fragments of RI [7]. The results of structural studies have shown that, in the absence of cAMP, RI wraps around the large lobe of the C subunit, making intense contacts with the activation loop and covering the substrate-binding site. The binding of four cAMP molecules – two to each R subunit – leads to a gross conformational change in R. 434 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

As a consequence, the afﬁnity between R and C is reduced by a factor of between 104 and 105, and the holoenzyme dissociates into the dimer of the cAMP-bound R subunits and two monomers of C, which now become catalytically active. Full catalytic activity of C requires the phosphorylation of a threonine residue (Thr197) in the activation loop; this loop functions as a major binding surface for the R subunit. The consensus sequence for the phosphorylation of proteins by PKA is Arg-Arg-X- Ser/Thr, Arg-Lys-X-Ser/Thr, Lys-Arg-X-Ser/Thr, or Lys-Lys-XSer/Thr. The RII subunit contains such a sequence, and is therefore subject to phosphorylation by the C subunit in the holoenzyme, but without the release of inhibition. The R subunits have a similar modular structure (Figure 9.9). A dimerization domain at the N terminus forms a four-helix bundle that also provides a docking surface for the AKAPs. At the C terminus of each protomer are located the A and B binding sites for cAMP. Both types of R subunit contain an inhibitory region that mediates an inhibition of the C-subunit; however, RI and RII are functionally nonredundant and differ in the nature of their inhibitory site. Typically, RII

Figure 9.9 Domain structure and phosphorylation sites of the RIa, RIIb, and C-subunits of protein kinase A. M, myristoyl anchor. 9.3 Protein Kinase A 435 subunits have a phosphorylation site in their inhibitory motif and thus serve as both substrates and inhibitors. The inhibitory motif on RI is a true pseudosubstrate that functions as a substrate mimetic by binding to the active site cleft of the C þ subunit. Furthermore, RI subunits require ATP and Mg2 to form a stable holoenzyme complex, whereas RII subunits do not. The C subunit has a myristic acid residue of unknown function at the N terminus, and shows the typical kinase fold. In addition, the C subunit has speciﬁc Ser/Thr phosphorylation sites, namely Thr197 and Ser338. Of these, Thr197 is located in the activation loop and is phosphorylated by an autophosphorylation mechanism.

9.3.2 Regulation of PKA

PKA is subject to multiple regulatory influences. Whereas, the regulation by cAMP is the primary determinant of PKA regulation, other regulatory influences ensure a high specificity of PKA with regards to tissue distribution and compartment- specific actions. The activity of PKA can be controlled by the following mechanisms, both in a temporal and spatially regulated manner (Figure 9.8):

Changes in cAMP concentration: The changes in concentration of cAMP that lead to the activation of PKA in the cell are relatively small. In many tissues, a two- to threefold increase in cAMP concentration is sufficient to bring about the maximum physiological effect. The cell has different mechanisms available that limit the increase in cAMP concentration to a relatively narrow range and contribute to a damping of signal transduction via PKA. An example of a mechanism with a damping effect in signal transduction by PKA is a feedback control by a cAMP phosphodiesterase. In this case, the activated PKA phosphorylates and activates a phosphodiesterase that hydrolyzes cAMP to AMP. This mechanism enables PKA to control its own steady-state activity. It also ensures that, as the external signal diminishes, the cAMP signal rapidly subsides. Binding of inhibitor proteins: Regulation of PKA also takes place via the binding of specific inhibitor proteins. There are three natural inhibitors of PKA. The inhibitor PKI, for example, is involved in the subcellular transport of C subunits and is considered a major regulator of C subunit activity. Targeting to subcellular sites by AKAPs: Given the fact that PKA is widely distributed and is activated by a large variety of external signals, the question arises how – apart from isoenzyme patterns, differential phosphorylation and inhibitor binding – the high specificity of PKA action is achieved in the cell. The specific compartmentalization of PKA enzymes has now been recognized to be a major determinant of PKA specificity. By binding to subcellular structures via AKAPs, isoenzymes of PKA can be assembled at distinct subcellular sites 436 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

in the vicinity of their substrates. Currently, more than 50 different AKAPs are known that are located to different compartments of the cell. Most of the interactions between AKAPs and PKA holoenzymes seem to be mediated by RII subunits.

& Main regulation of PKA is by: — cAMP — Inhibitor proteins — Subcellular localization via AKAPs.

9.3.3 A-Kinase Anchor Proteins (AKAPs)

Signaling by PKA occurs in a temporally and locally highly regulated manner. A large part of the compartmentalization and dynamics of PKA signaling is controlled through association with AKAPs, which are scaffolding proteins that target PKA to specific subcellular compartments so as to provide spatial and temporal regulation of the PKA-signaling events. The AKAPs comprise a structurally diverse family of functionally related proteins that include more than 50 members when splice variants are included [8]. AKAPs are defined on the basis of their ability to bind to PKA. This binding is mediated by a stretch of 14–18 amino acids forming an amphipathic helix that interacts with the dimerization motif of the R subunit dimers. In addition to the PKA binding domain, AKAPs contain specialized targeting domains responsible for tethering the complex to different subcellular localizations through protein–lipid or protein–protein interactions (Figure 9.10). AKAPs have been identified in association with a variety of cellular compartments, including centrosomes, endoplasmic reticulum, the Golgi apparatus, mitochondria, microtubules, cell membranes, nuclear matrices, secretory granules, and the cytoskeleton. The AKAPs immobilize the PKA isoforms at specific subcellular sites by binding the R subunits (Figure 9.8). The binding of cAMP to the regulatory subunit releases the catalytic subunit, which can then phosphorylate substrates in close vicinity; the released catalytic subunit can also be transferred to other compartments of the cell. In parallel with the increase in cAMP, translocation of the catalytic subunit from the Golgi apparatus to the nucleus is observed in many cells, and this leads to a stimulation of transcription.

9.3.3.1 AKAPs as Multivalent Scaffolds Another important functional characteristic of AKAPs is their ability to form multivalent signal transduction complexes by interaction with phosphoprotein phosphatases, other kinases, PDEs, and other proteins involved in signal transduction (Figure 9.10). Several AKAPs with this property have been 9.3 Protein Kinase A 437

Figure 9.10 Signal transduction through that direct AKAP signaling complexes to preassembled AKAP complexes. (a) A-kinase distinct subcellular locations. After Ref. [9]; anchoring proteins (AKAPs) organize PKA, (b) PKA anchoring to various AKAP isoforms PKC, guanine nucleotide exchange factors targets the kinase to defined subcellular (Epac) and phosphodiesterases (PDE) into locations. Diagram of a prototypic cell showing cAMP-responsive complexes. Anchoring of the targeting of PKA via AKAP18, AKAP350/ certain protein phosphatases (PP2B) and PKC 450, and mAKAP variants. NMDA, N-methyl- broaden AKAP function. Furthermore, AKAPs D-aspartate. Scott 2009 [9], figure 3C. contain unique subcellular targeting domains Reproduced with permission of Elsevier. 438 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

identiﬁed that provide a precise spatiotemporal regulation of the cAMP– PKA pathway and integration with other signaling pathways in one signal complex.

& AKAPs:

Scaffolding and targeting proteins that organize signals mediated by second messengers. AKAP isoforms AKAP250/79 bind to: — R subunits of PKA — b 2-adrenergic receptor — PKC isoforms — Protein phosphatases, PP2B — Ion channels — Src kinase.

For example, the AKAP isoform AKAP79 is a versatile scaffolding protein that can associate – in addition to the R subunits of PKA – protein kinase C, b the protein phosphatase 2B (calcineurin), the 2-adrenergic receptor, Src kinase (Section 9.3.2), and the GPCR kinase 2. Furthermore, this AKAP harbors targeting motifs that direct PKA to speciﬁc sites such as GPCR signaling complexes. These properties open up the prospect of a coordinated and layered regulation of multiple activities in a multiprotein complex anchored around a GPCR at a distinct subcellular site. Another more complex example is the muscle-speciﬁc, mAKAP, that coordinates two integrated cAMP effector pathways (Figure 9.11). Both, PKA and phosphodiesterase PDE4-D3 are present in the mAKAP-signaling complex, thus creating a negative feedback loop where the PKA phosphorylation of PDE4-D3 increases its activity and facilitates a more rapid signal termination. Moreover, PDE4-D3 serves as an adapter protein for the cAMP-regulated exchange factor Epac (Section 8.2.2) and the extracellular signal-regulated kinase (ERK), ERK5 (Chapter 12). The ERK5- mediated phosphorylation of PDE4-D3 decreases the PDE activity, thereby favoring a local increase in cAMP and successive PKA and Epac activation. In this system, two coupled cAMP-dependent feedback loops are synchronized within the same AKAP complex. Overall, the composition of the AKAP signaling complex and the function of its components appear to be regulated in a dynamic fashion. In these complexes, several signals can be integrated and directly transmitted to the substrates, allowing regulation of both the forward and backward steps of a given signaling process. 9.4 The PI3 Kinase/Akt Pathway 439

Figure 9.11 Signal complexes involving PKA and PDE4D3, whereas PDE4D3 scaffolds muscle-specific AKAP. The graphic shows a an Epac---Rap1 pathway that coordinates a signaling complex consisting of mAKAP, PKA, MEKK/MEK5/ERK5 module (see Chapter 12). PDE4D3, Epac, and a MEKK/MEK5/ERK5 Red-filled circle ¼ cAMP; blue-filled module. For details, see text. mAKAP anchors circle ¼ phosphate. After Ref. [8].

9.4 The PI3 Kinase/Akt Pathway

Summary PI3 kinase catalyzes the phosphorylation at the 30 position of the inositol part of

various PtdIns derivatives. The products of the reaction, such as PtdIns(3,4,5)P3, serve as membrane-localized second messengers that help to localize various signaling proteins to membranes. There are three classes of PI3Ks, termed I, II, and III. The best-investigated member of class I, PI3Ka, is a heterodimer, composed of a catalytic subunit (p110a) and a regulatory subunit of 85 kDa (p85a) that functions as an adapter for the association of upstream partner proteins. PI3 kinase is activated mainly via three pathways, namely transmembrane receptor pathways involving Tyr-kinase signaling, the Ras pathway, and Gbc signaling. The major downstream effector of PI3 kinase is the protein kinase

Akt (PKB). The PtdIns(3,4,5)P3 formed by PI3 kinase binds to Akt and other protein kinases, inducing their membrane association and activation. Signals mediated by the PI3K/Akt pathway have proliferation-promoting and antiapoptotic effects, and deregulation of the PI3K/Akt pathway is observed in many cancers. 440 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

The Ser/Thr-speciﬁc protein kinase Akt, also known as protein kinase B (PKB), is at the center of a major signaling pathway of the cell regulated mainly by

phosphoinositide-3 kinase (PI3K) and its reaction product, PtdIns(3,4,5)P3. Often, this pathway is collectively termed the PI3K/Akt pathway. Fundamental cellular functions such as cell proliferation and survival are regulated by the PI3K/Akt pathway, and alterations to the PI3K/Akt signaling pathway are a frequent occurrence in human cancers. Furthermore, many physiological functions of insulin involve PI3K/Akt signaling, and this pathway is therefore of great importance for the regulation of glucose metabolism.

9.4.1 PI3K

The reactions catalyzed by PI3 kinase have been presented in Section 8.6.

9.4.1.1 Classification and Properties of PI3K The family of PI3Ks comprises at least 15 kinases that differ in substrate speciﬁcity, the nature of the associated subunits, and the modes of regulation. There are three classes of PI3Ks, termed I, II, and III, and also an additional class IV that includes the PI3K- related kinases. The latter kinases are large enzymes with a catalytic core similar to that of the PI3Ks. Important examples of PI3K-related kinases are the “mammalian target of rapamycin” (mTOR), and kinases implicated in DNA repair such as DNA-dependent protein kinase (DNA-PK) and the ATR and ATM kinases (see Section 15.6). The class I PI3Ks are heterodimeric enzymes composed of a range of four catalytic (p110a, p110b, p110d, and p110c) and ﬁve regulatory (p85a, p85b, p55, p101, and p84) subunits [10]. These enzymes are activated by tyrosine kinases or

GPCRs to generate PtdIns(3,4,5)P3, which engages downstream effectors such as the Akt pathway and the Rho family GTPases (Chapter 10). The class II and III PI3Ks play a key role in intracellular trafﬁcking through the synthesis of PtdIns(3)P

and PtdIns(3,4)P2. Of the three classes of PI3Ks, only class I will be presented in more detail here. Most members of class I are associated with a subunit that functions as an adapter in signal transduction. The best-investigated enzyme, PI3Ka, is a heterodimer with adapter function, composed of a catalytic subunit (p110a) and a regulatory subunit of 85 kDa (p85a). The PI3Ka phosphorylates various PtdIns derivatives at the 30-

position (Figure 8.5). A physiologically important substrate is PtdIns(4,5)P2, which is converted to the second messenger PtdIns(3,4,5)P3 by PI3K. The p85a subunit has an SH3 domain, two SH2 domains and two Pro-rich domains that function as binding modules, which the PI3K uses for speciﬁc protein–protein interactions in the process of signal transduction and for association with other signal proteins. When bound to the p85 subunit in the cytosol, the catalytic activity of p110 is inhibited. Interaction of the SH2 domain of p85 with p-Tyr residues on a receptor Tyr kinase releases the inhibition on p110 and places this subunit close to its lipid substrates at the membrane (Chapter 10). 9.4 The PI3 Kinase/Akt Pathway 441

Other members of the class I PI3Ks, such as PI3K of the c-subtype, are stimulated by interaction with Gbc-complexes (Section 7.5.5) and have their own regulatory subunit. It is interesting that both a lipid kinase activity and a protein kinase activity have been identiﬁed in the catalytic domain of the PI3Kc subtype. This property allows for the formation of bifurcated signals, via formation of the second messenger PtdIns(3,4,5)P3 and via phosphorylation of substrate proteins. An important function in growth regulation is attributed to the PI3K. PtdIns

(3,4,5)P3 is not detectable in resting cells, but on stimulation of the cells with a growth factor there is a rapid increase in PtdIns(3,4,5)P3 levels and an associated translocation of PI3K to the membrane is observed. In accordance with its central growth-regulating function, mutants of the p110a subunit have been shown to be oncogenic both in vitro and in vivo [11]. The primary role of PI3Ks is to produce membrane-localized messenger substances via their lipid kinase activity. In addition, a scaffolding function has been identiﬁed for some PI3Ks [12].

9.4.1.2 Activation of PI3K PI3K is activated mainly via three pathways (Figure 9.12):

Activation by transmembrane receptors: The catalytic subunits of class I PI3Ks are recruited to activated transmembrane receptors through their regulatory subunits. For example, the SH2 domain of the p85 regulatory subunit mediates an interaction with phosphotyrosine residues on signal proteins involved in transduction of growth-regulating signals as for example, the activated PDGF receptor (Section 10.1.4). This interaction is accompanied by an allosteric activation of the catalytic p110a subunit. Another important binding partner is the insulin receptor substrate (IRS; Section 10.5) that becomes Tyr-phosphorylated upon insulin receptor activation. In both cases, binding of the SH2 domain of p85 to the tyrosine residue of the signal protein serves two goals: (i) activation of p110; and (ii) targeting to its membrane-localized substrate (Figure 9.12). The interaction between PI3K and IRS links insulin signaling to the PI3K pathways. Accordingly, many of the physiological functions of insulin are mediated by the PI3K/Akt kinase pathway. Activation in the Ras pathway: PI3K has also been identiﬁed as a part of the Ras signaling pathway (Chapter 10). Signals originating from transmembrane receptors can be transmitted from the Ras protein to PI3K. The catalytic subunits of class I PI3Ks contain a Ras-binding domain that mediates the selective binding of Ras to PI3Ks, generating a synergy between Ras and PI3K signaling. In this way, PI3K serves as an effector molecule of Ras. Activation by the Gbcdimer: Gbc dimers (see Section 7.5.5) directly activate the PI3K b- and c-subtypes. In this way, a variety of extracellular signals can be transmitted via GPCRs and G proteins to PI3K and its effectors. 442 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Figure 9.12 Pathways of PI3-kinase activation. is PtdIns(3,4,5)P3, which binds to PH domains PI3-kinase can be activated by growth factor of various signaling proteins. Overall, the receptors, either by direct interaction, via activation of PI3-kinase stimulates cell growth adaptors such as insulin receptor substrate and proliferation and inhibits apoptosis. A (IRS; Chapter 10), or via the Ras pathway suppressing effect is exerted by the tumor (Chapter 11). Another route of PI3-kinase suppressor PTEN, which hydrolyzes and thus activation employs the bc-subunits of inactivates PtdIns(3,4,5)P3. All reactions heterotrimeric G proteins liberated upon shown are closely associated with the inner activation of G protein-coupled receptors side of the membrane (this aspect is not (GPCR). The product of the PI3-kinase reaction addressed in the figure).

9.4.2 Akt Kinase (PKB)

A major downstream effector of PI3 kinase is the protein kinase Akt (also termed PKB). The domain structure of Akt kinase of which three isoforms are known is shown in Figure 9.13. Akt kinase contains a PH domain at the N terminus, a typical kinase domain, and a C-terminal regulatory domain. 9.4 The PI3 Kinase/Akt Pathway 443

Figure 9.13 Domain structure and main regulatory phosphorylation sites of PKB/Akt. PDK1, phosphoinositide-dependent kinase1; mTORC2, mammalian target of rapamycin complex 2; PP2A, protein phosphatase 2A.

9.4.2.1 Activation of Akt Kinase The main regulatory inputs for activation of Akt kinase are:

Binding of the second messenger PtdIns(3,4,5)P3, the product of the PI3 kinase reaction, to the PH domain. Phosphorylation of a speciﬁc threonine residue (Thr308) in the activation loop, catalyzed by phosphoinositide-dependent protein kinase 1 (PDK1). Phosphorylation of Thr473 in the regulatory region, catalyzed by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Dephosphorylation of P-Thr473 by the protein phosphatase 2A family members.

The central stimulus for activation of Akt kinase is provided by PI3K. The PtdIns

(3,4,5)P3 formed by PI3 kinase binds to the PH domains of two protein kinases next in sequence, PKB/Akt kinase and PDK1, inducing the membrane translocation of both enzymes.

& Akt kinase (PKB) signaling is: — proliferation-promoting; — anti-apoptotic; and — deregulated in many cancers. Akt phosphorylates: — Transcription factors: CREB — Protein kinases: IkB kinase — Proapoptotic proteins: Bad — Others: Mdm2.

The membrane recruitment of Akt kinase is a prerequisite for a subsequent phosphorylation of Thr308 in the activation loop, and this reaction is catalyzed by ﬁ PDK1 which also contains a PH domain with a high af nity for PtdIns(3,4,5)P3.In 444 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

order to be fully activated, Akt kinase requires a further phosphorylation (Thr473) within the regulatory region [13]. Ser/Thr protein kinase PKB/Akt is a key regulator of a wide range of cellular processes including growth, proliferation, and survival. PKB is clearly a crucial signaling molecule, and extensive research efforts have been aimed at understanding its regulation and action. Recent studies of the regulation of PKB activity by hydrophobic motif phosphorylation have yielded several exciting ﬁndings regarding members of the PI3-kinase-like family of kinases (PIKKs) acting as PKB regulators. Both, mammalian target of rapamycin complex 2 (mTORC2) and DNA- dependent protein kinase (DNA-PK) can phosphorylate Ser473 and activate PKB. Membrane targeting and the double phosphorylation activate Akt, allowing the phosphorylation of downstream substrates.

9.4.2.2 Signaling by Akt Kinase The signaling pathway for Akt kinase shown in Figures 9.14 and 9.15 illustrates the central roles of PI3K and Akt kinase in growth factor-controlled signal paths that lead from the cell membrane into the cytosol and the nucleus. In the PI3K/Akt signaling pathway, an extracellular growth factor ﬁrst activates the corresponding transmembrane receptor (e.g., PGDF receptor; Section 10.1) which transmits the signal further to the downstream effector, PI3 kinase. The associated membrane translocation of PI3K is synonymous

with its activation. The PtdIns(3,4,5)P3 formed induces membrane targeting and the activation of two protein kinases next in sequence, Akt kinase, and PDK1 (Figure 9.14). The latter is also central to the activation of other protein kinases as, for example, PKC. The importance of Akt kinase for cellular physiology can be illustrated from the large number of key signaling proteins that have been identiﬁed as substrates of Akt phosphorylation, and Akt kinase has been shown to regulate three major cellular processes:

Stimulation of protein synthesis and cell growth: This effect is mediated by a complex signaling cascade involving the protein kinases TSC and mTORCII (for details, see Ref. [14]). Promotion of cell survival and inhibition of apoptosis: Many substrates of Akt kinase are directly or indirectly involved in the promotion of cell proliferation and the prevention of apoptosis (Figure 9.15), and this is why deregulation of the Akt pathway is frequently associated with the development of cancer. Among the substrates of Akt kinase involved in the regulation of cell proliferation and apoptosis are cell cycle regulators (p27, p21, cyclin D1; see Chapter 15), transcriptional regulators such as the Fork head (FH) transcription factor and NFkB (Section 2.8.5.3), the pro-apoptotic protein Bad, pro-caspase 9, and the Mdm 2 protein (Chapters 16 and 17). 9.4 The PI3 Kinase/Akt Pathway 445

Figure 9.14 Activation of PKB/Akt kinase by accompanied by the release of an membrane translocation. Stimulation of RTKs autoinhibition, leading to the activation of upon growth factor binding induces RTK PDK1 and Akt kinase activities. Full activation autophosphorylation at Tyr residues. The p-Tyr of Akt requires phosphorylation by mTORC2. residues serve as attachment points for the Activated Akt phosphorylates a variety of target p85 subunit of PI3K, leading to PI3K activation. proteins that prevent apoptotic death, regulate PtdIns(3,4,5)P3 (PIP3) generated by PI3K transcription and other metabolic processes. serves as a binding site for the PH domains of PIP3 formation and the subsequent Akt PDK1 and Akt triggering membrane activation is antagonized by PTEN translocation of PDK1 and Akt. This is phosphatase.

Overall, Akt-mediated phosphorylation of these key regulators promotes cell survival and inhibits apoptosis. Regulation of cellular metabolism: Akt kinase is a major regulator in insulin- dependent metabolic pathways. The PI3-kinase/Akt pathway is activated by insulin and thereby mediates many of the metabolic effects of insulin, including glucose transport, lipid metabolism, glycogen synthesis, and protein synthesis. An important substrateofthisbranchofAktsignalingis GSK3 kinase.

& Phosphatase and tensin homolog (PTEN): — Lipid phosphatase — Tumor suppressor function — Hydrolyzes PtdIns(3,4,5)P3 — Downregulates Akt. 446 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Figure 9.15 Multiple mechanisms of cell Section 4.4.3.1. For IKK, see Section 2.8.5.3. survival regulation by Akt kinase. For Bad and FH, forkhead transcription factors; for JNK, see caspase 9 signaling, see Chapter 17. For Chapter 12. For mTORC1, see Section 5.2.5. Mdm2/p53, see Chapter 16. For CREB, see

9.4.2.3 Phosphatase and Tensin Homolog PTEN Phosphatase The great importance of the PI3kinase/Akt pathway for growth regulation is

illustrated by the observation that an enzyme of PtdIns(3,4,5)P3 metabolism, phosphatase and tensin homolog (PTEN) lipid phosphatase, has been identiﬁed as a tumor suppressor protein [15]. PTEN has lipid phosphatase activity that is speciﬁc

for the hydrolysis of PtdIns(3,4,5)P3. It is a negative regulator of the Akt pathway, acting by lowering the concentration of PtdIns(3,4,5)P3 and counteracting the stimulation of Akt kinase (Figure 9.12). Because of the strong cell proliferation- promoting and antiapoptotic activity of the Akt kinase pathway, low concentrations

of PtdIns(3,4,5)P3 have antiproliferative and pro-apoptotic effects and inhibit tumor formation. In accordance with this, a functional inactivation of the PTEN phosphatase is observed in a multitude of tumors. A wide spectrum of mechanisms has been identiﬁed that contribute to a lowered PTEN activity, including PTEN gene mutation, aberrant transcription and microRNA regulation and aberrant posttranscriptional protein modiﬁcation as, for example, phosphorylation, acetylation, and ubiquitination [16]. 9.5 Protein Kinase C 447

9.5 Protein Kinase C

Summary Protein kinase C (PKC) enzymes include a family of Ser/Thr-specific protein þ kinases that are regulated by DAG, Ca2 and phosphorylation, among others. There are three PKC subfamilies with different structural and regulatory properties. Interest in PKC enzymes has been fueled by the identification of the tumor promoter tetradecanoyl phorbol acetate (TPA) as a specific activator of some PKC enzymes. Most functions of PKC are intimately linked to association with the cell membrane or other membrane compartments. The regulated localization of PKC to distinct subcellular sites is mediated by binding of the cofactors þ DAG, Ca2 and by association with adaptors, the RACK proteins.

9.5.1 Classification and Structure of PKC

The family of PKC enzymes includes Ser/Thr-specific protein kinases, which were þ first identified by the requirement of the cofactors DAG, Ca2 and phospholipid for activity.

& PKC subfamilies: — Classical PKC: a, bI, bII, c — Novel PKC: d,e,r,g — Atypical PKC: f,l,t.

9.5.2 The Protein Kinase C Family

There are more than 10 different subtypes of protein kinase C that are classiﬁed into three subfamilies according to the structure of the N-terminal regulatory domain and their respective cofactor requirements.

Classical or conventional PKCs: The members of this subfamily (cPKCs, a, bI, þ bII, c-subtypes) are activated by Ca2 and DAG and phorbol esters. Novel PKCs: The novel PKCs (nPKCs, subtypes d,e,r,g) are not responsive to þ Ca2 but are activated by DAG and phorbol esters. þ Atypical PKCs: These isoforms (aPKCs, f, l, t-subtypes) are Ca2 - and diacylglycerol-independent, and require adapter proteins for enhancement of their enzymatic activity.

In addition to the different cofactor requirements, the PKC isoenzymes are distinguished by different cellular localizations and a different pattern of substrate 448 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

proteins. For example, the a, d and f subtypes are widespread in almost all tissues, whereas the other subtypes only occur in specialized tissues.

& C1 domains bind: — DAG — Phorbol ester. C2 domains may bind: þ — Ca2 , phospholipids — Phosphotyrosine.

The members of the protein kinase C family are composed of a polypeptide chain with a molecular weight of 68–83 kDa. The N-terminal regulatory domains C1 and C2 and a C-terminal catalytic domain can be differentiated in the primary structure (Figure 9.16) of the conventional PKCs. The C1 (conserved domain 1) domain, which is present in the classical and novel PKC isoenzymes, contains two cysteine-rich motifs (also known as C1A- and C1B þ elements), each with two bound Zn2 ions, that mediate the binding of DAG and phorbol esters. The atypical PKC enzymes contain a C1-like domain with only a single Cys-rich motif that is unable to bind DAG and phorbol esters. The C2 domain of the classical protein kinase C subfamily is a module of about þ 120 amino acids that binds phospholipids in a Ca2 -dependent manner, resulting in membrane association of PKC. The C2 domains are very versatile regarding their function and ligands. PKCd, a member of the novel PKC enzyme subfamily, þ contains a variant of the C2 domain that does not bind Ca2 but binds to the phosphotyrosine residues of phosphorylated proteins. Furthermore, C2 domains

are found in proteins other than PKC as, for example, phospholipase A2 mediating protein–protein interactions.

þ Figure 9.16 Domain structure of protein nPKCs are Ca2 -independent. V, variable kinase C isoenzymes. The phorbol esters and region; PS, pseudosubstrate region; CR, the second messenger diacylglycerol bind to cysteine-rich domain; TM, transmembrane the cysteine-rich motif present in conventional domain; PH, pleckstrin homology domain. PKCs (cPKC) and novel PKCs (nPKC). The 9.5 Protein Kinase C 449

Figure 9.17 Structure of tetradecanoyl phorbol acetate (TPA), which functions as a tumor promoter and is a specific activator of protein kinase C.

9.5.2.1 Stimulation by Phorbol Esters A property of the classical and novel PKC subfamilies that is highly valuable for their identification and characterization is their activation by tumor promoters such as phorbol esters (Figure 9.17). These subfamilies bind to the tumor promoter TPA with high affinity. TPA binds to the C1 domain of PKC isoforms, leading to membrane attachment of PKC and activation of its kinase activity. The specific activation of PKC by externally added phorbol esters is an important tool for demonstrating their involvement in signal transduction pathways.

& Phorbol esters (TPA): — Are tumor promoters — Activate PKC — Bind to C1 domain — Induce membrane attachment.

9.5.3 Activation of PKC

In the absence of activating cofactors, the catalytic domain is subject to autoinhibition. All PKCs have a pseudosubstrate region in the C1 region, in which the phosphorylatable Ser/Thr is replaced by an Ala, which maintains the inactive state in the absence of an activating signal. In order to overcome autoinhibition and þ become sensitive to the activating cofactors DAG, Ca2 and phospholipid, PKC must be primed for activation by phosphorylation of three residues. First, the activation loop Ser/Thr is phosphorylated by PDK1, after which two Ser/Thr residues of the C-terminal region are phosphorylated by the protein kinase mTORC2. The primed PKCs are then activated for phosphorylation of their 450 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

substrates when their regulatory domains bind the appropriate combination of þ signals – that is, Ca2 , DAG and phospholipids for the conventional PKCs. Signal engagement triggers the release of the pseudosubstrate sequence from the active site, allowing access of the substrates. þ Two functions are attributed to the activating cofactors Ca2 , DAG and phospholipid:

Promotion of membrane association: Many functions of PKC are intimately linked to association with the cell membrane or other membrane compartments. A large part of the membrane recruitment of PKC is mediated by the þ C2 domain that binds Ca2 and interacts with anionic phospholipids of the cell membrane. Relief of autoinhibition: Binding of DAG and phospholipid to the C1 domain leads to an allosteric activation of PKC, by release of the pseudosubstrate region from the active site and association of C1 with the cell membrane.

Use of the two membrane-targeting domains C1 and C2 helps to ensure high affinity, specificity, and regulation of the membrane interaction. Important insights into the activating functions of the cofactors could be obtained from structural studies of PKC bII that carries two C1 domains, C1A and C1B [17]. These studies suggest that C1A clamps the NFD helix of the catalytic domain to the active site, fixing an inactive state. Relief of the inhibition requires binding of the cofactors DAG and phospholipid to the C1A and C1B domains that are induced to reorient and associate with the cell membrane. As a consequence, autoinhibition is released and substrate phosphorylation is possible. Use of the two membrane-targeting domains C1 and C2 apparently helps to ensure high affinity, specificity, and regulation of the membrane interaction. A model of the pathway of PKC activation is shown in Figure 9.18.

9.5.4 Regulation of Protein Kinase C

The main regulatory inputs that control protein kinase C activity are:

DAG signals þ Ca2 signals Phosphorylation Binding to signaling proteins that have enzyme, adapter, or scaffolding function.

As shown by the different domain organizations, the various subtypes may differ widely in their sensitivity to these signals. In most cases, PKC enzymes are sensitive to and require the input of several regulatory signals, and therefore the regulation of PKC is complex and each subtype exhibits a distinct pattern of regulation. 9.5 Protein Kinase C 451

Figure 9.18 Functions and regulation of protein kinase C; receptors for protein kinase protein kinase C. Receptor-controlled signal C, the RACK proteins, are also involved. pathways lead to formation of the intracellular Substrates of protein kinase C are the MARCKS þ messenger substances Ca2 and diacylglycerol proteins and other proteins associated with the (DAG) that, like phorbol ester (TPA), activate cytoskeleton. Other substrates are the Raf protein kinase C (PKC). Translocation to the kinase (see Chapter 10) and the receptor for cell membrane is linked with activation of vitamin D3 (VDR; see Chapter 5).

þ 9.5.4.1 Regulation by Ca2 and DAG þ The cofactors Ca2 , DAG and phosphatidyl serine are required for membrane association, substrate access and activation of the classical PKC subtypes. þ Ca2 and DAG are second messengers released upon the induction of a variety of þ signaling paths (Chapter 8). A main component of Ca2 /DAG signaling pathways is a PLC which, when activated, produces the messenger substances Ins(3,4,5)P3/ þ Ca2 and DAG. Activation of PLC, and hence of PKC, may take place via several central pathways such as receptor tyrosine kinase-, GPCR-, and Ras-signaling pathways. 452 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

& PKC activation is linked to: — GPCR signaling via PLC-b — Receptor tyrosine kinase signaling via PLCc — Ras signaling via PLCe.

Signaling pathways starting from receptor tyrosine kinases trigger the stimulation of PKC by activating PLC-c. An activating signal may also be dispatched in the direction of PKC – via activation of PLC-b – from G protein-coupled membrane receptors (Figure 7.7). Furthermore, the activation of PLCe (Section 7.7.2) and the þ generation of Ca2 /DAG signals may occur via the Ras pathway (see Section 11.8), þ providing a link between Ras signaling, Ca2 /DAG signals, and PKC activation. In þ addition, the cell has available a broad palette of other tools for producing Ca2 þ signals (Section 8.5) and, accordingly, many ways of activating PKC via Ca2 exist.

& PKC is regulated by: þ — Ca2 signals — DAG — Phosphorylation in activation loop, catalyzed by PDK1 — Phosphorylation at Tyr-residues — Localization by RACK proteins.

9.5.4.2 Regulation by Phosphorylation: PDK1 All PKC subtypes contain several Ser/Thr- and Tyr- phosphorylation sites, the phosphorylation of which is an essential step in the maturation and activation of PKC þ enzymes. A pivotal role in priming PKC for reception of DAG and Ca2 signals is attributed to the PDK1 kinase [18] that is upstream of both atypical and conventional PKC isoforms. PDK1 is partly regulated by phosphoinositide 3-kinase (PI3-kinase). The PI3K-generated lipids activate PDK1, which then phosphorylates and activates several members of the conserved AGC kinase superfamily including PKA, cGMP- dependent kinase, Akt kinase, and PKC. An initial activating phosphorylation of PKC by PDK1 takes place on Ser/Thr residues in the activation loop of the catalytic domain. Another two Ser/Thr phosphorylations are introduced in the C-terminal region by the mTORC2 protein kinase complex [19]. The participation of this kinase links PKC activation to signaling pathways that mediate growth signals. A number of PKC isoforms, as for example PKCd and PKCe, are phosphorylated on Tyr residues in response to a variety of signals. These phosphorylations are mostly catalyzed by members of the Src or Lyn family of nonreceptor tyrosine kinases (see Section 10.3). How PKC is targeted to Src kinase has become clear only after the discovery of the C2 domain of PKCd to be a phosphotyrosine-binding domain. The C2 domain binds speciﬁcally to a phosphotyrosine on a transmembrane protein named CDCP1 which binds to, and is phosphorylated by, Src kinase. The CDCP1 protein has the function of an adapter in this case that promotes association of PKCd with Src kinase allowing Tyr-phosphorylation of PKCd by Src kinase (Figure 9.19). 9.5 Protein Kinase C 453

Figure 9.19 Signaling complexes at the cell allowing phosphorylation of membrane- membrane involving PKCd and Src kinase. associated substrates. Phosphorylation of PKCd becomes activated upon binding to CDCP1 is catalyzed by the nonreceptor phosphotyrosine residues on the tyrosine kinase Src that in turn binds to transmembrane protein CDCP1. Membrane phosphotyrosine residues of CDCP1 via its recruitment is thought to activate PKCd, SH2 domain.

9.5.4.3 Protein Kinase C-Interacting Proteins and Regulation by Localization A large number of proteins have been found to interact with PKC enzymes during signal transduction. The PKC-interacting proteins are mostly isoform-speciﬁc and include proteins that:

target PKC to its upstream activators; direct PKC to intracellular compartments; are substrates of PKC; and modify PKC as for example, other protein kinases.

The PKCs are thought to reside in the cytoplasm in an inactive conformation and, after activation, these proteins translocate to the cell membrane, cytoplasmic organelles, or nucleus. The proteins that direct PKC enzymes to speciﬁc subcellular sites will be discussed in more detail in the following subsections.

9.5.5 Receptors for Protein Kinase C, RACK Proteins

A major regulatory aspect of PKC enzymes is the regulated localization to distinct subcellular sites. The stimulation of cells with phorbol esters or with hormones that activate phospholipases Cb or Cc leads to a translocation of PKC isoenzymes from the cytoplasm to the cell membrane or cytoskeleton, or into the nucleus. The differential localization of the PKC isoenzymes is mediated by PKC-targeting proteins, among which the RACK proteins are predominant. 454 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

& RACK proteins: — Target PKC to distinct sites — Direct the trafficking of PKC — Assemble PKC into multiprotein complexes.

The RACK proteins (RACK ¼ receptors for activated protein kinase C) specifically interact with PKC enzymes and anchor them to the membrane, thus providing access to membrane-localized substrates. Furthermore, subtypes of the RACK proteins are involved in the intracellular trafficking of PLC enzymes. Most of the PKC–RACK interaction is mediated by the C2 domain of the PKC enzymes. In addition to anchoring activated PKCs, the RACK proteins anchor other central signaling proteins. As an example, RACK 1, the anchoring protein for activated PKCbII, targets Src tyrosine kinase (see Section 10.3.2), cAMP-specific phosphodiesterase and integrin b-subunits (Section 12.4) to distinct subcellular sites. RACK 1 contains several WD motifs that are thought to be responsible for the protein– protein interactions and for its scaffolding function. Similar to the AKAPs, the RACK proteins appear capable of organizing various signaling enzymes into multiprotein signaling complexes. Members of the A kinase-anchoring protein (AKAP) family have also been identified as binding partners of PKC enzymes. As an example, AKAP79 assembles PKC into protein signaling complexes, keeping it in an inactive state. On the þ receipt of Ca2 /DAG signals, the PKC is released from the inhibitory complex.

9.5.6 Functions and Substrates of PKC

The members of the PKC family are central signal proteins and, as such, are involved in the regulation of a multitude of cellular processes including cell proliferation, subcellular transport, cell migration, cytoskeleton reorganization, immune receptor signaling, apoptosis, and memory formation. A large number of substrates have been identified for which each PKC isoenzyme has a distinct substrate specificity. Of the many substrates of PKC isoenzymes, the MARCKS (myristoylated, alanine-rich C-kinase substrate) proteins are highlighted as very well-characterized and specific substrates of protein kinase C, and their phosphorylation is used as an indicator of the activation of protein PKC in vivo. The MARCKS family of proteins is heavily involved in the restructuring of the actin cytoskeleton, which involves a PKC-mediated phosphorylation of the MARCKS proteins.

& PKC substrates: — MARCKs proteins — Raf kinase — EGF — NMDA receptor. 9.6 Ca2þ/Calmodulin-Dependent Protein Kinases, CaM Kinases 455

Further selected examples of substrates of PKC are the epidermal growth factor þ þ receptor (EGFR; Chapter 10), an Na /H exchanger protein, the GTPase K-Ras (Chapter 11), Raf kinase (Chapter 11), and N-methyl-D-aspartate (NMDA) receptors. The activation of PKC may, as these examples show, act on other central signal transduction pathways of the cell; it may have a regulating activity on transcription processes, and it is involved in the regulation of transport processes and in neuronal communication. Many substrates of PKC are membrane proteins, and it is evident that the membrane association of PKC is of great importance for the phosphorylation of these proteins.

9.6 þ Ca2 /Calmodulin-Dependent Protein Kinases, CaM Kinases

Summary þ þ Protein kinases activated by Ca2 /calmodulin are classified as Ca2 /calmodulin protein kinases (CaM kinases). The most interesting member of this kinase þ family is CaM kinase II (CamKII), which plays a central role in deciphering Ca2 signals, especially in the processes of learning and memory formation. CamKII is þ an oligomeric enzyme that is regulated by Ca2 /calmodulin and phosphoryla- þ tion. Most interesting is the ability of CamKII to conserve the stimulatory Ca2 / calmodulin signal over a longer period of time and to remain in an activated state, even when the initiating stimulus has died away. This “memory” function þ allows the CaMKII to decode the frequency and amplitude of Ca2 signals.

9.6.1 General Function of CaM Kinases

þ þ The signal-mediating function of Ca2 is performed as a Ca2 /calmodulin complex þ in many signaling pathways. Ca2 /calmodulin can bind speciﬁcally to effector proteins and modulate their activity (Section 8.7). In the ﬁrst role as effector proteins þ þ of Ca2 /calmodulin are the Ca2 /calmodulin protein kinases (CaM kinases) [20].

& CaM kinases (CaMK): þ — Activated by Ca2 /calmodulin — Specialized CaMKs — Multifunctional CAMKs: CAMK I, II, IV.

A rough categorization of the CaM kinases differentiates between specialized CaM kinases and multifunctional CaM kinases. An example of a specialized CaM kinase is myosin light-chain kinase (MLCK), the primary function of which is to phosphorylate the light chain of myosin and thus to control the contraction of smooth musculature. 456 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Table 9.2 Examples of substrates of CaMKs (Source: Ann. Rev. Physiol., 1995, 57).

Protein Function

Acetyl CoA carboxylase biosynthesis of fatty acids Glycogen synthase glycogen synthesis HMG CoA reductase biosynthesis of cholesterol NO synthase biosynthesis of NO þ þ Ca2 channel (N-type) presynaptic Ca2 inﬂux þ þ Ca2 ATPase (heart) storage of Ca2 Synaptogamin release of neurotransmitters þ Ryanodin receptor release of Ca2 p56LCK tyrosine kinase activation of T cells EGFR growth control Cyclic nucleotide phosphodiesterase cAMP and CGMP metabolism PPA2 hydrolysis of phospholipids Ribosomal protein S6 protein biosynthesis CREB transcription control

The multifunctional CaM kinases include the CaM kinases of types I, II and IV, all of which phosphorylate a rather broad spectrum of substrate proteins. These enzymes regulate many processes including, among others, glycogen metabolism, activity of transcription factors, microﬁlament formation, synaptic release of neurotransmitters from storage vesicles, and biosynthesis of neurotransmitters. Some important substrates of CaM kinases are listed in Table 9.2. CaM kinases of type II are oligomeric enzymes which exhibit outstanding regulatory properties and will therefore be discussed below in more detail. The two other multifunctional CaM kinases of types I and IV are, apart from þ Ca2 /calmodulin control, monomeric enzymes regulated via phosphorylation by an upstream CaM kinase, CaMKK. The CaMKK phosphorylates CaM kinase I and IV in the activation loop and thereby greatly enhances the activity of these enzymes.

9.6.2 CaM Kinase II

CaM kinase II (CamKII) enzymes are divided into subtypes a, b, c, and d. The principal difference between these gene products is in the linker connecting the kinase domain to the hub domain, which is variable both in sequence and in length. The a and b subtypes of CaMKII only occur in the brain, whereas the other subtypes are also found in other organs. In the hippocampus, CaMKII constitutes up to 2% of the total cellular protein. From a regulatory point of view, CaMKII is of particular interest, as it has the characteristic of an enzyme with a built-in “memory switch.” The “memory” allows the CaMKII to conserve a stimulatory signal over a longer period of time and to remain in an activated state, even when the initiating stimulus has died away (Figure 9.20). Due to these properties, CaMKII is 9.6 Ca2þ/Calmodulin-Dependent Protein Kinases, CaM Kinases 457

Figure 9.20 (a) Linear representation of the Thr307 on neighboring subunits, which functional domains of CaM kinase II of type b; converts these subunits into an autonomous, þ (b) The various autoregulatory stages of Ca2 /CaM-independent active state and CamKII. The multimeric holoenzyme structure prepares the enzyme for phosphorylation of of CaMKII is depicted as a 6-mer for simplicity, the physiological substrates. The active state is with activated catalytic subunits illustrated in terminated when the activating phosphate þ blue. Ca2 /CaM binding triggers residue is cleaved off by a protein autophosphorylation of Thr286, Thr306 and phosphatase. considered an important element of memory formation and storage in the brain [21].

9.6.2.1 Structure and Activation of CaMKII

& CAMK II: — Oligomeric kinase of 12–14 subunits þ — Activated by: Ca2 /calmodulin, autophosphorylation — Inactivated by phosphatases þ — Memory effect for Ca2 signals — Important for memory and learning.

The domain structure of CaMKII shows an N-terminal catalytic domain, a regulatory domain, and a C-terminal association or “hub” domain (Figure 9.20). þ The Ca2 /calmodulin binding site and an autoinhibitory region carrying three 458 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

autophosphorylation sites (T286, T306, T307) are located within the regulatory domain. Phosphorylation occurs in trans, between two kinase subunits of the same þ holoenzyme, and releases autoinhibition, even in the absence of Ca2 :

Structure of the holoenzyme: Electron microscopic images and X-ray studies have shown that CaMKII has an oligomeric structure (Figure 9.21) in which two hexameric rings, each containing six copies of the monomeric enzyme (a, b, c, or d subtype) are configured in the form of a cylinder [22,23]. The catalytic domains are oriented outwards, and the association domains form a central hub of each ring. The aggregated form of CaMKII represents the holoenzyme form, which performs the catalytic functions and is subject to a sophisticated control by þ Ca2 /calmodulin concentration, autophosphorylation and dephosphorylation by CamKII phosphatases. þ Autoinhibition: In the absence of Ca2 /calmodulin and phosphorylation of the regulatory segment, CaMK II exists in an autoinhibited state. X-ray analysis of the autoinhibited state of CamKII holoenzyme shows a tightly packed assembly with a compact arrangement of kinase domains docked against the central hub. þ In this arrangement, the Ca2 /calmodulin-binding sites of the regulatory segment are inaccessible. Furthermore, parts of the regulatory segments form a coiled-coil structure that blocks peptide and ATP binding to the otherwise intrinsically active kinase domains (Figure 9.21). In this arrangement, the linker between the hub and the regulatory segment domain is ascribed an important þ role in determining the sensitivity to Ca2 /calmodulin signals. There is experimental evidence that an equilibrium exists between the compact state and þ a more extended state that allows the binding of Ca2 /calmodulin and subsequent phosphorylation. This equilibrium appears to depend on the length of the linker. Over 30 distinct mammalian splice variants have been generated, lengthening or shortening this region, and changes in the linker length are þ correlated with the sensitivity to Ca2 -signals, shorter linkers leading to a þ stabilization of the autoinhibited state, and a reduced sensitivity to Ca2 . 2þ þ Ca /calmodulin binding: An increase in the Ca2 concentration leads to þ binding of Ca2 /calmodulin to the regulatory domain, which releases the enzyme from its autoinhibited state (Figure 9.21). This triggers a large conformational change and disengages the kinase domain from the tightly packed structure. Furthermore, the phosphorylation of T286 increases the þ affinity of CaMKII for Ca2 /calmodulin by more than 10 000-fold, an effect þ known as CaM-trapping.Ca2 /calmodulin only dissociates very slowly from this high-affinity complex. The activated state is thus preserved over a longer period of time. Phosphorylation: The kinase domains now are directed upwards and downwards of the ring midplane, which allows transphosphorylation of other kinase subunits on T286. Once T286 is phosphorylated, rebinding of the autoinhibitory segment to the kinase domain is prevented. There are two other critical phosphorylation sites (T306,T307) on the regulatory domain. T306 and T307 lie at the heart of the calmodulin-binding region of the regulatory segment. These 9.6 Ca2þ/Calmodulin-Dependent Protein Kinases, CaM Kinases 459

Figure 9.21 CaMKII subunit architecture and domains tightly arranged about the central hub activation. (a) Activation of subunits in the domain. Each kinase domain occupies a holoenzyme proceeds via regulatory segment position between two hub domain subunits, þ displacement by Ca2 /CaM binding to enable with its active site pointed towards the center access and presentation of Thr286 for of the assembly. The arrangement forms two phosphorylation by other subunits; (b) separate hexameric rings of kinase “petals” Domain architecture of the dodecameric which fold against the central hub. Chao 2011 CaMKII holoenzyme (subunits A---L). The [23], figure 2. Reproduced with permission of holoenzyme assembly comprises kinase Elsevier. 460 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

þ residues may be autophosphorylated in trans, which prevents Ca2 /calmodulin þ binding and makes the enzyme Ca2 -insensitive. It is unclear which conditions initiate autoinhibitory T306/T307 phosphorylation. 2þ þ Ca /calmodulin-independent activity: When Ca2 declines to base-line level and þ Ca2 /calmodulin dissociates, the CamKII phosphorylated at Thr286 still has 20– þ 80% of the activity of the Ca2 /calmodulin-bound form. This ensures that þ signiﬁcant activity remains after the Ca2 /calmodulin signal has died away. This þ autonomous, Ca2 /calmodulin-independent state of CaMKII is only terminated when CamKII phosphatases cleave off the activating phosphate residue and thus leads the enzyme back into the inactive state. Substrate binding: The autophosphorylation of CamKII exposes a binding site þ for target proteins such as the NMDA receptor, a Ca2 -channel regulated by glutamate and voltage changes. In the receptor-bound state, the requirement of þ CaMKII for Ca2 /calmodulin is reduced and the kinase may stay active even without being autophosphorylated. A major regulatory aspect of CamKII substrate recruitment is the colocalization with distinct subcellular structures. Subtypes of CaM kinase II have been found to be speciﬁcally localized in the nucleus and at the cytoskeleton, especially at postsynaptic structures called postsynaptic densities (PSD) that contain large amounts of associated CaM kinase II. By interaction with scaffold proteins such as PDZ proteins, CaM þ kinase II is assembled in the vicinity of Ca2 entry sites and of potential substrates. Examples of neuronal substrates are the ligand-gated ion channels such as the AMPA receptor, the NMDA receptor, and neuronal NO synthase. Deactivation: The switching into an autonomous activity state is thought to be an essential feature of CamKII participation in memory formation. However, this function also requires that the kinase activity can be adequately switched off. Two mechanisms appear to be involved in the deactivation of CamKII: (i) dephosphorylation by phosphatases; and (ii) the binding of inhibitor proteins. The protein phosphatases PP1 and PP2A have been implicated in removing the phosphorylations at Thr287, Thr306, and Thr307.

All of these features are dependent on the oligomeric state of the holoenzyme. Speciﬁcally, the formation of the large kinase aggregate endows the holoenzyme with exquisite regulatory properties.

9.6.2.2 Memory Function of CaMKII An outstanding feature of regulation of CaM kinase II is the memory effect within the activation process. Activation of the enzyme is initiated by a generally transient þ þ increase in cellular Ca2 that activates CaM kinase II in the form of the Ca2 / calmodulin complex. Moreover, the kinase may remain active for up to 30 min after þ the Ca2 signal has died away because the enzyme is converted into an autonomous activated state upon autophosphorylation. This property is illustrated þ by the ﬁnding that CaMKII is able to respond in a switch-like fashion to Ca2 - þ signals over a very narrow Ca2 -concentration range, and that this range is sharpened further by the presence of protein phosphatase. Cooperative 9.7 Ser/Thr-Specific Protein Phosphatases 461 autophosphorylation within the oligomer and the action of protein phosphatases þ þ provide for an ultrasensitive Ca2 -switch that responds to changes in Ca2 - þ concentrations over a range of 300 nM Ca2 , a range that allows the sensing of þ speciﬁcCa2 signals. A special importance is attributed to this property, particularly for the detection þ and differentiation of repetitive Ca2 signals in neuronal cells. The magnitude of constitutive CaM kinase II activity in the oligomeric, autophosphorylated form has þ been shown to depend on the duration, amplitude, and frequency of elevated Ca2 . þ For example, the interval between the occurrence of staggered Ca2 signals is a þ determining factor for the intensity of activation. If the Ca2 signals occur with a higher frequency, a longlasting and effective activation is possible, as the kinase remains in the activated state between signals because of the memory effect. Based on these special properties, it is assumed that CaMKII actively participates in synaptic plasticity and memory formation. The ability of CaMKII to decode the þ frequency of Ca2 oscillations during synaptic stimulation and to give a prolonged response beyond the initial stimulus enables it to provide two characteristics required for a molecule involved in synaptic plasticity and in memory formation [21]. In agreement with this is the observation that elimination of CamKII phosphorylation interferes with learning and memory functions.

9.7 Ser/Thr-Specific Protein Phosphatases

Summary Protein phosphatases are the antagonists of protein kinases, and their action can reverse all functions that are coupled to the phosphorylation status of a protein. Both, Ser/Thr-phosphate-specific protein phosphatases (PSPs) and Tyr- phosphate-specific protein phosphatases (PTPs; see Chapter 10) have been identified. About 30 genes have been found for PSPs that are classified according to their substrate specificities, metal requirements, and sensitivities to natural or synthetic inhibitors. Among these, the protein phosphatases (PPs) 1, 2A, and 2B (calcineurin) are most abundant and have been studied to the greatest degree. One key defining feature of all PSPs is that they are multimeric enzymes, assembled from only a small number of catalytic subunits combining with hundreds of regulatory subunits. The regulatory subunits mediate the subcellular localization, substrate specificity, and fine-tuning of phosphatase activity.

Under physiological conditions, phosphate esters of Ser and Thr residues are stable and show only a low rate of spontaneous hydrolysis. Thus, the cell requires its own tools for the regulated cleavage of phosphate residues to terminate and dampen signals mediated by protein phosphorylation. This role is performed by 462 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

speciﬁc protein phosphatases that are frequently essential components of signaling pathways.

9.7.1 Classification and Structure of Ser/Thr Protein Phosphatases

Currently, Ser/Thr-phosphate-specific protein phosphatases (PSPs) and Tyr-phosphate-specific protein phosphatases (PTPs) have been identified. About 30 genes for PSPs and about 107 genes for protein PTPs exist in the human genome (the latter are described in Chapter 10). The regulatory most important class within the PSP family are the phosphoprotein phosphatases (PPPs). A key defining feature of all PPPs is that they are multimeric enzymes, assembled from only a small number of catalytic subunits combining with hundreds of regulatory subunits. The members of the PPP family share a common mechanism of metal-catalyzed hydrolysis of phosphate esters, þ þ with two metal ions, Mn2 and Fe2 , in the active site. Only 13 human genes encode PPP catalytic subunits, which is a low number when compared to the approximately 500 protein kinase genes. It is the regulatory subunits, rather than the catalytic subunits that provide the essential determinants for subcellular localization, substrate specificity, and fine-tuning of phosphatase activity. Unlike phosphatase and protein kinase catalytic subunits, the phosphatase regulators do not share an extensive sequence conservation. Rather, they are identified by their physical association and function. An important feature that distinguishes PPPs from protein kinases is substrate recognition. Unlike protein kinases, the PPPs do not recognize well- defined linear sequences or consensus motifs within their substrates. Instead, structural studies indicate that substrates interact with both the regulatory and the catalytic subunits [24]. Thus, substrate recognition appears to be determined to a large part by interactions with the regulatory subunits, of which a large number exist. This feature ascribes the regulatory subunits a key role in substrate selection, and it provides an explanation for the differing specificity of phosphatase multimers.

9.7.2 Function and Regulation of Ser/Thr Protein Phosphatases

Protein phosphatases are the antagonists of protein kinases, and their action can reverse all functions that are coupled to the phosphorylation status of a protein (for the general aspects of protein phosphorylation, see Section 2.5). In signaling pathways, these enzymes can perform a dual function: by diminishing and terminating a signal created by protein phosphorylation, they can have a dampening effect on protein kinase-mediated signal transduction. However, the protein phosphatases can also have a positive, reinforcing effect in signaling pathways. The dephosphorylation of a signal protein by a protein phosphatase can lead to its activation and thus to an ampliﬁcation of the signal (Figure 9.22a) . 9.7 Ser/Thr-Specific Protein Phosphatases 463

Figure 9.22 (a) The dual function of protein five different binding sites for the regulatory kinases and protein phosphatases. (R) subunits. The R subunits act as activity- Phosphorylation of proteins (P1, P2) can fix the modulators (R1), targeting proteins (R2) and/ latter into an active or inactive state. In the or substrates (R3). It is suggested that the R case of P1, protein kinases have an activating subunits have multiple contacts with PP1C and effect and protein phosphatases are that they can share binding sites. Specificity is inactivating; the reverse is true for P2; achieved by interaction with specific subsets of (b) Combinatorial control model of the binding pockets on PP1C. Chao 2011 [23], catalytic subunit of protein phosphatase 1 figure 2. Reproduced with permission of (PP1). The catalytic subunit is represented with Elsevier.

& Protein phosphatases: — Important examples: PP1A, PP2A, PP2B — Contain catalytic, regulatory subunits — Regulation by: Phosphorylation Localization Inhibitors.

The protein phosphatases (PPPs) serve as active parts of the signal transduction processes, and as such are subject to diverse and complex regulation. A large part of this regulation is exerted via the subunits that associate with the catalytic subunit to form the active holoenzyme. These subunits serve to transmit incoming signals to the catalytic subunit and to target the catalytic subunit to distinct subcellular sites. 464 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Regulation of the Ser/Thr phosphatases takes place predominantly by the following mechanisms:

Posttranslational modification: Protein phosphatases are subject to distinct PTMs as for example, phosphorylation, methylation, and ubiquitination. Targets of phosphorylation are both the catalytic and the regulatory/localization subunits. These phosphorylation events can change subunit composition, catalytic activity, and subcellular localization. Targeted localization: Much of the specificity of substrate dephosphorylation is achieved by targeting or scaffolding subunits that serve to localize the phosphatase in proximity to particular substrates, and also to reduce its activity towards other potential substrates. Specific inhibitor proteins: Specific inhibitor proteins for Ser/Thr phosphatases exist which can control the activity of the protein phosphatases. These inhibitors are generally subject to regulation themselves, for example, by phosphorylation.

9.7.3 Protein Phosphatase 1, PP1

PP1 is a major class of eukaryotic Ser/Thr-specific protein phosphatases that regulate diverse cellular processes such as cell cycle progression, muscle contraction, carbohydrate metabolism, protein synthesis, transcription, and neuronal signaling. Each functional PP1 enzyme consists of a PP1 catalytic subunit and a regulatory subunit R [24,25]. There are four PP1 isoforms, derived from three genes, expressed in mammalian tissues; this low number is contrasted by the large number of R subunits of which more than 100 have been identified. These PP1 regulators show tissue-specific expression and exhibit preferential associations with individual PP1 isoforms. The interactions between the catalytic subunit and specific R subunits are central to the functions of PP1. R subunits may target the PP1 catalytic subunit to specific subcellular compartment, modulate substrate specificity, or serve as substrates themselves (Figure 9.22b). Based on their main effects on PP1c, the R subunits can be roughly divided into three groups:

Activity-modulating proteins: This group includes inhibitor proteins that, in their phosphorylated form, block the activity of PP1c towards its substrates. Targeting subunits: This type of R subunits binds both PP1c and one of its substrates. For example, the G-subunits target PP1c to the substrate glycogen synthase, and this binding is modulated by phosphorylation of glycogen synthase. PP1c substrates: A subset of PP1c substrates bind directly to PP1c and may also function as targeting subunit. 9.7 Ser/Thr-Specific Protein Phosphatases 465

9.7.4 Protein Phosphatase 2A, PP2A

The protein phosphatase 2A (PP2A) is one of the major Ser/Thr phosphatases implicated in the regulation of many cellular processes, including the regulation of signaling pathways, cell-cycle progression, apoptosis, DNA replication, gene transcription, and protein translation. PP2A enzymes show both a Ser/Thr- phosphate-specific and a weaker Tyr-phosphate-specific protein-phosphatase activity. The latter activity can be specifically stimulated by a distinct protein named protein tyrosine phosphatase activator (PTPA).

& PP2A: — Ser/Thr phosphatase — Weak Tyr phosphatase — Subunits A, B, and C — Many ABC holoenzymes — Tumor suppressor function — Inhibited by okadaic acid — Regulation by: Phosphorylation Methylation Nature of B subunit.

PPA2 enzymes are oligomeric enzymes composed of a conserved catalytic subunit and one or more additional regulatory subunits (Figure 9.23). The PP2A catalytic (or C) subunits – of which two genes are encoded in mammalian genomes – are usually associated with a scaffolding A-subunit and one of a large array of regulatory B-subunits. There are four families of B-subunits, each with two to five genes, and many with multiple splice variants. A huge diversity of biochemically distinct PP2A complexes can assemble in cells, due to a combinatorial association of the three subunit types and due to the association of the other cellular proteins. Organisms express different B-subunits in a tissue-specific and developmental stage-specific manner, leading to the presence of distinct PP2A complexes in different tissues and at different times. The aberrant expression, mutation or deletion of PP2A subunits are involved in cellular processes leading to tumor formation, and both the A and B subunits have been identified as tumor suppressors; this indicates an involvement of PP2A in the dephosphorylation of key proteins of cell-cycle progression and apoptosis. Of great importance for the study of PP2A was the discovery of okadaic acid as an inhibitor of PP2A. Okadaic acid (Figure 9.24) is a tumor promoter and inhibits PP1 and PP2A. Regulation of PP2A activity is mainly mediated by:

Phosphorylation: A PTM of the C subunit by phosphorylation of speciﬁc Thr and Tyr residues inactivates the enzyme. 466 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

Figure 9.23 Subunit structure and regulation The heterodimer can associate three types of of protein phosphatase A (PP2A). The core of regulatory subunit, PR55B, PR55B0 or PR55B00, PP2A is heterodimer consisting of a catalytic of which different subtypes exist. In addition, subunit PP2AC and a structural subunit PR65A. various regulatory modifications are indicated.

C-terminal methylation: A unique PTM is found at the highly conserved C terminus of the catalytic C-subunit. The C-terminal Leu309 is methylated by a novel type of methyltransferase (leucine carboxymethyl transferase; LCMT ) and demethylated by a specific esterase, named PME-1. C-terminal methylation seems to be a requirement for the association of the B-subunits, and is thus a determinant of holoenzyme formation. Nature of associated B-subunit: A major determinant of PP2A activity is the identity of the associated B-subunit. The nature of the B-subunit assembled in the heterotrimer of PP2A influences substrate specificity, catalytic activity, and subcellular distribution of the holoenzyme. The striking features of the B- subunits are their diversity, which stems from the existence of entire subunit

Figure 9.24 Okadaic acid, an inhibitor of Ser/Thr protein phosphatases. 9.7 Ser/Thr-Specific Protein Phosphatases 467

families, and the apparent lack of sequence similarity between these gene families.

9.7.5 Protein Phosphatase 2B, Calcineurin

Calcineurin (protein phosphatase 2B; PP2B) is a Ser/Thr phosphatase that is controlled by cellular calcium and regulates a large number of biological responses including lymphocyte activation, neuronal and muscle development, and the development of vertebrate heart valves.

& PP2A, calcineurin: — Composed of calcineurin A and calcineurin B þ — Regulated by Ca2 /calmodulin — Inhibited by: Cyclosporine/cyclophilin FK506/FK506-Bp. — Regulates nucleocytoplasmic shuttling of NF-AT members.

Like the other major classes of protein phosphatases, calcineurin is an oligomeric enzyme composed of a catalytic subunit, calcineurin A, and a regulatory subunit, calcineurin B. Calcineurin A harbors an N-terminal phosphatase domain and three regulatory domains, which have been identiﬁed as the calcineurin B binding site, þ the Ca2 /calmodulin-binding domain, and an autoinhibitory domain that blocks access to the catalytic center. In the active site of calcineurin A, a binuclear metal þ þ center containing Fe2 and Zn2 is found. Both ions are thought to participate directly in phosphate ester hydrolysis [24]. Calcineurin B is highly homologous to calmodulin, containing four EF-hands as þ binding sites for Ca2 . Calcineurin is inactive alone and only gains phosphatase þ þ activity upon binding of Ca2 /calmodulin. Whilst it is not yet known how Ca2 / þ calmodulin activates calcineurin, it is possible that Ca2 /calmodulin induces a dimerization of calcineurin which might be required for activation. þ þ Both, Ca2 binding to calcineurin B and the requirement of Ca2 /calmodulin þ cause calcineurin activity to be strongly Ca2 -dependent, allowing it to function as a þ Ca2 sensor. The central regulatory function of calcineurin was recognized during the quest for the cellular target of the immunosuppressant drugs cyclosporine and FK506, which are often used following organ and tissue transplantation. Both drugs achieve their immunosuppressive effect via inhibition of calcineurin, in an indirect fashion. Cyclosporine and FK506 bind speciﬁcally to two proteins known as cyclophilin and FK506-binding protein, respectively, that belong to the family of immunophilins and function as peptidyl prolyl cis/trans isomerases. The complexes of cyclosporine/cyclophilin and FK506/FK506-binding protein bind to calcineurin and inhibit the phosphatase activity of the latter. 468 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

During the process of T lymphocyte activation, calcineurin forms part of a signaling pathway that is activated by a rise in intracellular calcium upon ligand binding to the T-cell receptor (see Section 13.3), and this ultimately leads to an activation of the transcription factors, named nuclear factor of activated T cells (NF- AT) (Figure 9.25). Members of the NF-AT family of transcription factors control the expression of a large number of proteins, including cytokines, ion channels, cell- surface proteins, and proteins involved in apoptosis. A subset of the NF-AT transcription factors (NF-ATc members) is found in the cytosol and undergoes nuclear translocation upon calcineurin activation, allowing the subsequent

Figure 9.25 Immunosuppressants and stimulates the transcription of corresponding calcineurin signaling cyclosporine A (CsA) and genes. Among the genes controlled by NF-AT FK506. Model of the function of calcineurin in is the gene for the cytokine interleukin-2 (IL-2). T lymphocytes. Antigenic peptides are Following secretion into the extracellular presented to the T lymphocytes by an antigen- space, the IL-2 so formed binds to IL-2 presenting cell (APC) within a cell---cell receptors (pink) of the same cell or cells of the interaction (see also Chapter 11). Antigen same type. A proliferation signal is created by binding activates the T-cell receptor (blue) that the activated IL-2 receptor, leading to a starts a signal chain leading to an increase in proliferation of the T lymphocytes. Complexes þ cytosolic Ca2 and the activation of of the immunosuppressants CsA or FK506 calcineurin. The activated calcineurin cleaves with their binding proteins cyclophilin and an inhibitory phosphate residue from the FK506-binding protein (FK506B), respectively, transcription factor NF-AT. This causes NF-AT inhibit calcineurin and disrupt the signal to be transported into the nucleus where it transmission to NF-AT. Questions 469 activation of target genes. The cytoplasmic form of NF-ATc is phosphorylated in its nuclear localization signal and requires dephosphorylation by phosphatase action þ in order to gain access to its cognate genes. At this point, Ca2 -regulation of calcineurin comes into play. þ The rise in Ca2 caused by ligand binding to T-cell receptors activates calcineurin’s phosphatase activity, which in turn dephosphorylates cytoplasmic NF- ATc proteins. The dephosphorylated NF-ATc enters the nucleus and binds to DNA in cooperation with other transcription factors, such as AP-1. In this way, many target genes in a wide diversity of tissues can be activated, allowing for the transcriptional regulation of a large number of genes by calcineurin. One of the target genes in lymphocytes is the gene for the cytokine interleukin (IL)-2, which is required to stimulate the proliferation of these cells. Transcription of IL-2 is inhibited as one of the consequences of calcineurin inhibition by cyclosporine/cyclophilin and FK506/FK506-binding protein and, as a result, the proliferation of lymphocytes is severely impeded. Following the discovery of the role of calcineurin in transcription regulation in T lymphocytes, a large number of calcineurin substrates other than NF-AT have more recently been discovered; these include, among many others, NO synthase, ion channels and adenylyl cyclase. þ In addition to Ca2 signals, calcineurin is also regulated and targeted by other cellular proteins, such as AKAP79. The inhibitory effect of calcineurin on the nuclear translocation of NF-ATc proteins may be counteracted by the action of central protein kinases that include PKA, glycogen synthase kinase 3, and the stress-activated kinases JNK and p38 (see Chapter 12).

Questions 9.1. Name the principal mechanisms by which protein phosphorylation participates in cellular signaling. Give at least one example of each mechanism. 9.2. Describe the main structural elements of protein kinases and their function in the protein kinase reaction. 9.3. Give examples of autoinhibition in protein kinases. What mechanisms are used to relieve autoinhibition? 9.4. What mechanisms may be used to couple protein kinases to their substrates? Give examples. 9.5. Name the major mechanisms used for control of protein kinase activity. Give examples. 9.6. Name the second messengers involved in protein kinase control. 9.7. What mechanisms are available for directing protein kinases to distinct subcellular sites? Give examples. 9.8. Describe the regulation of PKA by cAMP. What other regulatory mechanisms contribute to PKA control? 470 9 Ser/Thr-Specific Protein Kinases and Protein Phosphatases

9.9. What may be the advantage of using AKAPs as adaptors in PKA signaling? Name examples of signaling enzymes that may associate with AKAPs. 9.10. Explain the signaling function of the muscle-speciﬁc mAKAP. 9.11. What signals are used for activation of PI3K? Which enzyme counteracts the reaction of PI3K? How may this enzyme participate in tumor formation? 9.12. Name the major cellular processes that are under control of Akt kinase. Describe the principal function of Akt in these regulations. 9.13. Name the major structural elements of classical PKC enzymes and the function of these elements. 9.14. What signals are used for the control of PKC, and how are these signals generated? 9.15. Describe the characteristic structural and functional features of CamKII! 9.16. Explain the functional basis of the memory effect in CamKII activation. 9.17. Name examples of signaling events where protein phosphatases have an activating or inactivating effect on signaling. 9.18. Name the major mechanisms involved in the control of protein phosphatases. Give some examples. 9.19. Explain the role of calcineurin in immune suppression.

References

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9 Scott, J.D. and Pawson, T. (2009) Cell PubMed PMID: 21215369. Pubmed Central signaling in space and time: where proteins PMCID: 3104240. come together and when they’re apart. 18 Bayascas, J.R. (2010) PDK1: the major Science, 326 (5957), 1220–1224. transducer of PI 3-kinase actions. Curr. Top 10 Vanhaesebroeck, B., Stephens, L., and Microbiol. Immunol., 346,9–29. PubMed Hawkins, P. (2012) PI3K signalling: the PMID: 20563709. Epub 2010/06/22. eng. path to discovery and understanding. Nat. 19 Bononi, A., Agnoletto, C., DeMarchi, E., Rev. Mol. Cell. Biol., 13 (3), 195–203. Marchi, S., Patergnani, S., Bonora, M. et al. PubMed PMID: 22358332. Epub 2012/02/ (2011) Protein kinases and phosphatases in 24. eng. the control of cell fate. Enzyme. Res., 2011, 11 Chalhoub, N. and Baker, S.J. (2009) PTEN 329098. PubMed PMID: 21904669. Pubmed and the PI3-kinase pathway in cancer. Central PMCID: 3166778. Annu. Rev. Pathol., 4, 127–150. PubMed 20 Swulius, M.T. and Waxham, M.N. (2008) PMID: 18767981. Pubmed Central PMCID: Ca(2þ)/calmodulin-dependent protein 2710138. kinases. Cell. Mol. Life Sci., 65 (17), 12 Costa, C. and Hirsch, E. (2010) More than 2637–2657. PubMed PMID: 18463790. just kinases: the scaffolding function of 21 Lucchesi, W., Mizuno, K., and Giese, K.P. PI3K. Curr. Top Microbiol. Immunol., 346, (2011) Novel insights into CaMKII function 171–181. PubMed PMID: 20563705. Epub and regulation during memory formation. 2010/06/22. eng. Brain Res. Bull., 85 (1–2), 2–8. PubMed 13 Bozulic, L. and Hemmings, B.A. (2009) PMID: 21070840. PIKKing on PKB: regulation of PKB activity 22 Rellos, P., Pike, A.C., Niesen, F.H., Salah, by phosphorylation. Curr. Opin. Cell Biol., 21 E., Lee, W.H., vonDelft, F. et al. (2010) (2), 256–261. PubMed PMID: 19303758. Structure of the CaMKIIdelta/calmodulin Epub 2009/03/24. eng. complex reveals the molecular mechanism 14 Zoncu, R., Efeyan, A., and Sabatini, D.M. of CaMKII kinase activation. PLoS Biol., 8 (2011) mTOR: from growth signal (7), e1000426. PubMed PMID: 20668654. integration to cancer, diabetes and ageing. Pubmed Central PMCID: 2910593. Nat. Rev. Mol. Cell. Biol., 12 (1), 21–35. 23 Chao, L.H., Stratton, M.M., Lee, I.H., PubMed PMID: 21157483. Pubmed Central Rosenberg, O.S., Levitz, J., Mandell, D.J. PMCID: 3390257. et al. (2011) A mechanism for tunable 15 Salmena, L., Carracedo, A., and Pandolﬁ, autoinhibition in the structure of a human þ P.P. (2008) Tenets of PTEN tumor Ca2 /calmodulin-dependent kinase II suppression. Cell, 133 (3), 403–414. holoenzyme. Cell, 146 (5), 732–745. PubMed PMID: 18455982. PubMed PMID: 21884935. Pubmed Central 16 Singh, G. and Chan, A.M. (2011) Post- PMCID: 3184253. translational modiﬁcations of PTEN and 24 Shi, Y. (2009) Serine/threonine their potential therapeutic implications. phosphatases: mechanism through Curr. Cancer Drug Targets, 11 (5), 536–547. structure. Cell, 139 (3), 468–484. PubMed PubMed PMID: 21486223. Epub 2011/04/ PMID: 19879837. 14. eng. 25 Virshup, D.M. and Shenolikar, S. (2009) 17 Leonard, T.A., Rozycki, B., Saidi, L.F., From promiscuity to precision: protein Hummer, G., and Hurley, J.H. (2011) phosphatases get a makeover. Mol. Cell, Crystal structure and allosteric activation of 33 (5), 537–545. PubMed PMID: protein kinase C betaII. Cell, 144 (1), 55–66. 19285938.

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10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Summary One of the fundamental mechanisms by which multicellular organisms communicate is the binding of extracellular protein ligands to transmembrane receptors that use Tyr-phosphorylation as a tool for signal transduction on the cytoplasmic side. Coupling of an extracellular ligand binding to tyrosine phosphorylation in the interior of the cell can occur via two schemes, and involves two different types of receptor (Figure 10.1): — Transmembrane (TM) receptors with intrinsic tyrosine kinase (TK) activity (RTKs): Ligand binding to an extracellular domain of the receptor is coupled to the stimulation of TK activity localized on a cytoplasmic TK domain. The ligand-binding and TK domains are part of one and the same protein. Upon ligand binding, the TK activity of the receptor is activated. Consequently, tyrosine phosphorylation is initiated at the receptor itself and also on associated substrate proteins; these in turn trigger the biological response of the cell by switching on further chains of reaction. The response can reach as far as the cell nucleus, where transcription of particular genes is activated. It can also affect the reorganization of the cytoskeleton, cell–cell interactions, and reactions of intermediary metabolism. In particular, the RTKs regulate cell division activity, differentiation, and cell morphogenesis by this mechanism. Activation of RTKs is triggered, in particular, by signals that control cell growth and differentiation. Extracellular signals are often protein hormones, which – if they have a regulating influence on cell proliferation – are also classed as growth factors. In addition to the RTKs, there exists a large family of non-RTKs which are integral components of signaling cascades triggered by RTKs and other transmembrane receptors. — TM receptors with associated TK activity: A second type of TM receptor is associated, on the cytoplasmic side, with a TK that is activated when a ligand binds to the extracellular receptor domain. The TK and the receptor are not located on the same protein in this case. This type of receptor will be discussed in Chapter 13.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 474 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

(a) (b) L L

Regulatory domain

Tyr kinase- domain Tyrosine Kinase Signal Signal Effector proteins Effector proteins

Figure 10.1 Scheme of signal transmission by are also located on the cytosolic side; receptors with intrinsic and associated tyrosine (b) Receptors with associated tyrosine activity kinase activity. (a) Tyrosine kinase receptors pass the signal on to a tyrosine kinase that is possess a tyrosine kinase domain in the not an intrinsic part of the receptor, but is cytoplasmic region. Binding of a ligand L to the permanently or transiently associated with the extracellular domain of the receptor produces a cytoplasmic receptor domain. The receptor signal on the cytoplasmic side by activating the shown has been simplified as a dimer. tyrosine kinase. Regulatory sequence segments

10.1 Structure and Function of RTKs

Summary Receptors with intrinsic tyrosine kinase (TK) activity (RTKs) have a ligand- binding domain on the extracellular side, a TM portion composed of a single a-helical element, and a cytosolic part that harbors a conserved TK domain. Most RTKs are found as single polypeptide chains and are monomeric in the absence of ligand. Ligand binding to the RTK then triggers an association of the monomers to dimers or higher oligomers. One exception is the insulin receptor and its family members, which exist as preformed dimers comprising two extracellular a-chains and two membrane-spanning b-chains. In the absence of an extracellular stimulus, the kinase domain of RTK monomers or dimers exists in an inactive, autoinhibited state with no or very low basal activity. RTK activation occurs by ligand-induced conformational changes within the receptor oligomer that convert the TK domain from the inactive into the active state. The mechanisms of TK domain activation are diverse, 10.1 Structure and Function of RTKs 475

and include autophosphorylation at Tyr-residues within the TK domain and at segments outside of the TK domain. Autophosphorylation occurs “in trans” within the receptor oligomer. The P-Tyr formed at the receptor then serve as docking sites for downstream effector proteins that harbor P-Tyr- specific interaction domains, such as SH2, phosphotyrosine-binding (PTB) and C2 domains. In addition, downstream effectors can be Tyr-phosphorylated by the activated RTK to transmit the signal further.

10.1.1 General Structure and Classification

RTKsareintegralmembraneproteinsthathavealigand-bindingdomainonthe extracellular side and a TK domain on the cytosolic side (Figure 10.1). A common characteristic is the presence of just one a-helical element in the transmembrane (TM) portion. In the activated form, all receptor TKs exist either as a dimer or – occasionally – as a higher oligomer. On the cytoplasmic side, in addition to the conserved TK domain, there are also further regulatory sequence portions at which autophosphorylation and phosphorylation/dephosphorylation by other protein kinases and by protein phosphatases can take place. Furthermore, other signaling proteins with scaffolding or enzymatic function can associate with the cytoplasmic part.

& Domains of RTKs: — Extracellular domain: Ligand binding Glycosylation — Transmembrane domain: Single a-helix Dimerizes upon ligand binding — Cytosolic domain: Kinase activity Binding of effectors Regulatory modifications.

The human genome encodes a total of 58 genes for RTKs, the most important members of which are listed in Table 10.1, together with the corresponding natural ligands. The family of mammalian RTKs can be divided into different subfamilies, which are named according to their naturally occurring ligands. The subfamilies are classiﬁed according to the structure of the extracellular ligand-binding domains, in which different sequence portions can be differentiated (Figure 10.2). In the extracellular domain, for example, there are Cys-rich sequences that occur as multiple repeats and sequences with an immunoglobulin-like structure. Most receptors are found as single polypeptide chains and are monomeric in the absence of ligand. One exception is the insulin receptor and its family members, which exist as preformed 476 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Table 10.1 Selected mammalian growth factors and their receptors.

Growth factor Characteristics Receptors

Platelet derived growth Dimers, A (17 kDa)- and B 2 types of receptor tyrosine factor, PDGF, types AA, (16 kDa)-chains, B-chain is a kinases, PDGFRa (170 kDa), AB and BB product of c-sis proto-oncogene PDGFRb (180 kDa) Epidermal growth factor, ca 6 kDa, EGF and TGF-a are up Receptor tyrosine kinase, EGF Transforming to 40% identical EGFR is a product of the growth factor-a, TGF-a c-ergB protooncogene Transforming growth Homodimers of 25 kDa TGFb receptor I und II, factor-b, TGF-b1, -b2, -b3 contains Ser/Thr-speciﬁc protein kinase activity Insulin-like growth factor, 7 kDa, related to proinsulin Receptor tyrosine kinase, IGF-1 and IGF-2 IGFR Fibroblast Growth Factor, related proteins of 16-32 kDa Receptor tyrosine kinase FGF-1, FGF-2, FGF-3, FGF-4 Granulocyte colony 24 kDa 150 kDa, G-CSFR, receptor stimulating factor, G-CSF with associated tyrosine kinase Granulocyte/makrophage 14 kDa 51 kDa, GM-CSFR, receptor colony stimulating factor, with associated tyrosine GM-CSF kinase Interleukins (IL): IL-1R---IL-7R, IL-9R, IL-10R, IL-1---IL-7, IL-9, IL-12, IL-12R, IL-15R, IL-15 IL-20R, IL-22R, IL-10, IL-19, IL-20, IL-22, Receptors with associated IL-24, IL-26 tyrosine kinase Interleukin 8, IL-8 IL-8R, G-protein-coupled receptor Erythropoietin 34 kDa EpoR, receptor with associated tyrosine kinase Tumor necrosis factor, 26 kDa TNFR, receptor with TNF associated tyrosine kinase Interferon a, b, c INFRa, INFRb, INFRc, receptors with associated tyrosine kinase

dimers comprising two extracellular a-chains and two membrane-spanning b-chains. a b ﬁ This 2 2heterotetramer is stabilized by various disul de linkages. In the absence of an extracellular stimulus, the kinase domain of RTK monomers or dimers exists in an inactive state, with no or very low basal activity. In the ligand- bound receptor, self-association of the extracellular domain triggers a conformational change in the dimer that activates their kinase domains. Thereby, two crucial events are triggered at the cytosolic side: 10.1 Structure and Function of RTKs 477

Figure 10.2 Domain organization for a variety receptor; InsR, insulin receptor; IGF1R, insulin- of receptor tyrosine kinases. The extracellular like growth factor 1 receptor; CSF1R, colony- portion of the receptors is at the top, and the stimulating factor 1 receptor; KDR and Flt: cytoplasmic portion is at the bottom. The receptors for the vascular endothelial growth lengths of the receptors is only approximately factor; FGFR, fibroblast growth factor receptor; to scale. Domains: L, homology domain; Ig, Trk, receptor for neurotrophins, such as nerve immunoglobulin-like domain; SAM, sterile growth factor; MuSK, muscle-specific receptor alpha motif; Cadherin, cell---cell adhesion tyrosine kinase; Met, receptor for hepatocyte domain. Explanations for some receptors: growth factor; Eph, receptor for ephrin ligands; PDGFR, platelet-derived growth factor DDR, discoidin domain receptor. receptor; EGFR, epidermal growth factor

First, a Tyr-autophosphorylation of the receptor takes place in trans, that is, between the partner kinase domains of the dimeric receptor, leading to kinase activation (Figure 10.3). Subsequently, downstream signaling partners associate with P-Tyr residues of the activated receptor. Often, the downstream effectors are Tyr-phosphorylated by the activated RTK for further signal transduction.

10.1.2 Ligand Binding and Receptor Dimerization

Ligand-induced dimerization of RTKs is the ﬁrst step in transducing growth factor signals into the interior of the cell. There are two scenarios that an extracellular ligand may face upon encountering the receptor (Figure 10.4): 478 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.3 Ligand-induced The P-Tyr residues created serve as attachment autophosphorylation and substrate points for downstream effectors carrying phosphorylation of receptor tyrosine kinases. specific phosphotyrosine-binding domains Upper left: In the absence of a ligand, the RTK (SH2 in the figure or PTP domains; is in an inactive, autoinhibited state; Upper Section 10.1.5). The bound effectors are thus right: Ligand (L) binding induces kinase activated for further signaling. activation and autophosphorylation; Lower:

Most RTKs exist as a monomer, and ligand binding to the extracellular portions of the receptor induces the noncovalent dimerization of monomeric receptors leading to the formation of homodimers or heterodimers. A subset of RTKs exists as a dimer in solution, and ligand binding induces a conformational change in the preassembled dimer that leads to activation of the kinase activity. 10.1 Structure and Function of RTKs 479

Figure 10.4 Mechanism of activation of RTKs a receptor which exists in a monomeric form by ligand binding. The activation of RTKs is without the ligand; (b) A dimeric receptor is based on a ligand-induced oligomerization activated via an allosteric mechanism by ligand and/or conformational change of the receptor. binding. In the absence of the ligand, the two An example is shown of a dimeric receptor; kinase active sites are not close enough for however, activation can also occur in a higher mutual activation by phosphorylation (trans- receptor oligomer. (a) A bivalent ligand activation). (monomer or dimer) induces a dimerization of

& Ligand binding induces: — Dimerization and/or allosteric changes and subsequently: Autophosphorylation Substrate phosphorylation Substrate docking.

In ligand-induced dimerization of RTKs, the ligand itself is a dimer and interacts simultaneously with two receptor molecules and crosslinks them into a dimeric 480 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.5 Activation of RTK dimers. In upon receptor activation, interact across the general, RTKs associate into dimers when dimer interface. Thus, KIT combines ligand- ligand (red) binds to their extracellular regions. mediated and receptor-mediated dimerization The bound ligand, which can form all, a modes; (c) Two fibroblast growth factor portion, or none of the dimer interface, receptor (FGFR) molecules contact one activates the receptors by stabilizing a specific another through the Ig-like domain D2, and relationship between two individual receptor the accessory molecule heparin or heparin molecules. (a) A nerve growth factor dimer sulfate proteoglycans (white sticks) also (red) crosslinks two TrkA molecules without contacts this domain. In addition, each FGF any direct contact between the two receptors; molecule (red) contacts Ig-like domains D2 (b) A stem cell factor dimer (red) also and D3 of both FGFR molecules. Lemmon crosslinks two KIT molecules. In addition, two 2010 [2], figure 2. Reproduced with permission Ig-like domains (D4 and D5), which reorient of Elsevier.

complex. This “ligand-mediated” mode of receptor dimerization is supported by crystal structures of several fragments of the ligand-binding domains from RTKs bound to their relevant ligands. The structural studies suggest the following mechanisms for ligand-induced dimerization, as illustrated in Figure 10.5:

Receptor dimerization is entirely ligand-mediated: The interaction surface of the dimeric receptor is entirely provided by the bound growth factor, and the extracellular domains of the receptor do not contact one another (Figure 10.5a). This mode of dimerization has been demonstrated for binding of nerve growth factor (NGF) to the receptor TrkA. Dimerization involves both ligand-mediated and receptor-mediated contacts: Examples of this mechanism of dimerization include the binding of the bivalent Kit ligand to the Kit receptor and the binding of ﬁbroblast growth factor (FGF) to FGF receptor (FGFR; see Figure 10.5b). In the latter case, an accessory molecule, namely heparin, is also involved in complex formation. The additional heparin ligand has been shown to bridge two receptor molecules in the dimer and the 10.1 Structure and Function of RTKs 481

adjoining FGF ligands, allowing for a stable dimerization of the ligand-bound receptor. Receptor–ligand, receptor–heparin, ligand–heparin and receptor– receptor interactions all cooperate to stabilize the FGFR dimer. In the absence of ligand, FGFR monomers exist in a “closed” or autoinhibited conﬁguration. This autoinhibited state is thought to be in equilibrium with an “open” conﬁguration, that is poised to interact with FGF and heparin, allowing FGFR activation. Activating ligands make no direct contribution to the dimerization interface: Receptors in the epidermal growth factor (EGF) receptor (EGFR or ErbB) family represent this mode of activation mechanism. The dimer interface of EGFR is composed entirely of receptor–receptor contacts that are made possible by a ligand-induced structural rearrangement of the extracellular domains, as illustrated in the model of Figure 10.5c.

For further details of these receptors, see Ref. [1]. Such receptors also display a dramatic form of intramolecular autoinhibition (see Section 10.1.3).

10.1.2.1 Receptor Heterodimerization One aspect of ligand-induced receptor oligomerization of regulatory importance is the possibility of forming heterodimers. Protein families composed of closely related members can be identiﬁed for a number of growth factors and corresponding receptors, and heterologous dimerization is observed within the different members of the receptor family. A certain growth factor can thus bind to and activate different dimeric combinations of the members of a receptor family. Possibilities for the heterodimerization of receptors, using the platelet-derived growth factor (PDGF) receptor as an example, are shown in Figure 10.6. The heterodimerization of receptor molecules is a mechanism that can increase and modulate the diversity and regulation of signal transduction pathways. As the various members of a receptor family differ in the exact structure of their autophosphorylation sites and other regulatory sequences, it is assumed that activity and regulation are different for the various combinations of receptor subtypes. The tissue-speciﬁc expression of receptor subtypes enables the organism to process growth factor signals in a differential fashion.

10.1.3 Structure and Activation of the TK Domain

In the nonsignaling state, most RTKs possess a low basal kinase activity that increases substantially upon ligand binding, and this activation is accompanied by the phosphorylation of tyrosine residues within the kinase domain. The crucial question is how the ligand-induced dimerization of the extracellular regions of RTKs leads to an activation of the intracellular TK domain. In general, the structures of the kinase domain of protein kinases in its active state are all very similar (see Section 9.2.3), and this is also true for the kinase domain of RTKs. However, the inactive states differ substantially between different RTKs, and this variation reﬂects the diversity in their regulatory mechanisms. Each RTK kinase 482 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.6 Heterodimerization of PDGF growth factor, composed of chains A and/or B. receptors. (a) There are a and b subtypes of The protein may exist as a homodimer (AA, platelet-derived growth factor receptor (PDGF- BB) or a heterodimer (AB). The AA homodimer R); these are induced by ligand binding to form of PDGF binds to the aa dimer of PDGF-R, AB homodimers and heterodimers; (b)Platelet- binds to the aa and ab types, and BB binds all derived growth factor (PDGF) is a dimeric three combinations.

domain is uniquely cis-autoinhibited (i.e., between monomers of the receptor dimer) by a set of intramolecular interactions speciﬁc for its receptor. The release of cis-autoinhibition, following ligand-induced receptor dimerization, is the key event that triggers RTK activation (for a review, see Ref. [2]. This includes a reorientation of the kinase domains to allow activation loop phosphorylation and/or phosphorylation in the structural parts outside of the kinase domain, namely in those regions immediately following the transmembrane segment, namely the juxtamembrane domain and the C-terminal regulatory region. In the following, three modes of autoinhibition and one mechanism of allosteric activation of RTKs will be discussed.

& Kinase domain of RTKs: — Classical kinase fold — Activation requires phosphorylation of Tyr on activation loop by autophosphorylation or by other Tyr kinases 10.1 Structure and Function of RTKs 483

Autophosphorylation of RTKs: — Occurs in trans and produces Tyr-P within kinase domain and neighboring sequences — Activates kinase — Creates docking sites for effectors.

10.1.3.1 Autoinhibition by the Activation Loop: The Insulin Receptor The activation loop is the most critical structural element in protein kinase regulation (Section 9.2.1). In most protein kinases (including RTKs), an inactive state of the kinase domain is maintained by an unfavorable orientation of the activation loop, and activation of kinase activity has been linked to autophosphorylation of the activation loop in all RTKs studied so far, except for some members of the EGF receptor family. It is generally assumed that autophosphorylation takes place by a trans- mechanism, triggered by ligand binding and dimerization. Accordingly, two neighboring TK domains in the receptor dimer perform a mutual phosphorylation (Figure 10.3), leading to activation of the kinase activity. Activation of the insulin receptor represents an excellent example of activation by a b loop phosphorylation. The insulin receptor is a heterotetrameric RTK of an 2 2 structure (Figure 10.2) in which the a- subunit is completely extracellular and is bound to the b-chain via disulfide bridges. The b-chain has a transmembrane portion, and an intracellular TK domain. On binding insulin at the extracellular side, the TK activity of the b-chain is activated, and the autophosphorylation of a total of seven Tyr residues takes place in the cytoplasmic domain. Essential for kinase activation is the phosphorylation of a key tyrosine (Y1162) in the activation loop of the insulin receptor. Y1162 projects into the active site (see Figure 10.7), and this interaction stabilizes an activation loop configuration that occludes the active site, blocking access to both ATP and protein substrates. Thus, the TK domain of the insulin receptor is autoinhibited in cis by its own activation loop. When insulin activates the receptor, Y1162 in one TK domain becomes phosphorylated by its partner (together with two additional tyrosines), and this trans-phosphorylation disrupts the cis-autoinhibitory interactions within the dimer. As a consequence, the activation loop is free to adopt the “active” configuration of the kinase domain seen in all other activated TK domains. Upon this dramatic reorientation, the steric hindrance to substrate binding is removed and the residues involved in substrate binding and catalysis can now be correctly positioned. The alternative conformation of the activation segment is fixed by ionic interactions involving the P-tyrosine residues. A similar activation mechanism has been proposed for the FGF receptor (FGFR).

& Autoinhibition of RTKs is mediated via: — Activation loop orientation — Juxtamembrane inhibition — C-terminal sequences — Allosteric mechanisms. 484 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.7 Comparison of the activation loop pink. The rest of the protein in each case is conformation in unphosphorylated insulin represented by a semitransparent molecular receptor kinase (IRK) and tris-phosphorylated surface. Also shown is the ATP analog AMP- IRK (IRK3P). The figure illustrates the dramatic PNP, which is partially masked by the amino- repositioning of the activation loop upon terminal lobe of IRK3P. Carbon atoms are autophosphorylation. Note, that in IRK, shown in white, nitrogen atoms in blue, oxygen Tyr1162 is positioned in the active site atoms in red, phosphorus atoms in yellow, and competing with protein substrates. The magnesium ions in purple. Hydrogen bonds activation loop is shown in green, the catalytic between the substrate tyrosine Y(P) and loop in orange, and the peptide substrate in Asp1132 (IRK3P) are indicated by black lines.

10.1.3.2 Juxtamembrane Autoinhibition In addition to autoinhibition by the activation loop, many RTKs are cis- autoinhibited by elements outside the TK domain itself. In each case, sequences in the juxtamembrane region make extensive contacts with several parts of the TK domain (including the activation loop) and stabilize an autoinhibited conformation. The relief of autoinhibition involves the phosphorylation of key tyrosines in the juxtamembrane region. Ligand-induced receptor dimerization promotes transphosphorylation of these tyrosines, which disrupts the cis-autoinhibitory interactions and promotes receptor activation.

10.1.3.3 Autoinhibition by C-Terminal Sequences Another mechanism of autoinhibition has been described for the Tie2 receptor. The autoinhibited state of this receptor is thought to be caused, at least in part, by a region in the C-terminal tail that docks to the active site blocking substrate access. The C-terminal tail of Tie2 contains autophosphorylation sites, and it is assumed that ligand-induced autophosphorylation disrupts the inhibitory interactions leading to receptor activation. 10.1 Structure and Function of RTKs 485

10.1.3.4 Allosteric Mechanism of Activation Some RTKs, such as members of the EGFR/ErbB family (EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), do not require trans-phosphorylation for activation. For EGFR, structural studies have proposed an allosteric mechanism of activation and, in the absence of ligand, EGFR monomers exist in an inactive conformation due to cis-autoinhibitory contacts in the kinase domain. Ligand-induced dimerization then converts the inactive conformation into an active conformation. In the active state, the kinase domain forms an asymmetric dimer (Figure 10.8) in which the C-lobe of one kinase domain, called the “Activator,” makes intimate contacts with the N-lobe of the second kinase domain, called the “Receiver.” These contacts induce conformational changes in the N-lobe of the Receiver kinase that disrupt cis-autoinhibitory interactions seen in the inactive monomer. As a result, the Receiver kinase can adopt the characteristic active conﬁguration without activation loop phosphorylation. The reaction is analogous to the activation of cyclin-dependent kinases (CDKs) by cyclins (see Chapter 15), where the CDK adopts a similar inactive conformation until binding of the cyclin induces the transition into the active state.

10.1.4 RTK Activation and Downstream Signaling

The first and primary substrates that RTKs phosphorylate are the receptors themselves. This autophosphorylation occurs in trans, and the autophosphorylation sites are phosphorylated in a precise order with at least two major phases of reactions. A first phase of autophosphorylation primarily serves to enhance the catalytic activity of the kinase once the receptor binds its activating ligand. Autophosphorylation sites in the kinase domain and adjacent regions (see Section 10.1.3) are used to relieve the autoinhibited state in most RTKs, with EGFR and Ret as exceptions. A second phase of autophosphorylation requires prior (first- phase) activation of the kinase and creates the P-Tyr-based binding sites that recruit cytoplasmic signaling molecules containing binding sites for P-tyrosines. In this way, downstream signaling molecules are recruited to the receptor and activated in response to growth factor stimulation.

10.1.4.1 Nucleation of Signaling Complexes The ﬁrst response to the autophosphorylation of RTKs is the recruitment and activation of diverse effectors, that is, signaling molecules (Figure 10.9) that carry enzymatic activity or function as adaptors for the recruitment of further signaling proteins. As illustrated in Figure 10.10, the proteins involved in downstream signaling are of a multidomain architecture. Recruitment to the activated receptor is mediated by binding of their SH2 or PTB domains to the P-tyrosines on the receptor. In many cases, the effector proteins are substrates for Tyr-phosphorylation catalyzed by the TK activity of the receptor, and this phosphorylation activates the effector for further signal transmission. The colocalization of the effectors and the kinase via SH2/PTB-P-tyrosine interaction provides for a high efﬁciency and 486 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.8 The mechanism of EGFR kinase and correct positioning for catalysis of the activation. Shown are the N-lobes and C-lobes activation loop. In the absence of such an of the EGFR kinase domains, the b-sheet in the interaction (kinase colored purple), the N-lobe containing Lys721 (orange; K), a-helix activation loop is stabilized in a Src/CDK-like C in the N-lobe containing Glu738 (green; E), inactive state in which a short a-helix in the and the activation loop in the C-lobe activation loop (containing Leu834) and containing Leu834 (gray; L). The C-lobe of one a-helix C are stabilized in an inactive kinase domain (purple) activates the second configuration. The two kinase domains are kinase domain (cyan) by interacting with presumed to reverse roles in a dynamic a-helix C, which facilitates formation of the fashion. Lys721---Glu738 salt bridge (red dashed line) 10.1 Structure and Function of RTKs 487

Figure 10.9 Functions of autophosphorylation (SH2 or PTB domains) at phosphotyrosine of receptor tyrosine kinases (RTKs). residues of the activated receptor. A critical Autophosphorylation of RTKs takes place in factor for further signal transduction is the trans, that is, between neighboring protomers membrane association of the effector proteins of the receptor. The catalytic domain of the that enter into binding with the activated receptor is shown as a shaded segment. As a receptor. Details of the effector proteins can consequence of autophosphorylation, the be found as follows: phospholipase Cc intrinsic tyrosine kinase activity of the receptor (Section 7.7.2); Src kinase (Section 10.3.2); is stimulated. Effector proteins can also bind to p120 GAP (Section 11.2); Grb2, Shc, IRS the activated receptor. Binding takes place with (Section 10.2.1.2); PI3-kinase (Section 9.4); specific phosphotyrosine-binding domains Syp tyrosine phosphatase (Section 10.4).

specificity of phosphorylation, and this is often a first step towards the assembly of larger signaling complexes in the vicinity of the activated receptor. Most activated receptors contain multiple P-tyrosines (Figure 10.11) and involve numerous effectors. These are often multivalent and assemble a variety of further downstream signaling proteins. By these mechanisms, activated RTKs can recruit and influencealargenumberofdifferentsignaling molecules within a complex signaling network where an activated RTK (see Figure 3.10, Section 3.3, and Figure 10.18) transmits information from the exterior to the interior of the cell.

& Coupling of effectors to RTKs:

Tyr-P of RTK functions as docking site for — SH2 domain — PTB domains — C2 domains 488 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.10 The modular nature of signaling illustrated. For an explanation of the protein proteins. Representative members from modules, see the text. C1, C2, cysteine-rich various SH2 domain families and the domains; PI3Kp85, p85 subunit of PI3 kinase. positional organization of these domains are 10.1 Structure and Function of RTKs 489

Figure 10.11 Phosphotyrosine residues in the domain. The phosphotyrosine residues are PDGF receptor and specificity of binding of found in different sequence environments and SH2-containing signal proteins. The figure are recognized by the SH2 domains of the illustrates the diversity of the different effector assigned effector proteins. The filled rectangles proteins that can interact with an activated indicate the two-part tyrosine kinase domain of receptor. The tyrosine residues of platelet- PDGF-R. Src, members of the Src tyrosine derived growth factor receptor (PDGF-R), for kinase family; Sh2, Grb2, Nck, adapter which autophosphorylation has been proteins; PI3 kinase, phosphatidyl inositol-3 demonstrated, are designated according to kinase; GAP, GTPase-activating protein; PTP- their position in the receptor sequence. 1D, protein tyrosine phosphatase 1D; PL-Cc, PDGF-R has at least nine different tyrosine phospholipase C-c. phosphorylation sites in the cytoplasmic 490 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

of effectors such as: — PI3 kinase — PLC-c — Src kinase — Adaptors Grb2, Shc — Phosphatase SHP2.

10.1.5 Recruitment of Effectors via Interaction Domains

A key step in signaling by RTKs is the binding of effector proteins or adapter proteins to P-tyrosines on the activated receptor. At present, three interaction domains are known – the SH2, PTB and C2 domains – that recognize P-tyrosine residues in a sequence context-dependent manner [3]. As determined from binding studies with Tyr-phosphorylated peptides, selection between different Tyr-phosphates is mostly affected by sequences of one to six amino acids located C- and/or N-terminal to the P-tyrosine residue. The structures of the three domains bound to P-tyrosine-containing substrates are shown in Figure 10.12. Often, multiple P-tyrosine residues with different sequence contexts are found in signaling proteins. An example is the b-subtype of the PDGF receptor on which several tyrosine phosphorylation sites have been identified allowing, at least in theory, the docking of at least eight different effector molecules (Figure 10.11). The same receptor may convey signals to very different signaling pathways, as shown by this example. Which pathway is used will depend on the availability and activity of the different effector proteins, a situation that is regulated in a cell-specific and tissue-specific manner. Typically, the downstream effectors of RTKs are composed of multiple interaction domains and are subject to multiple posttranslational modifications (PTMs). In

Figure 10.12 P-Tyr binding by (a) SH2, PTB domain from the adapter protein Shc (see (b) PTB and (c) C2 domains. Shown are P-Tyr Section 8.5) and the C2 domain from PKCd. (green) containing phosphopeptides (white) in Residues involved in the recognition of P-Tyr complex with the SH2 domain from v-Src; the are shown in blue. 10.1 Structure and Function of RTKs 491

Figure 10.13 Targets of interaction modules involved in RTK signaling. For an explanation, see the text and Section 2.4.4. addition to the P-Tyr binding domains, effectors may harbor binding domains for membrane attachment and for binding to PTMs on further protein partners (Figure 10.13) which allows for the formation of large signaling complexes at the activated receptor. Furthermore, the presence of multiple interaction domains serves to integrate different signals on the RTK–effector complex (see also Figure 10.17). These signals may control signal transmission by, for example, dampening or enhancing any further signaling.

10.1.5.1 SH2 Domains The SH2 domains were first discovered as a sequence motif showing homology with a sequence of the Src tyrosine kinase (Section 9.3): hence the name SH, derived from Src homolog. More than 100 different SH2 domains have since been identified with different requirements for the sequences surrounding the P-tyrosine residue. It has long been thought that the primary sequence surrounding the P-tyrosine is the major determinant of specificity in SH2 domain interactions. Accordingly, most studies on SH2 domain interactions have used short peptides containing P-tyrosines as binding substrates, and the data obtained this way were used to categorize the specificity of P-Tyr binding by a SH2 or PTP domain. 492 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

However, the conclusions drawn from this approach have more recently been challenged by the structural investigations of an N-terminal SH2 domain from PLC-c bound to the Tyr-phosphorylated TK domain of FGFR [4]. The results of these studies showed that SH2 domains may use two sites for high-afﬁnity binding to the intracellular domain of the FGF receptor: one site comprises the P-tyrosine and its neighboring sequence, while the other site uses extensive contacts to key regions of the C-lobe of the kinase domain. This secondary binding site is crucial for the activation of PLC-c from which the SH2 domain was derived. Consequently, short synthetic peptides may not serve as good model substrates for SH2 binding, and this may be true also for other interaction domains such as SH3 domains. The great variability of SH2 domains is emphasized by the observation that two SH2 domains occur in many signal proteins, and these mostly have different substrate-binding preferences. P-tyrosine residues as the targets of SH2 domains are found in a variety of signaling proteins. In addition to the RTKs and cytokine receptors, T-cell receptors and non-RTKs utilize P-tyrosine-SH2 interactions for signal transmission.

& SH2 domains: — Recognize P-Tyr within N-terminal sequence context — Many variants are known — Can engage in intramolecular and intermolecular binding of P-Tyr — Transmit signals by: Allosteric activation Membrane localization Inducing Tyr-phosphorylation.

The activation of signal proteins by P-tyrosine-SH2 interactions can be achieved in different ways, which are discussed below:

Access to membrane-localized substrates: Via binding of an SH2-containing signal protein to an activated RTK, the signal protein is brought to the membrane and into the vicinity of the corresponding target protein or substrate. Examples are PLC-c and PI3-kinase, which have substrates in the phospholipid membrane. Activation by tyrosine phosphorylation: Many SH2-containing signal proteins are brought, via interaction of their SH2 group with P-tyrosine residues, into the neighborhood of the catalytic center of the TK and are themselves substrates for tyrosine phosphorylation. By this mechanism, new attachment sites can be generated for other SH2-containing signal proteins. In this way, several components of a signaling pathway can be sequentially linked. Relief of autoinhibition: Several cases have been described in which the binding of an SH2-containing enzyme to an activated RTK leads to an increased catalytic activity of the enzyme. Often, SH2 domains mediate autoinhibition by binding to the active site of the signaling enzyme. One such example is PI3-kinase, which exists in an autoinhibited state due to binding of the SH2 domain on the 10.1 Structure and Function of RTKs 493

regulatory subunit p85 to the active site on the catalytic p110 subunit. Binding of the SH2 domain to a tyrosine-phosphorylated PDGF receptor releases the autoinhibition and leads to a stimulation of the PI3-kinase activity. The intramolecular binding of P-tyrosine residues by SH2 domains is another mechanism for regulating the activity of signaling enzymes. A prominent example is the non-RTK Src that is controlled allosterically by SH2-P-tyrosine interactions (Section 9.3.2). Another example is PLC-c, which is activated by RTKs such as PDGF via phosphorylation on tyrosine residues. This activation is based on the intramolecular binding of the P-tyrosine residue to one of the SH2 groups of PLC-c, which relieves the enzyme from an autoinhibited state.

10.1.5.2 P-Tyrosine-Binding (PTB) Domain PTB domains are found almost exclusively in proteins that act at membranes and possess a docking or adapter function by recruiting various signaling proteins to the vicinity of an activated receptor. A clear functional dichotomy exists within the family of PTB domains that is based on the requirement of a P-tyrosine in the peptide ligand for binding. One group of PTB domains is P-tyrosine-dependent, while the other group does not require P-tyrosine for binding to the target protein.

& PTP domains: — Two subgroups: P-Tyr-dependent P-Tyr-independent — P-Tyr-dependent PTPs: Recognize P-Tyr within C-terminal sequence context Occur mostly on adapter proteins May also bind phosphoinositides.

Well-studied examples of P-tyrosine-dependent PTBs are found on the adapter proteins Shc and the insulin receptor substrate IRS1 (see Section 10.5). Both adaptors bind via their PTB domain to P-tyrosine residues of the activated insulin receptor recognizing P-tyrosine residues in context with sequence sections toward the N terminus. Interestingly, most P-Tyr-dependent PTB domains also show high- afﬁnity binding to phosphoinositides, though the functional relevance of this dual binding speciﬁcity is unclear. P-tyrosine-independent PTB domains are also known that have been found on adapter proteins that bind to physiologically important receptors, such as the low-density lipoprotein (LDL) receptor and on adaptors associated with the Alzheimer precursor protein (APP).

10.1.5.3 C2 Domains The C2 domains are long-known modules of approximately 120 residues that bind þ phospholipids in a Ca2 -dependent manner. However, one subtype of the C2 domains that was ﬁrst discovered for protein kinase Cd mediates protein–protein interactions by binding to P-tyrosine residues (see Section 7.5). 494 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

10.2 Downstream Effector Proteins of RTKs

Summary Ligand-induced activation of the TK activity serves to recruit a multitude of effector proteins into RTK signaling. Two ways of recruiting the effectors have been identified: (i) An SH2 or PTB domain of the effector binds to P-tyrosines on the RTK, linking the effector directly to the RTK; and (ii) adapter or scaffolding protein serve to recruit the effector to the RTK for further signal transmission. These adaptors bind via SH2 or PTB domains to the activated RTK; the adapter is then Tyr-phosphorylated by the RTK, after which its P-Tyr residues serve to guide SH2-domain-containing effectors to the receptor. Important adaptors in RTK signaling are the IRS, FRS, Grb2 and Gab proteins, which interact with downstream effectors to organize larger signaling complexes at the activated receptor. In this way, RTKs can become part of large signaling networks. Examples of downstream effectors of RTKs include PI3 kinase, PLC-c, small GTPases such as Ras protein, non-RTKs such as Src kinase, and protein-Tyr phosphatases. Signaling by RTKs is tightly regulated via positive and negative feedbacks, inhibitor proteins, ubiquitination, endocytosis, and trafficking.

10.2.1 Adapter Proteins in RTK Signaling

The adapter proteins function as essential signaling intermediates downstream of activated RTKs (and other cell-surface receptors) by assembling larger signaling complexes at the activated receptor. Adapter proteins of RTK signaling typically contain a membrane targeting site at their amino (N-) terminus, SH2 or PTB domains for association with Tyr-P on the activated receptor, and an array of tyrosine phosphorylation sites that serve as binding sites for a distinct repertoire of downstream signaling proteins. In this way, the phosphorylation of adapter proteins is functionally equivalent to second- phase RTK autophosphorylation. Membrane association and SH2/PTB- mediated binding to the receptor are essential steps for Tyr-phosphorylation of the adapter and recruitment of further downstream signaling proteins. Although a number of adapter proteins (such as Gab1) are recruited by multiple RTKs, others are restricted to particular subsets of receptors. For example, the two members of the FRS2 family, FRS2a and FRS2b,mediate signaling primarily by FGF and NGF receptors, respectively [5]. Importantly, the signaling function of adaptors may be controlled by other PTMs such as Ser/Thr phosphorylation, acetylation and ubiquitination, and these modiﬁcations serve to feed multiple signals into the RTK signaling network. Overall, the adapter proteins are major branching points that mediate diversiﬁca- tion and feedback regulation of RTK signaling. 10.2 Downstream Effector Proteins of RTKs 495

Some important adaptors in RTK signaling will be highlighted in the following sections.

10.2.1.1 Insulin Receptor Substrate (IRS) The members of the IRS family (IRS-1, -2, -4 in humans) play crucial roles in mediating signaling by the insulin and IGF1-receptors, which rely entirely on these adapter proteins for the recruitment of downstream signaling molecules. It is a major task of the IRS proteins to shape insulin signaling in response to multiple microenvironmental stimuli. These proteins are essential elements in the insulin- mediated control of growth, development and for the normal homeostasis of glucose, fat and protein metabolism [6]. Insulin receptor and IGFR are not the only upstream effectors that use IRS as essential signaling intermediate; other cell- surface receptors such as growth hormone receptor, vascular endothelial growth factor receptor (VEGFR) and integrins (Chapter 13) have also been shown to signal through IRS proteins.

10.2.1.2 Tyr-Phosphorylation: Effector Recruitment The domain structure of IRS-1 and its link to central signaling paths are shown in Figure 10.14. An outstanding feature of IRS protein structure is the presence of a set of Tyr-phosphorylation sites at the C-terminal part that act as on/off switches to recruit the downstream effectors. There are approximately 20 potential Tyr- phosphorylation sites on IRS-1 and IRS-2, and these serve to bind to SH2 domains of the downstream effectors. On binding of insulin to the insulin receptor (or the binding of IGF to IGFR), the tyrosine kinase activity of the receptor is stimulated and the IRS protein binds via its PTB domain to autophosphorylated tyrosine residues of the receptor. Subsequently the IRS protein is phosphorylated by the activated insulin receptor at multiple Tyr residues, which then serve as attachment points for sequential effector molecules. Effectors that have been characterized to bind to the IRS proteins include PI3K, the nonRTKs Fyn and Nck, protein tyrosine phosphatase SHP-2, and the adaptors Grb2 and Crk. As illustrated in Figure 10.14, these multiple interactions put the IRS molecules on center stage in insulin action, and also link insulin signaling to the major signaling paths of the cell. PI3K is one of the best-characterized downstream effectors of IRS proteins that binds to Tyr-phosphorylated IRS via the SH2 domain of the regulatory p85 subunit (Section 9.4.1). By recruiting and activating PI3-kinase, a signal in the direction of the Akt kinase pathway is generated, and many of the biological inﬂuences of insulin have been linked to activation of this pathway.

& IRS proteins: — Function as adaptors of insulin receptors — Couple to activated InsR via a PTB domain — Become Tyr-phosphorylated by activated InsR — Harbor multiple Tyr-P sites for recruitment of effectors — Become multiply phosphorylated on Ser/Thr sites to dampen signaling. 496 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.14 IRS signaling. (a) Schematic of termini. Once bound, they are phosphorylated the IRS protein family. Interaction domains of on tyrosine residues in their C termini. The the IRS proteins are indicated. PH, pleckstrin phosphorylation of tyrosine residues (pY) homology domain; PTB, phosphotyrosine- creates docking sites for the recruitment of binding domain; PI3 K, region containing downstream signaling effectors. Subsequently, multiple PI3 K binding motifs; Grb-2, Grb-2 signaling cascades are activated that can binding site; SHP-2, SHP-2 binding site; regulate gene expression, protein synthesis, (b) Signaling via the IRS proteins. The IRS glycolysis, cell proliferation, survival, and proteins are recruited to activated cell-surface motility/invasion. After Ref. [6]. receptors via PH/PTB domains in their N

10.2.1.3 Ser/Thr Phosphorylation: Signal Dampening Another distinct feature of IRS proteins is the presence of multiple serine/threonine phosphorylation sites. IRS proteins are substrates for many serine/threonine kinases downstream of other signaling networks, and become Ser/Thr phosphorylated in response to various conditions such as inﬂammation, stress and excess nutrients 10.2 Downstream Effector Proteins of RTKs 497

[7]. It is the unique structure of IRS-proteins that renders them ideal molecules to fulfill the task of sensing distinct environmental cues and integrating them into insulin sensitivity through serine/threonine phosphorylation. The Ser/Thr phosphorylation of IRS-proteins alters the capacities of the IRS- proteins to be Tyr-phosphorylated, and interferes at multiple points with insulin signaling. For example, IRS-1 has been shown to be phosphorylated on at least 20 different Ser/Thr residues, and these sites are placed at the regions of IRS proteins that mediate the three major functions of IRS proteins, namely membrane association, receptor binding and downstream effector recruitment (Figure 10.15). For instance, Ser/Thr phosphorylation at the pleckstrin homology (PH) domain and PTB domain interferes with membrane association and receptor recruitment, respectively. Ser/Thr phosphorylation in the vicinity of Tyr-phosphorylation sites impedes binding of the SH2 domains of downstream effectors, thus inhibiting insulin signaling and leading, for example, to insulin resistance. The protein kinases responsible for Ser/Thr phosphorylation can be divided into two groups. The first group includes kinases that are mediators of insulin action, such as mTor, S6 kinase, MAPK and PKC-d; these kinases negatively regulate IRS proteins upon prolonged insulin stimulation. The second other group consists of kinases that are activated along unrelated pathways, and includes IKKb (Sec- tion 2.8.5.3), c-Jun-terminal kinase (JNK; Chapter 12) and glycogen synthase kinase (GSK)-3b. Altogether, these kinases transmit to the IRS proteins a diverse set of signals that modulate and dampen insulin action, and any malfunction of the regulatory inputs into IRS signaling has been shown to be associated with – and even causative of – insulin resistance and type 2 diabetes. Overall, IRS proteins illustrate in an impressive manner the multiplicity of signals that converge on and are distributed further from a seemingly simple signaling intermediate. The large number of distinct Tyr- and Ser/Thr phosphorylation sites that can be used by incoming signals in a specific fashion enables the IRS proteins to be sensitive not only to many internal and external stimuli but also to the triggering of distinct biological responses.

10.2.2 Adaptor Proteins: FRS, Grb2, Gab, Shc, LAT, and p130 Cas

10.2.2.1 Fibroblast Growth Factor Receptor Substrate (FRS) The FRS2 family of adapter or scaffolding proteins has two members, FRS2a and FRS2b [5], both of which contain N-terminal myristoylation sites for localization on the plasma membrane and a PTB domain for binding to distinct RTKs, including the FGF receptor, the nerve growth factor receptor, and the anaplastic lymphoma kinase (ALK). Activation of these RTKs allows FRS2 proteins to become Tyr-phosphorylated and then bind to the Grb2 adapter and protein tyrosine phosphatase SHP2.

10.2.2.2 Grb2 The Grb2 protein (Grb: growth factor receptor binding protein) contains one SH2 domain flanked by two SH3 domains, and was first identified as a component of 498 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.15 Regulation of IRS-1 by shown in pink mediate signaling events that phosphorylation. Serine residues that are result in the phosphorylation of S789. Kinases phosphorylated in IRS-1 and the kinases that that initiate signaling events that result in target these sites are indicated. Kinases shown phosphorylation of S1223 and interfere with in blue mediate signaling events that impede SHP-2 binding are unknown (yellow). IRS-1 localization to the membrane or Exogenous stimuli that have been implicated upstream receptors by disrupting PH and/or in cancer and that can activate kinases to PTB domain function. Kinases shown in regulate IRS-1 serine phosphorylation are orange mediate signaling events that interfere indicated. AMPK, AMP-dependent kinase; For with PI3 K recruitment and activation. Kinases details, see Ref. [6]. 10.2 Downstream Effector Proteins of RTKs 499 signal transduction of growth factors and the Ras signaling node (Chapter 10). The adapter proteins Gab1, Shc, the EGF receptor, the PDGF receptor and the phosphatase SH-PTP2 have each been described as binding partners of the SH2 domain of Grb2 protein. Grb2 protein is tightly bound via its SH3 domain to the Pro-rich domain of the GTP–GDP exchange factor mSos, which can pass the signal by nucleotide exchange to the Ras protein (Chapter 11). In the form of the Grb2– mSos complex, Grb2 protein functions to generate a coupling between the activated RTK and the Ras protein. The Grb2-mediated membrane association of the Sos protein is necessary for its function as a GEF in the Ras signaling pathway (Chapter 11). In addition to the mSos protein, other Pro-rich signaling proteins have been known to bind to the SH3 domain of Grb2, indicating a broad spectrum of downstream effector proteins of Grb2.

10.2.2.3 Gab The Grb2-associated binder (Gab) family of adapter proteins comprises three members (Gab1–3) in mammals [8]. The domain structure shows a PH domain, Pro-rich sequences interacting with SH3 domains, several potential Tyr-phosphorylation sites, and Ser-phosphorylation sites. In response to the activation of transmembrane receptors with intrinsic or associated tyrosine kinase activity, the Gab proteins are recruited to the cell membrane and become phosphorylated on Tyr sites. The phosphorylated Gab then serves as a scaffold for the assembly of further signaling enzymes such as serine phosphatase PP2A, tyrosine phosphatase SHP2 and PI3K. Furthermore, functional interactions with the adaptors FRS and Grb2 have been demonstrated.

10.2.2.4 Shc The Shc (Src homology and collagen) protein was one of the ﬁrst characterized cellular adaptors. Mammalian genomes harbor four genes for Shc, namely ShcA, B, C, and D [9]. The best-characterized family member is ShcA, of which three alternatively spliced forms are known. All of the homologs share a common architecture deﬁned by an N-terminal PTB domain and a C-terminal SH2 domain, both of which can independently coordinate phosphorylated tyrosine residues of active receptors. The domains are connected by a central CH (collagen homology) 1 region containing consensus tyrosine residues that are subject to phosphorylation upon engagement of the adapter with an RTK, and subsequently serve as recognition motifs for proximal signaling effectors. The PTB and SH2 domains mediate interaction of the adapter with P-Tyr residues on RTKs and on receptors with associated Tyr-kinase activity such as B- and T-cell receptors and integrins. In the vertebrate Shc proteins, the PTB and SH2 domains are connected by a CH1 region rich in glycine and proline residues, that can bind SH3 domains of nonRTKs such as Src, Fyn, and Lyn. The best-characterized features of this region, however, are conserved tyrosine residues in positions that become phosphorylated upon engagement of Shc proteins with active kinases. The canonical role of Shc adaptors is to coordinate signaling complexes at activated transmembrane receptors. The interaction of Shc with EGFR is 500 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

considered as a prototype of Shc function, and in this system Shc binds to phosphorylated tyrosine residues of activated EGFR, becomes Tyr-phosphorylated by the TK activity of EGFR, after which the P-Tyr residues serve as docking sites for the adapter Grb2. This leads to an activation of Ras and the MAPK cascade (see Chapter 11). Furthermore, Tyr-phosphorylated Shc can serve as docking site for nonRTKs such as Src kinase, allowing for the parallel signaling of RTKs and nonRTKs. Another link to major signaling pathways employs the activation of Akt kinase through participation of the Gab adapter. Surprisingly, the 66 kDa isoform of Shc harbors oxidoreductase activity, and this form has been implicated in oxidative stress responses and in apoptosis.

10.2.2.5 PDZ-Containing Adapter Proteins The protein PSD-95 is an example of a PDZ-containing protein. PSD-95 is found in postsynaptic cells where, via its PDZ domains, it mediates interactions with intracellular domains of receptors such as the NMDA receptor. The InaD protein, which is composed solely of PDZ domains, has an adapter function in the vision process in Drosophila (see Section 3.1.1).

10.2.2.6 LAT The adapter protein LAT (Linker for activation of T-cells) is an example of a transmembrane adapter [10]. LAT spans the membrane with a single a-helix, and becomes Tyr-phosphorylated in response to the activation of T-cell receptors so as to provide a platform for the association of further downstream components of T-cell receptor signaling (see Section 13.3).

10.2.2.7 p130Cas This adapter is a multifunctional protein that is multiply phosphorylated on Tyr and Ser residues [11]. p130Cas is involved in cell motility, survival and proliferation, and its participation in the integrin signaling pathway is well established (Section 13.4). Of note is the presence of multiple copies of the sequence YxxP that, when Tyr-phosphorylated, serve as binding sites for SH2- and PTB-containing signaling enzymes.

10.2.3 Downstream Effectors of RTK Signaling

With multiple P-tyrosines in most receptors and the involvement of numerous adapter proteins, activated RTKs can recruit and inﬂuence a large number of different signaling molecules and signaling pathways. Selected classes of signaling enzymes used for downstream signal transduction include:

PI3-kinase (Section 9.4.1): A subgroup of PI3-kinase enzymes containing a p85 subunit can bind via its SH2 domain to the autophosphorylated receptor or to phosphorylated adapter proteins, as for example, IRS-1, assembled on the

activated receptor. Activated PI3-kinase generates PtdIns(3,4,5)P3, which 10.2 Downstream Effector Proteins of RTKs 501

mediates the membrane association of a variety of signaling proteins (Section 9.4.1 and Figure 9.12). One important response is the stimulation of cell survival. Ras: Ras proteins (Chapter 11) function as central signaling nodes that convey signals from Tyr-phosphate-based receptors to a variety of central signal paths.OnewayoflinkingRTKsignalingtoRassignalingrequiresthe recruitment of adapter proteins such as Grb2, Gab and Shc to the activated receptor. A multitude of signals can be delivered via Ras proteins, and this resultsinanactivationoftheMAKkinasecascadeandtheactivationof transcription factors. Phospholipase C-c: Activation of RTKs leads to a stimulation of phospholipid metabolism and to the generation of multiple second messengers. PLC-c (Section 7.7.2) binds through its SH2 domain to P-tyrosine sites on the receptor þ molecules and is thereby activated. As a consequence of PLC-c activation, Ca2 and DAG signals are produced leading, for example, to the activation of PKC isoenzymes and of CaM kinases. NonRTKs of the Src family (Section 10.3.2). Tyr-speciﬁc protein phosphatase SHP-2 (Section 10.4).

Many of the signaling pathways activated by RTKs ultimately lead to the activation of transcription factors, inﬂuencing central differentiation and developmental programs of the cell. The ﬂow of signals from activated receptors through central signaling pathways is summarized in Figure 10.16.

10.2.4 RTKs as Part of Signaling Networks

Earlier studies on growth factor signaling had suggested a linear signal transfer from activated RTKs to downstream components. A key example of this mode of signaling was the pathway that leads from the EGF receptor via Grb2, mSos, Ras to the MAP kinase pathway (see Chapters 11 and 12). However, there is now compelling experimental evidence supporting the existence of much more complicated and intertwined signaling networks of which the RTKs form a part. The complexity of signaling by RTKs is due to the following features of RTKs, their adaptors and the effectors:

Most RTKs harbor multiple autophosphorylation sites, each with different neighboring sequences. For example, 10 autophosphorylation sites have been identiﬁed on the PDGF receptor, allowing for the binding of a variety of effectors (Figure 10.11). Most adaptors are multidomain proteins that have many interaction partners (see for example, IRS; Section 10.2.1.1). Furthermore, most adaptors are multiply phosphorylated upon recruitment to the activated receptor and can therefore interact with a variety of effectors. Typically, effector proteins of RTKs are composed of multiple interaction domains that specify for example, membrane attachment or interaction with 502 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.16 Signaling pathways activated by and PI3-kinase signaling pathways also receptor tyrosine kinases. Different signaling regulate the activity of transcriptional factors pathways are presented as distinct signaling by phosphorylation and other mechanisms. cassettes (colored boxes). Not all known STAT, see Section 13.2.2; PLC, see Chapter 7; components of a given pathway are included. PI3-K, see Section 9.4; Ras, see Chapter 11; Examples of stimulatory and inhibitory signals MAPK, Cdc42/JNK, see Chapter 12. PTP, for the different pathways are also shown. The protein tyrosine phosphatase; FKHR, Forkhead signaling cassettes presented in the figure transcription factor; S6-K, ribosomal protein S6 regulate the activity of multiple cytoplasmic kinase; GSK-3, glycogen synthase kinase 3. targets. However, the Ras/MAPK, STAT, JNK

further, more downstream positioned signaling proteins. Often, the downstream interaction partners of RTKs are of a multidomain structure. As illustrated in Figure 10.17, by the example of PLCc, these domains may serve a coincidence detector function where different signals must cooperate for correct signaling. In addition, effectors often carry PTMs such as acetylation, phosphorylation or ubiquitination, and these modiﬁcations contribute to a further diversiﬁcation of downstream signaling.

It is now well established that RTKs are at the center of a large and complicated signaling network. All components of RTK signaling appear to be multivalent, and each component can interact with a host of different partners within a signaling network built up by a huge number of reactions and components. For example, the network inﬂuenced by the EGF receptor has been estimated to comprise more than 200 reactions involving over 300 components. A subset of reactions of the signaling network of the EGF receptor is shown in Figure 10.18. As quantitative information is scant for the vast majority of these reactions, it is not yet possible to model the network in a deterministic fashion. Several key organizational features 10.2 Downstream Effector Proteins of RTKs 503

Figure 10.17 Domains of PLCc involved in tyrosine phosphorylation of PLCc leads to RTK signaling. The multiple domains of PLCc intramolecular binding of the C-terminal SH2 cooperate to integrate multiple signals at the domain to P-Tyr783. This stimulates enzymatic plasma membrane. The N-terminal SH2 activity of PLCc, leading to hydrolysis of PtdIns domain is responsible for complex formation (4,5)P2 (PIP2) and consequently leads to the with activated RTKs. The C2 and PH domains formation of Ins(1,4,5)P3 (IP3) and cooperate with the SH2 domain to target PLCc diacylglycerol (DAG). Lemmon 2010 [2], to the plasma membrane. One or both of the figure 3B. Reproduced with permission of PH domains may also specifically recognize Elsevier. products of RTK-activated PI3 K. RTK-mediated have emerged from the modeling of complicated biological networks that have provided also some insight into the structure and behavior of the network underlying RTK signaling (see Section 3.3). According to one key concept, the network has an “hourglass structure” (see Figure 3.10), where diverse inputs and outputs are linked through a conserved processing core [12]. In the EGFR/ErbB receptor signaling network, the four ErbB receptors (EGFR, ErbB2, ErbB3, ErbB4) are regulated by multiple ligands to provide multiple inputs into the network. These inputs converge on a limited set of highly conserved “core processes” such as PI3 kinase, PLC enzymes, regulatory GTPases and the MAPK pathway, and this small group of downstream signaling intermediates propagate signaling from RTKs. The core processes are also regulated via other signaling pathways and thereby receive a large number of different inputs. The “hourglass” then widens again due to an ability of the core components to create a highly diverse number of outputs – that is, further downstream reactions that affect many cellular processes such as transcription, proliferation, differentiation and apoptosis. A key feature of the RTK signaling network is the existence of positive and negative feedbacks loops that control the ﬂow of information through the hourglass, providing for high sensitivity and robustness of signaling. 504 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.18 A coarse outline of the signaling its behavior. For example, the inhibition of Ras network regulated by EGFR. A subset of by Ras-GAP or EGFR by protein kinase C (PKC) intracellular signaling components influenced serve a negative feedback function. On the by EGFR activation are intertwined in a other hand, H2O2 inhibits protein tyrosine complex network. Through a combination of phosphatases (PTPs) and thus, prolongs or stimulatory (black arrows) or inhibitory (red increases the activity of EGFR by a positive- lines) signals, several key positive feedback feedback mechanism. Lemmon 2010 [2], loops (blue circular arrows) and negative figure 4. Reproduced with permission of feedback loops (red circular arrows) emerge in Elsevier. the network and exert a significant influence on

10.2.5 Attenuation and Feedback Regulation in RTK Signaling

Signaling by RTKs must be tightly regulated and correctly balanced in order to produce the normal cellular responses [2,13]. Many examples are known where the aberrant expression or dysfunction of RTKs is responsible for developmental disorders and diseases including tumor formation. In order to function correctly, the RTK signaling network must contain controls that ensure speciﬁcity, robustness and protection against inappropriate stimulation. These controls operate by feedback mechanisms and other reactions such as antagonistic ligands, the binding of inhibitor proteins, endocytosis, and degradation. Numerous feedback controls have been identiﬁed in RTK signaling (Fig- ure 10.18), including:

Positive feedback: One example of a positive feedback loop has been identiﬁed in EGFR/ErbB signaling, with the adapter protein Gab1 as a main player. In this 10.2 Downstream Effector Proteins of RTKs 505

system, Gab1 and PI3 kinase cooperate to create a positive feedback loop that stimulates Akt-dependent anti-apoptotic signaling. Negative feedback: This control can operate at many levels of RTK signaling, and serves to dampen and terminate RTK signaling in response to a variety of signals.

The negative control of EGFR signaling can be achieved in several ways:

Receptor dephosphorylation: Protein tyrosine phosphatases (PTPs) may be directly activated by RTKs. For example, the SH2 domain-containing phosphatase SHP2 (or PTPN1) is recruited to activated EGFR and promotes dephosphorylation of the P-tyrosines on the receptor. Receptor degradation: Recruitment of the E3 ligase Cbl to the activated RTK induces receptor degradation via the Ub/proteasome pathway (Section 2.8). Inhibitory receptor phosphorylation: EGFR activation promotes the activation of PKC via PLC-c; the activated PKC can then phosphorylate EGFR on T654 of its juxtamembrane region. This eliminates high-afﬁnity binding of EGF to EGFR and thus inhibits EGFR activation in a negative feedback loop. Negative regulation of downstream signaling components: The RTK-induced activation of MAPK enzymes leads to inhibitory phosphorylations of PI3K, mSos, and Raf kinase.

For further examples, see Ref. [2].

10.2.5.1 Antagonistic Ligands, Heterodimerization In several systems, natural antagonistic ligands have been identiﬁed that bind on the extracellular side to the receptor and inhibit receptor activation. Furthermore, certain tissues express naturally occurring receptor variants that are deﬁcient in TK activity. The expression of these mutants may lead to a dominant negative inhibition of full-length receptors through the formation of inactive heterodimers or heterooligomers.

& Negative regulation of RTKs is mediated by: — Tyr-Phosphatases — Ser/Thr-phosphorylation — Antagonistic ligands — Inhibitory proteins: SOCS, Sprouty — Ubiquitination, degradation — Endocytosis.

10.2.5.2 Inhibition by Inhibitor Proteins In another negative control mechanism, inhibitory proteins such as SOCS (suppressor of cytokine signaling; Section 13.2.3) bind via their SH2 domains to P-tyrosine residues in the tyrosine kinase domain of the receptor and directly inhibit the kinase activity. This type of protein regulates mainly the insulin receptor 506 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

and cytokine receptors. Another class of antagonistic proteins includes the family of Sprouty proteins [14].

10.2.6 Endocytosis and Trafficking of RTKs

An early and general response in the activation of RTKs, is receptor “downregulation” (Section 7.2.4). This involves the ligand-stimulated endocytosis of occupied receptors and subsequent intracellular degradation of both ligand and receptor molecules following the clathrin-coated pit pathway (Section 7.3.3) [15]. After internalization, the activated RTKs are dephosphorylated, ubiquitinated, and the activating ligand dissociates from the receptor in the lower pH in the endosome. However, endocytosis not only serves to terminate RTK activation; rather, activated RTKs can continue to recruit and activate intracellular signaling pathways from within intracellular vesicles following their internalization. The stage in the endocytic pathway at which these events occur varies with both ligand and receptor. In addition, RTKs may be recycled from endosomes to the plasma membrane or sorted for degradation, which affects receptor numbers and thus signaling. Intracellular signaling of internalized receptors has been demonstrated for several RTKs but, interestingly, the signaling molecules that have been shown as being associated with an activated receptor may be significantly different at the plasma membrane compared to those seen in the endosomal (and other) compartments. Furthermore, certain RTKs – notably Eph receptors – require to be internalized to effect their signaling function [16]. These features add another dimension to the RTK network, namely the space- dependence of signaling. Clearly, the precise subcellular location of the receptor can define signaling specificity, although how this is achieved is not yet understood.

10.2.6.1 Ubiquitination A major role in the downregulation of RTKs is ascribed to ubiquitination. One of the RTK-proximal proteins that associates with phosphorylated EGFRs and other RTKs is the E3 ligase Cbl (see Section 2.8), a modular protein that triggers the ubiquitination, subsequent endosomal internalization and proteasomal degradation of a variety of RTKs. Cbl contains a SH2 domain that mediates binding to P- tyrosine residues of the activated receptor, and a RING ﬁnger domain that brings a ubiquitin-conjugating E2 enzyme to the vicinity of the receptor. Receptor ubiquitination results in an accelerated removal from the cell surface and entry into endosomal pathways, leading ﬁnally to receptor degradation, thereby terminating RTK signaling.

10.2.7 RTK Dysfunction in Disease

The dysfunction of RTKs has been linked to many diseases. Indeed, any genetic changes or abnormalities that alter the activity, abundance, cellular distribution or 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 507 regulation of RTKs may lead to an aberrant function of RTKs and of the downstream signaling pathways, and this has been causally linked to cancers, diabetes, inﬂammation, severe bone disorders, arteriosclerosis, and angiogenesis. Aberrant RTK activation in human cancers is mediated by four principal mechanisms: autocrine activation; chromosomal translocations; RTK overexpression; and/or gain-of-function mutations. The oncogenic activation of RTKs will be discussed in Section 16.4.2.1, using an example of the EGF receptor.

10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs

Summary In addition to RTKs, the cell also contains a number of tyrosine-specific protein kinases that are not an integral component of transmembrane receptors. These non-RTKs are localized in the cytoplasm at least occasionally, or they are associated with transmembrane receptors on the cytoplasmic side of the cell membrane; they are therefore also known as cytoplasmic tyrosine kinases. The non-RTKs perform essential functions in signal transduction via cytokine receptors and T-cell receptors, and in other signaling pathways. Most non-RTKs show a characteristic SH3-SH2-TK domain structure, whereby the SH3 and SH2 domains mediate autoinhibition and are responsible for signal-dependent binding to substrate proteins. Other sequence motifs are involved in association with specific subcellular sites and structures. Due to the multidomain structure, the activation mechanisms of the non-RTKs are varied, with activation typically requiring the relief of autoinhibition by phosphorylation of the activation loop and Tyr-phosphorylation at other Tyr-residues within the multidomain structure. Of the non-RTK families, the subfamilies of Src kinases and Abl kinases are the best-studied, as members of both subfamilies have been implicated in tumorigenesis.

10.3.1 Structure and General Function of Non-RTKs

The primary clade of human non-RTKs has 22 members, all but three of which share a characteristic SH3-SH2-TK domain structure, as shown in Figure 10.19 for the major subfamilies of the non-RTK superfamily. The SH3 and SH2 domains serve two main purposes, in that they mediate autoinhibition and are also responsible for signal-dependent binding to substrate proteins. Other sequence motifs mediate association with speciﬁc subcellular sites and structures. Structural studies on the inactive and active states of the non-RTKs failed to reveal a general mechanism of activation for all members of the superfamily. Due to their multidomain structure, different inactive states of non-RTKs have been 508 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.19 Domain organization for the nonreceptor tyrosine kinases: Csk, Src, Abl major subfamilies of nonreceptor tyrosine (Section 10.3); Zap 70 (Section 12.3); Jak kinases. The amino terminus is on the left, and (Section 13.2); Fak (Section 13.4). Btk, Bruton the carboxy terminus on the right. The lengths kinase. are only approximately to scale. Details of the

identiﬁed [17], and the mechanisms for activation appear to vary among the members studied so far. The structures of the active states also appear to be different, although the requirements for the structure of the active kinase domain (see Section 9.2) apply also to this kinase class.

10.3.1.1 Functions of non-RTKs A permanent or transient association with subcellular structures, and a variable subcellular distribution, are characteristics of the non-RTKs. These enzymes are intracellular effector molecules that can be ascribed the function of nodes in the large kinase signaling network. Typically, non-RTKs are activated by multiple signals and associate with speciﬁc substrates that become phosphorylated to generate a diverse number of different responses. Many of the functions of non- RTKs are performed in the immediate vicinity of the cell membrane, whether a signal is received from an activated membrane receptor or is passed on to a membrane-associated protein. To facilitate membrane association, many non-RTKs contain N-terminal lipid anchors. The details of two nonreceptor tyrosine kinases, Src kinase and Abl kinase, are highlighted in the following sections.

10.3.2 Src Tyrosine Kinase

Src tyrosine kinase (henceforth termed Src) was first identified as the cellular form of v-Src, the transforming gene product of the avian Rous sarcoma virus (see also Section 16.4). Additional enzymes with homology to Src have been identified in the human genome, are referred to collectively as Src family kinases (SFKs), and now 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 509 comprise 11 members. A review of the structure and regulation of SFKs is provided in Ref. [18]. These enzymes are tyrosine-specific protein kinases involved in the regulation of cell division, cell differentiation, and cell aggregation. Different tissues vary widely in their expression levels of SFKs, and aberrant functions of these kinases have been associated with a variety of diseases including several types of cancer and neurodegenerative disease.

& Src kinase structure: — N-terminal myristinic acid — SH3 domain — SH2 domain — Classical kinase domain

10.3.2.1 Structure of Inactive Src All SFK members exhibit the same N- to C-terminal arrangement of structural domains, an example of which is shown for Src in Figure 10.20a. At the N terminus, Src carries a myristinic acid residue as a membrane anchor, followed by

Figure 10.20 Structure and regulation of Src A disordered section of the activation segment kinase. (a) Domain structure of Src kinase; is shown as a dashed line. The amino- and (b) Structure of c-Src kinase phosphorylated at carboxy-terminal kinase lobes are shown in Tyr527. Ribbon diagram showing the structure light and dark green, respectively. The and organization of the “closed conformation” activation loop in the kinase domain, of c-Src kinase. Two aspects of the structure containing Tyr416, is shown in gray. The SH2 are important for the regulation of c-Src and SH3 domains are shown in dark blue and kinase: (i) The phosphorylated Tyr527 of the cyan, respectively. The SH2-kinase linker, which C-terminal tail is engaged in an intramolecular contains a short stretch of polyproline type II interaction with the SH2 domain; and (ii) the helix (PPII helix) is shown in red. The carboxy- SH3 domain binds to the linker between the terminal tail, which contains pTyr527, is shown SH2 domain and the kinase domain. Both in orange. interactions fix an inactive state of the kinase. 510 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.20 (Continued)

a unique domain of 50–80 residues the function of which is not well characterized. The unique domain is followed by modular SH3 and SH2 domains, a regulatory linker, the kinase domain, and a C-terminal negative regulatory tail. The SH3 domain contributes to substrate recruitment and is critical for the allosteric regulation of kinase activity by interacting with the regulatory linker and the SH2 domain. The SH2 domain functions in P-tyrosine binding, either intramolecularly within Src or with P-Tyr residues of phosphorylated substrates. The alternative interactions of the SH3 and SH2 domains form the basis of the regulation of Src activity. 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 511

& Inactive state of Src kinase: — Intramolecular binding of P-Tyr527 to SH2 domain — SH2 and SH3 domains clamp on kinase domain, displace C-helix of kinase domain stabilizing inactive conformation — Activation loop not phosphorylated.

10.3.2.2 Inactive State of Src Src regulation is closely linked to two Tyr-phosphorylation sites, namely Tyr416 in the activation loop and Tyr527 in the C-terminal tail (numbering of chicken c-Src). In the unstimulated, basal state, Src exists as an inactive, autoinhibited enzyme that is phosphorylated on Tyr527. The inactive state is maintained by intramolecular interactions involving the SH2 and SH3 domains and P-Tyr527 of the C-terminal tail. As predicted from mutation experiments, P-Tyr527 enters into intramolecular binding with the SH2 domain; in addition, the SH3 domain binds to the linker between the catalytic domain and the SH2 domain. The linker contains proline residues and adopts a polyproline II helix in the intramolecular complex, providing a binding motif for the SH3 domain. The protein kinase responsible for the repressive Tyr527- phosphorylation is Csk kinase, a nonreceptor tyrosine kinase that can shuttle between cytoplasmic and membrane-bound states. The intramolecular interactions clamp the SH2 domain and the SH3 domain to the reverse side of the catalytic domain (Figure 10.20b), as a result of which the C- helix of the catalytic domain is displaced and misaligned when compared to the structures of other active protein kinases. A catalytically unfavorable position of the catalytic residues in the kinase domain is stabilized by the clamp, and ATP binding is prevented. Furthermore, Tyr416 of the activation loop is sequestered and is not available for phosphorylation.

& Activation of Src occurs via: — The binding of competing ligands to SH2 and SH3 domains induces unclamping and transition into the active conformation — Activation loop phosphorylation by Csk kinase.

10.3.2.3 Activation of Src The relief of autoinhibition requires an unlocking of the clamp that ﬁxes the catalytic domain in an inactive conformation, and this can be achieved in several ways (Figure 10.21a and b):

Binding of the SH3 domain to target sequences on substrate proteins Binding of the SH2 domain to P-Tyr residues on substrate proteins Removal of the P-Tyr527 by tyrosine phosphatases Downregulation of Csk activity.

Through the binding of high-afﬁnity ligands, the SH2 and SH3 domains can be displaced from the P-Tyr527 and the linker region, respectively. For example, the 512 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.21 Activation of c-Src. (a) The left and SH2 domains are available for interaction panel represents the inactive conformation of target proteins. Recruitment to RTKs is Src in which Tyr527 (chicken c-Src) interacts mediated by SH2-P-Tyr interactions and by the with the Src homology 2 (SH2) domain, myristoylated N terminus of Src that promotes positioning the SH3 domain to interact with membrane association. Src recruitment to the linker between the SH2 and catalytic GPCR signaling complexes is thought to be domains. The middle panel illustrates different mediated by interaction of the SH3 domain mechanisms involved in the activation of Src, with Pro-rich sequences on arrestin. Wheeler and the right panel represents the open or 2009 [19], figures 2 and 3. Reproduced with active conformation; (b) In active Src, the SH3 permission of Wiley-VCH. 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 513 activation of Src kinase is achieved through SH2 binding to the autophosphorylated PDGF receptor or through SH3 domain binding of the HIV protein, Nef. Following unlocking of the clamp, the C-helix and the activation loop can switch to their active conformations. Tyr416 on the activation loop is now available for phosphorylation which is thought to occur in trans by another Src kinase molecule. Once autophosphorylated, the enzyme is stabilized in its active state. Furthermore, the dephosphorylation of P-Tyr527 by protein tyrosine phosphatases is now possible and will help to fix the active state. The structural design of Src kinase allows for a regulation at multiple levels. The internal interactions maintain an inactive state and external ligands promote an active state by binding to the SH2 and/or SH3 domain. Other regulatory inputs are provided by the action of tyrosine kinases as, for example, Csk kinase and by protein tyrosine phosphatases. The importance of strict regulation of Src is underscored by the occurrence of oncogenic Src variants [19]. One of the first viral oncogene products of retroviruses to be discovered was v-Src kinase, a variant of Src that is a product of the Rous sarcoma virus. Owing to a C-terminal truncation, v-Src kinase lacks the regulatory site Tyr527 and is constitutively active, resulting in an uncontrolled growth of infected cells. A selection of the major signaling pathways influenced by Src is shown in Figure 10.22. Src is involved, for example, in ion-channel regulation and in signal transduction via growth factor receptors, integrins and immunoreceptors. Func- tional interactions have been described, for example, with Tyr-P residues of PDGF receptors, EGF receptors, focal adhesion kinaseFAK (see Chapter 13), and scaffolding proteins of the N-methyl-D-aspartic acid (NMDA) receptor complex.

10.3.3 Abl Tyrosine Kinase

Abelson (Abl) tyrosine kinases are involved in the regulation of diverse cellular processes such as microﬁlament remodeling, cell motility and adhesion, receptor endocytosis, DNA damage response, and apoptosis. Much of the interest in Abl proteins [20] stems from their involvement in oncogenesis in rodents and in humans. Like many other non-RTKs, Abl proteins may be converted by mutation into a dominant oncoprotein and may thus contribute to tumor formation. The wild-type form of Abl enzymes is termed c-Abl (Abl in the following); the viral, oncogenic form is termed v-Abl. This mutated enzyme was ﬁrst discovered as the oncogene of murine Abelson leukemia virus.

10.3.3.1 Structure of Abl The complex multidomain structure is a distinctive feature of both Abl paralogs Abl1 and Abl2 (Figure 10.23). In addition to the N-terminal SH3-SH2-tyrosine kinase cassette, Abl1 and Abl2 carry Pro-rich sequences with capacity to bind SH3 and WW domains, and binding domains for microﬁlament proteins, namely G- actin and F-actin. Furthermore, the N terminus carries a myristinic acid residue as 514 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.22 Selected examples of Src signal activated protein kinase kinase MAPK/ERK transduction pathways. Abbreviations: CAS, kinase; MLCK, myosin light chain kinase; Crk-associated substrate; ERK, extracellular NFkB, nuclear factor kB; PI3 K, signal-regulated kinase; FAK, focal adhesion phosphatidylinositol-3-kinase; RhoGAP, Rho kinase; IKK, IkB kinase; IL-8, interleukin 8; JNK, GTPase-activating protein; SOS, son of Jun N-terminal kinase; MAPK, mitogen- sevenless. For details, see Ref. [19]. activated protein kinase; MEK, mitogen-

a membrane anchor. Vertebrates express two Abl enzymes, Abl1 and Abl2, which perform specialized functions. A distinctive feature of Abl1 is the presence of nuclear localization signals and of a DNA-binding domain through which it mediates functions in DNA repair. Furthermore, Abl1 carries a nuclear export signal in its C-terminal part which is likely responsible for the ability of Abl1 to 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 515

Figure 10.23 ABL domain structure. Linear nuclear export signal; magenta triangle, depiction of human ABL. my, myristoylation nuclear localization signal; green triangle, site; G BD, G-actin-binding domain; MT BD, proline-rich motif with capacity to bind SH3 or microtubule-binding domain. Blue triangle, WW domains.

shuttle between the cytoplasm and the nucleus. Abl2 is localized predominantly to the cytoplasm. It carries a second F-actin-binding domain which confers additional binding capacity for actin and for microtubules mediating cytoskeletal remodeling functions.

10.3.3.2 Activation of Abl The structure of the N-terminal half of Abl is very similar to Src in that there is the same arrangement of SH2, SH3, and kinase domains. Nevertheless, interesting differences in the regulation of kinase activity exist. Structural studies of the inactive states of Abl have demonstrated an unexpected function of the N-terminal myristinic acid, namely the autoinhibition of kinase activity. Unlike Src kinase, Abl does not possess an autoinhibitory P-tyrosine site at the C terminus; rather, the inactive state of Abl is stabilized by intramolecular binding of the N-myristoyl group to the large lobe of the kinase domain, inducing a clamping of the SH2 and SH3 domain onto the kinase domain (Figure 10.24), similar to that observed for Src kinase. Competing high-afﬁnity SH2 and SH3 ligands can unclamp this assembled state of Abl, and the kinase domain can then be switched into its active conformation by phosphorylation of the activation loop. How the myristinic acid anchor rearranges from its autoinhibiting position is unclear, but it is possible that the binding of SH2 and SH3 ligands and a membrane insertion of the lipid anchor can cooperate to relieve the autoinhibited state. Another interesting feature of Abl family members is the ability to self-associate. It is assumed that self-association in the form of dimers or higher oligomers enhances trans-phosphorylation, for example in the activation loop, leading to direct activation.

10.3.3.3 Substrate Selection and Interaction Partners of Abl Like the other non-RTKs, Abl interacts with a multitude of proteins, and a large number of putative substrates have been identiﬁed [20]. Many substrates are recruited via binding of the Abl SH2 domain to Tyr-phosphorylated sites on substrate proteins and, reciprocally, P-tyrosines on Abl proteins can mediate binding to SH2 domains on substrate proteins so as to allow substrate phosphorylation. The SH3 domain of Abl has also been shown to bind to PxxP motifs on substrates. Such motifs are also found on Abl proteins, and allow the 516 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.24 Structure of an autoinhibited the N-terminal myristate into the C-terminal state of Abl tyrosine kinase. (a) The lobe of the kinase domain. PD166326 indicates arrangement of a fragment of Abl kinase the binding site for the small-molecule comprising the SH3, SH2 and kinase domain inhibitor PD166326. Numbering for Abl kinase is shown. The autoinhibited state is from mouse; (b) Structure of Gleevec, a small- stabilized --- at least in part --- by an insertion of molecule inhibitor of receptor tyrosine kinases.

binding of SH3 domains on interaction partners. The long list of substrates includes signaling enzymes such as PLCc and PI3 kinase, and proteins involved in DNA repair such as RAD52, the large subunit of RNA Pol II, and the E3 ligase Cbl. Several of the Abl substrates (e.g., Rad52) are multiply phosphorylated in a processive manner, and it is assumed that the SH2 domain of Abl is involved in this cooperative phosphorylation. 10.3 Nonreceptor Tyrosine-Specific Protein Kinases, Non-RTKs 517

10.3.3.4 Regulation of Abl Abl kinases are subject to multiple regulatory modiﬁcations such as phosphorylation, acetylation, and ubiquitination. Phosphorylation on multiple Tyr and Ser/Thr sites appears to play a major role in regulating Abl activity. Tyr phosphorylation sites have been identiﬁed for example in the activation loop, in the linker between the SH2 and the kinase domain, and in the SH3 domain, leading to a relief of autoinhibition, activation, and increased substrate binding. Interestingly, Tyr sites may be phosphorylated in trans between Abl1 and Abl2, and Abl kinases are also phosphorylated by members of the Src kinase family and by PDGF receptors. At least some of the P-tyrosines are predicted to be binding sites for the SH2 domains of other signaling enzymes.

& Abl tyrosine kinase, domain structure: — SH2 domain — SH3 domain — N-terminal myristinic acid — Nuclear localization signals — DNA-binding domains.

Inactive Abl: — Autoinhibition by SH3, SH2 clamping to kinase domain and by myristinic acid binding to kinase domain.

Active Abl: — Binding of SH2, SH3 to competing ligands.

Medical importance of Abl: — Formation of Bcr/Abl hybrid protein in chronic myelogenic leukemia — Target of antitumor drug Gleevec.

The large number of PTMs, and the presence of several regulatory binding modules as well as various subcellular localization signals, is indicative of multiple functions and a complicated regulation of activity and localization of Abl proteins. Overall, the multivalency of Abl proteins mirrors the many links that these proteins have within the signaling network of the cell.

10.3.3.5 Cellular Functions of Abl The cytoplasmic functions of Abl are directed mainly towards the reorganization of the cytoskeleton, and the capacity of Abl proteins to bind the G- and F-actin microﬁlament is a deﬁning characteristic of this kinase family. This function is mediated by distinct cytoskeletal binding domains, namely an F-actin-binding domain on Abl1 and Abl2, and a G-actin-binding domain on Abl1. Abl family kinases bind for example, to F-actin and microtubules directly, and can use this 518 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

activity to control cytoskeletal structures. Possible upstream effectors are activated RTKs, as for example the PDGF receptors that provide high-affinity binding sites for the SH2 and SH3 domains of Abl. This serves to recruit Abl to receptor complexes and to induce escape from the autoinhibited state. The functions of Abl in the nucleus appear to be manifold. Abl has been implicated, for example, in apoptosis (programmed cell death), transcription regulation, DNA damage checkpoints, and cell-cycle control. A major regulation of nuclear functions of Abl is exerted by the 14-3-3 proteins that bind to phosphorylated Abl and sequester it in the cytoplasm. Upon activating signals, the 14-3-3 proteins (see Section 2.5.3) become phosphorylated and release Abl, allowing it to translocate into the nucleus. One important upstream effector of Abl in DNA damage responses is the ATM kinase (see Section 15.6.2) that phosphorylates Abl on specific Ser/Thr residues, thereby promoting its activation and transmission of the signal to downstream effectors. As an example, the p53- related protein p73 has been identified as a downstream effector of Abl in DNA damage control and cell-cycle control.

10.3.3.6 Oncogenic Activation of Abl The mutation of Abl proteins has been found to play a prominent role in the development of some types of leukemia. Mutated Abl kinase was first discovered as the oncogene of murine Abelson leukemia virus. Apart from the v-Abl enzymes, other oncogenic forms of the Abelson kinase exist. Chronic myelogenic leukemia in humans is caused by a chromosome translocation in which a fusion protein is created from c-Abl and a Bcr protein (see Section 16.4.2.2). The result is a greatly increased tyrosine kinase activity with very different regulatory properties, to which a causal role in the occurrence of this leukemia is attributed. Furthermore, c-Abl is an important target of antitumor drugs; the c-Abl inhibitor Gleevec (see Figure 10.24) has remarkable efficiency in treating chronic myelogenic leukemia. Gleevec binds specifically to the ATP site within the kinase domain of Abl kinase, thus stabilizing an inactive state of the kinase. By applying this action, Gleevec is able to interfere with Bcr/Abl signaling and suppress the proliferation-promoting action of the hybrid protein. The protein kinase activity of some receptor tyrosine kinases are also inhibited by Gleevec, which today is widely used to treat leukemias and other cancers.

& Functions of Abl: — Variable subcellular distribution with multiple functions in cytosol and nucleus — Involved in: Cytoskeletal reorganizations DNA repair Cell-cycle control RTK signaling. 10.4 Protein Tyrosine Phosphatases 519

10.4 Protein Tyrosine Phosphatases

Summary Protein tyrosine phosphatases (PTPs) antagonize the action of TKs. In doing this, PTPs can have a negative, dampening effect on cell signaling, or they can enhance cell signaling for example, by removing inhibitory Tyr-P residues. Therefore, PTPs play distinct roles in cell signaling, such as in the control of cell proliferation by RTKs, and several PTPs have been implicated in tumor suppression. The major family of PTPs comprises 99 members in humans that are subdivided as classical PTPs and dual-specificity PTPs. The classical PTPs hydrolyze P-Tyr, and these enzymes are further divided into two subfamilies: the poorly characterized receptor protein tyrosine phosphatases (PTPRs) and intracellular, nonreceptor protein tyrosine phosphatases (PTPNs). Members of the dual- specificity family hydrolyze both P-Tyr and also P-Ser or P-Thr. Like most of the PTPs, the PTPNs harbor a catalytic domain with Cys as an essential active site residue, as well as a variety of interaction domains that specify the intracellular localization and substrate or other protein interactions. The activity of PTPs is controlled on many levels, for example, by phosphorylation, targeted localization, and by oxidation of the active site cysteine.

Protein tyrosine phosphatases (PTPs) play a crucial role in the control of signaling pathways that use tyrosine phosphorylation as a regulatory mechanism. The importance of the tyrosine phosphatases in, for example, receptor tyrosine kinase signaling is illustrated by the observation that virtually all receptor tyrosine kinases can be activated, even in the absence of ligand, by treating cells with tyrosine phosphatase inhibitors, showing that the activity of tyrosine kinases is continuously controlled by inhibitory tyrosine phosphatase action. The function of PTPs is, however, not simply to scavenge P-Tyr and to reset the Tyr-phosphorylation clock to zero; rather, it is now firmly established that PTPs play specific roles in cell signaling and operate in concert with TKs to define specific cellular outcomes.

& Protein tyrosine phosphatases: — Essential elements of signaling paths — Function as positive or negative regulators — Dephosphorylation of substrates by PTPs may inhibit or activate signal transduction.

10.4.1 General Functions of PTPs

The PTPs are essential parts of the signaling pathways that control fundamental physiological functions such as cell–cell interactions, B-cell proliferation, 520 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

inflammatory responses, glucose homeostasis, organization of synapses, and the regulation of proliferation and of the cell cycle [21]. The biological importance of PTPs is underlined by the observation that defects in their activity have been shown to lead to phenotypically demonstrable errors in function in higher eukaryotes. Importantly, PTPs play a central role in the control of cell proliferation by RTKs. It is therefore not surprising that mutant PTPs are frequently found in cancers [22] such as colon cancer, and members of this enzyme class have been identified as tumor suppressors. For example, loss-of function mutations in receptor PTP-T (PTPR-T) are frequently found in various cancer cells, and PTPR-T is now considered a bona fide tumor suppressor [23]. However, some PTPs – such as SHP2 – also have oncogenic properties. Although members of the PTP family have crucial roles in a variety of cellular processes, the physiological functions of the individual PTPs are poorly understood owing to a lack of information regarding the “genuine” substrates of the respective PTPs. Hence, some selected aspects of PTP function will be presented in the following subsections.

10.4.2 Classification, Structure, and Mechanism of PTPs

The human genome encodes 107 genes for PTPs which are grouped into four families [22]. Family I PTPs comprises 99 members that are subdivided into “classical” PTPs and dual-specificity PTPs that hydrolyze both P-Tyr and also P-Ser or P-Thr. Interestingly, the latter subfamily includes lipid phosphatases such as the PTEN phosphatase (see Section 9.4.2.3). Family II PTPs encompasses the dual- specificity phosphatases CDC25A-C that play essential roles in cell-cycle regulation (see Chapter 15). PTP families I–III employ cysteine as a catalytic residue and are defined on the basis of a consensus catalytic signature (see below), while family IV PTPs are based on aspartate as the catalytic residue. The “classical” PTPs of class I are further divided into two subfamilies: receptor protein tyrosine phosphatases (PTPRs, 21 members) and intracellular, nonreceptor protein tyrosine phosphatases (PTPNs, 17 members). Both groups catalyze the hydrolysis of P-Tyr by a common mechanism and, correspondingly, both groups have a homologous catalytic domain. In addition to the phosphatase domain, PTPs typically harbor additional domains that serve to recruit ligands (resp. substrates) and target the PTP to specific subcellular locations. The domain structure of some important tyrosine phosphatases is shown in Figure 10.25.

10.4.3 Catalytic Mechanism of PTPs

The catalytic center of the PTPs includes approximately 230 amino acids and

contains the conserved sequence motif H/V-C-(X)5-R-S/T-G/A/P (where X is any amino acid), which is involved in phosphate binding and in catalysis. The catalytic 10.4 Protein Tyrosine Phosphatases 521

Figure 10.25 Domain organization of receptor-like and intracellular protein tyrosine phosphatases. For details of SH2, PTB, and PDZ domains, see Section 10.1.5.

mechanism of the classical PTPs is shown schematically in Figure 10.26. An invariant Cys is central to phosphate ester hydrolysis. The Cys residue exists as a thiolate (pKa 5.5) which is stabilized by an arginine residue. The thiolate carries out a nucleophilic attack on the phosphate of the P-tyrosine residue, and displaces the Tyr residue via an “in-line” attack such that an enzyme-bound Cys-phosphate is formed. Release of the phosphate from the intermediate Cys-phosphate is achieved by the nucleophilic attack of a water molecule.

10.4.3.1 Receptor Protein Tyrosine Phosphatases (PTPRs) The overall structure of PTPRs is very similar to the structure of RTKs, except that the former have a transmembrane and, in some cases, a large extracellular domain with a very variable structure (see Figure 10.25). 522 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.26 Mechanism of hydrolysis of Arg residue. During the course of the reaction, phosphotyrosine residues by tyrosine an enzyme---Cys---phosphate intermediate is phosphatases. Cleavage of phosphate from formed, which is hydrolytically cleaved to phosphotyrosine residues takes place by an phosphate and enzyme---Cys---SH. The figure “in-line” attack of a nucleophilic cysteine shows selected interactions. Other interactions thiolate of the tyrosine phosphatase at the in the active center involved in substrate phosphate of the phosphotyrosine residue. binding and catalysis are not shown. R, The negative charge on the thiolate is substrate protein. stabilized by the positive charge of a conserved

& Receptor PTPs: — Activated by binding of extracellular ligands to extracellular domain — Contain single transmembrane element — Carry PTPase activity on cytoplasmic domain.

Many, but not all, PTPRs have two catalytic domains in the cytoplasmic region that serve distinct roles. In the majority of cases, the membrane-proximal domain of PTPRs comprises the catalytic domain, the membrane-distal domain often 10.4 Protein Tyrosine Phosphatases 523 serves a negatively regulatory role. Much like the RTKs, PTPRs utilize their diverse extracellular domains to transduce extracellular signals through binding to soluble ligands. In addition, the extracellular domains serve to mediate cell-cell and cell-matrix interactions. Only for some PTPRs have the extracellular ligands been identified.Asanexample,thePTPRf has been found to be specifically inhibited by pleiotrophin, which is a cytokine implicated in tumor angiogenesis. The activity of PTPRs can be positively or negatively regulated by extracellular ligand binding. However, the mechanisms underlying regulation by extracellular ligands are not well defined, and there may be no universal ligand-mediated regulation mechanism for all PTPR members.

10.4.3.2 Nonreceptor Protein Tyrosine Phosphatases (PTPNs) The PTPN enzymes have a catalytic domain and a variety of interaction domains that specify the intracellular localization and protein interactions in substrate selection. These structural elements contain sequence signals for nuclear localization, for membrane association, and for association with the cytoskeleton.

& Nonreceptor PTPs: — Contain localization signals and modules for the interaction with other signaling proteins such as RTKs.

Other modules of PTPNs are Pro-rich sequences, SH2 and PDZ domains that allow targeting to substrates and the integration of phosphatase activity into larger signaling complexes. Speciﬁcally, the SH2 domains of PTPNs have been shown to mediate the association with tyrosine phosphates of activated RTKs and with Tyr- phosphorylated adapter proteins. By these interactions, PTPN enzymes are recruited to signaling complexes centered around activated transmembrane receptors (growth factor receptors, cytokine receptors, integrins) that employ Tyr- phosphorylation as a means of conveying signals into the cytosol. Depending on the nature of the phosphorylated protein component that serves as a substrate of the PTPN, dephosphorylation can have either a negative or positive effect on receptor signaling. Accordingly, some members of the PTPN family have been identiﬁed as tumor suppressors (e.g., PTP-Pest), whereas others (such as SHP2) can be activated by gain-of function mutation into an oncogene [24].

10.4.3.3 Autoinhibition of PTPN Enzymes The most well-studied SH2-containing PTPs are SHP1, which regulates signaling by hematopoietic receptors, and SHP2 (also known as SYP), which is involved in signaling by growth factor receptors and cytokines. Both phosphatases are subject to an intramolecular inhibition by the SH2 domains (Figure 10.27). It is assumed that these enzymes exist in an autoinhibited form where the N-terminal SH2 domain folds back to block the active sites. Signaling proteins containing P-tyrosine residues will bind to the SH2 domains, inducing the relief of autoinhibition. This 524 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.27 Function of SH2-containing N-SH2 to the phosphatase domain. PTPs in receptor tyrosine kinase signaling. Phosphorylation of C-terminal Tyr residues SH-PtPs contain two SH2 domains, a more relieves autoinhibition and allows binding of C-terminal domain (C-SH2) and a N-terminal substrates such as cytoplasmic proteins, SH2 domain (N-SH2). The ground state of receptor tyrosine kinases (RTKs) or adapter SH-PTPs is an inactive, autoinhibited state that proteins associated with activated RTKs. is stabilized by the intramolecular binding of

mechanism helps to activate the PTP enzymes and to target it to substrates. As an example, P-Tyr residues of autophosphorylated RTKs serve as docking sites for the SH2-groups of the PTP, directing the activity either towards the P-Tyr residues of the receptor or towards other signaling proteins such as GTPase-activating proteins (GAPs) of the Ras-signaling pathway or adapter proteins such as the IRS protein. Another mechanism for the activation of SHP1 and SHP2 employs the intramolecular binding of P-Tyr residues of the PTP to the N-terminal SH2 domain. Both, SHP1 and SHP2 are phosphorylated on speciﬁc tyrosine residues, and this modiﬁcation activates the enzyme. For example, SHP2 is phosphorylated on Tyr542 upon binding to the activated, autophosphorylated PDGF receptor. Two alternative functions may be ascribed to phosphorylation on Tyr542: (i) the relief of autoinhibition; and/or (ii) the creation of a docking site for SH2 domains of other signaling proteins. 10.4 Protein Tyrosine Phosphatases 525

10.4.4 Regulation of Cell Signaling by PTPs

The cellular functions of PTPs are closely associated with central growth-regulating pathways such as RTK signaling, cytokine signaling, the Ras pathway (see Chapter 11) and integrin signaling (see Section 13.4). The growth- and differentiation-promoting signals mediated by these pathways include multiple Tyr-phosphorylations on RTKs, adapter proteins, non-receptor tyrosine kinases and other signaling molecules such as GAPs, and transcription factors such as the Stat proteins (see Section 12.2.2). Because Tyr-phosphorylation can have either a positive (e.g., RTKs) or negative (e.g., Src) effect on the activity of signaling proteins (Figure 10.28), PTPs can have an

(a) Signal

S Inactive Active PTP PTK PTK

P P S PTP active Signal

Signal Signal

(b) Signal

PTP Inactive Active

PTK PTK Signal

P Signal

Figure 10.28 General functions of tyrosine pathways by tyrosine phosphatases. There are phosphatases in signal pathways. (a) Negative nonreceptor tyrosine kinases, such as Src regulation of signal pathways by tyrosine kinase, that are inactivated by signal-controlled phosphatases. Signal transduction of tyrosine Tyr phosphorylation. In this case, the kinases may be influenced in a negative dephosphorylating activity of tyrosine manner by tyrosine phosphatases in two ways. phosphatases can carry out a positive Tyrosine phosphatases may dephosphorylate regulation of signal transduction via tyrosine and inactivate both the activated, kinases. PTK, protein tyrosine kinase; PTP, phosphorylated tyrosine kinase and also the protein tyrosine phosphatase; S, substrate phosphorylated substrate proteins, disrupting proteins. the signal; (b) Positive regulation of signal 526 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

antagonistic effect on signal transduction, or they may positively cooperate with signal transduction [22,25]. Based on these dual roles, the PTPs form an essential part of the proliferation-regulating network of the cell, and dysregulation of their functions has frequently been linked to the process of carcinogenesis.

10.4.4.1 Negative Regulation by PTPs, and Tumor Suppression An elaborate interplay exists in the cell between the activity of PTPs and RTKs. Generally, the activity of RTKs is downregulated by the activity of PTPs such that, when cells are treated with inhibitors of PTPs, a ligand-independent activation of RTKs and activation of downstream signaling paths occurs that demonstrates the importance of PTPs in maintaining ligand-independent RTK signaling at low levels.

& Negative regulation of signaling by PTPs: — Removal of activating P-Tyr in, for example, autophosphorylated RTKs.

In the presence of their ligand, signaling by RTKs is also antagonized and dampened by PTPs. A dampening effect of PTPs on signaling by activated RTKs may occur, for example, via the cleavage of P-tyrosine residues required for receptor activation and signal propagation. Other potential targets of PTPs for controlling RTK signaling are phosphorylated effector molecules of RTKs, such as the IRS adapter. Signaling by RTKs generally has a proliferation-promoting effect on cells, and the downregulation of RTKs by PTPs is an essential element in controlling cell proliferation. A loss of the damping function of PTPs in signaling pathways involved in tumorigenesis is thought to bring about an uncoordinated increase in Tyr-phosphorylation and, ultimately, an uncontrolled growth. Accordingly, many studies have implicated the dysregulation of PTPs in the development of cancer, and several PTPs have been identiﬁed as tumor suppressors.

& PTPs may function as tumor suppressors.

Two examples of PTPRs that act on the transcription factor Stat (see Section 13.2.2) and which are assigned tumor-suppressing functions are illustrated in Figure 10.29. In both cases, Tyr-phosphorylation of Stat3 is downregulated by the PTPR, leading to a dampening of the growth-promoting effect of this transcription factor during cytokine signaling.

10.4.4.2 Positive Regulation by PTPs, and Oncogenic Functions An example of positive regulation by PTPs is Src kinase. As explained above (see Section 10.3.2), the phosphorylation of Tyr527 of Src kinase is linked with an inhibition of kinase activity. The SH2 domain of Src kinase binds in an intramolecular reaction to the Tyr phosphate at the C terminus, leading to a blockade of the active center. Activation of Src kinase may be brought about by cleaving off the inhibitory phosphate residue. 10.4 Protein Tyrosine Phosphatases 527

Figure 10.29 Examples of tumor suppression dephosphorylating interleukin-6 (IL-6)- by PTPs. (a) Protein tyrosine phosphatase activated STAT3 at Y705 in colorectal cancer. (PTP) receptor-type D (PTPRD) Decreased STAT3 phosphorylation inhibits the dephosphorylates signal transducer and nuclear translocation that is required for activator of transcription 3 (STAT3) that is transactivation of its target genes to promote aberrantly activated in human glioblastoma tumorigenesis. Julien 2011 [22], figure 4. cells; (b) Similarly, PTPR-T functions as a Reproduced with permission of Nature tumor suppressor by specifically Publishing Group.

& Positive regulation by PTPs: — Removal of inhibitory P-Tyr as for example, in autoinhibited kinases.

Importantly, dysregulation of the positive regulatory function of PTPs can lead to oncogenic activation. Currently, several PTPs are known that are activated to an oncogene upon a gain of function mutation. The best-studied oncogenic PTP is SHP2, which is a member of the PTPN family and has been identiﬁed as a positive regulator of the Ras signaling pathway. Moreover, somatic mutations of the gene PTPN11, which encodes SHP2, have been identiﬁed in individuals with pediatric leukemia in [26].

10.4.5 Regulation of PTP Activity

PTPs are regulated post-translationally through a variety of modiﬁcations, including proteolysis, phosphorylation, sumoylation ubiquitination and, importantly, oxidation. 528 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Proteolysis: Controlled proteolysis is a common PTM that regulates PTP activity. For example, the calcium-sensitive protease calpain elicits cleavage of negative regulatory domains of several PTPN enzymes, such as PTB-1B and SHP-1, resulting in their activation. In addition, several members of the PTPR family are subject to a limited proteolysis of their extracellular domain. Phosphorylation: Tyr-and Ser/Thr-phosphorylation is a common regulator of PTP function. Tyr-phosphorylation of several PTPs appears to modulate phosphatase activity, as well as to promote the recruitment of PTPs into larger protein complexes. For example, Tyr-phosphorylation of SHP-2 serves to create binding sites for SH2 domains of upstream partners such as activated RTKs, and it also provides a docking site for downstream partners such as the SH2 domain- containing adapter Grb2. This adapter protein functions as a platform from which to direct activation of the Ras/MAPK pathway. In this way, Tyr phosphorylation serves to integrate PTPs into central growth-regulatory signaling pathways

& PTPs are regulated by: — Extracellular ligand binding — Ligand binding to signaling modules — Subcellular targeting — Oxidation of catalytic cysteine — Ser/Thr phosphorylation.

10.4.6 Oxidation of PTPs

A major regulation of PTPs is provided by a reversible oxidation of the catalytic site cysteine (Figure 10.30) [27]. All class I–III PTPs contain an essential cysteinyl residue, which exists in the thiolate state (S ) to execute a nucleophilic attack on

P-tyrosines (see Section 10.4.1). The pKa values for the catalytic cysteine are in the range of 4–6 for different members of the PTP which makes these sites highly susceptible to oxidation and leading to inactivation of phosphatase activity.

Reactive oxygen species (ROS) such as H2O2 inactivate PTPs by oxidizing the –SH group of the active site cysteine to sulfenic acid –SOH, that may be oxidized further under conditions of high oxidants. The initially formed sulfenic acid can rapidly react further with amino acids present in proximity to yield secondary reaction products. For the most part, the cysteine oxidation of PTPs can be reversed by treatment with reducing systems such as the thioredoxin system. Oxidation/ reduction of PTPs is therefore suited to switch PTPs between inactive and active states. It is now well established that the regulated production of ROS serves a regulatory role in signaling pathways involving tyrosine phosphorylations by shutting down PTP activity. PTPs act as key mediators of a crosstalk between, on 10.4 Protein Tyrosine Phosphatases 529

Figure 10.30 Regulation of RTK signaling at activity/presence of ROS producers and/or low the level of PTP oxidation. A high-reducing activity/presence of ROS scavengers, leads to environment in the cell, through reduction in increased PTP oxidation and enhanced RTK activity/presence of ROS producers and/or signaling. The figure indicates direct oxidation elevated activity/presence of ROS scavengers, of PTPs by O2 and H2O2. However, indirect leads to decreased PTP oxidation and oxidation effects of H2O2 have also been attenuated RTK signaling. A high-oxidizing suggested. After Ref. [27]. environment in the cell, through elevated the one hand, the redox status/ROS and, on the other hand, RTK signaling (Figure 10.31). Cellular ROS production and the redox status are shaped by the action of redox enzymes that either increase (e.g., NADPH oxidase, ﬂavoprotein) or decrease (e.g., catalase, glutathione peroxidase) the levels of ROS, and these enzyme are targets of regulatory inputs to generate redox signals that impinge on RTK signaling. A quantitative study on the redox status of PTPs in normal cells and cancer cells has revealed a high level of control and speciﬁcity in PTP oxidation [28]. Growth factor treatment of cells has been shown to induce a moderate (<5%) 530 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

Figure 10.31 Four potentially general third mechanism involves the inhibitory mechanisms for growth factor-induced PTP phosphorylation of PrxI on Tyr194, leading to

oxidation. Growth factor stimulation can cause locally elevated levels of H2O2. A fourth the activation of NADPH oxidase (NOX) mechanism is the activation of cPLA2, which complexes, either through increased NOX produces arachidonic acid that in turn serves expression or accumulation of NOX complex as a substrate for lipooxygenase to produce cofactors, leading to superoxide and H2O2 peroxidized lipids. ecSOD, extracellular production. A second pathway is the activation superoxide dismutase; PLA, phospholipase A; through Ser36 phosphorylation of p66Shc, Prx, peroxiredoxin. After Ref. [27].

leading to mitochondrial H2O2 production. A

increase in PTP oxidation, which points to highly regulated and locally controlled PTP oxidation. The amount of PTP oxidation is sensitive to the cell type, with human cells and tissues – including cancer cells – displaying distinct proﬁles of oxidized PTPs evoked by exogenous and endogenous ROS. 10.4 Protein Tyrosine Phosphatases 531

A link between RTK signaling and PTP oxidation is now clearly established. Growth-factor binding to receptors can trigger an increased production of ROS in a controlled and spatially restricted fashion, with multiple effects on PTP activity and feedback to RTK signaling. Both, the general cellular redox-state and extracellular ligand-stimulated H2O2 (or other ROS) production can lead to PTP inactivation, and this can control P-tyrosine-dependent signaling in either a positive or a negative manner. Examples of transmembrane receptors that are involved in redox signaling include RTKs such as PDGFR, B-cell receptors, and TNFa receptors. The mechanisms by which ligand binding to these receptors triggers PTP oxidation are varied and not yet completely understood. The control of receptor signaling by PTP oxidation is shown in Figure 10.32, using the B-cell receptor as an example. The binding of an antibody to the B-cell receptor (Section 13.3) induces phosphorylation of associated protein kinases such as the Lyn and Syk non-RTKs. Further signaling to downstream effectors is dependent on Lyn and Syk phosphorylation and this leads, among others, to the þ þ creation of a Ca2 signal that activates a Ca2 -dependent transmembrane oxygenase. H2O2 is subsequently formed by the oxygenase, and this in turn leads to the oxidation and inhibition of a PTP. As a consequence, the PTP can no longer

Figure 10.32 Control of B-cell receptor redox signal triggered by the activated B-cell signaling by oxidation of PTPs. Signal receptor. The antigen-bound receptor þ transduction by activated B-cell receptors generates a Ca2 signal that activates a dual requires participation of the nonreceptor oxidase (Duox1). Activated Duox1 triggers the tyrosine kinases Lyn and Syk. The association production of reactive oxygen and H2O2, of Lyn with the receptor and further signaling leading to the oxidation and inactivation of the depends on Lyn phosphorylation that is PTP. Reduction of oxidized PTP is thought to controlled by the action of a PTP. A major use thioredoxin and glutathione (GSH). regulation of the PTP activity is exerted via a 532 10 Signal Transmission via Transmembrane Receptors with Tyrosine-Specific Protein Kinase Activity

remove P-tyrosines on the associated tyrosine kinases, the amount of active protein kinase is increased, and signaling is enhanced. Another example of redox regulation of PTP activity is provided by the regulation of dual-speciﬁcity phosphatases of the MAPK pathway. Here, the TNFa receptor- induced formation of ROS promotes MAP kinase phosphatase inactivation and thereby serves to sustain signaling through the mitogen-activated protein kinase, JNK (see Chapter 12).

10.4.7 Subcellular Localization of PTPs

The sequences of cytoplasmic PTPs frequently demonstrate sequence signals specifying a particular subcellular localization. This ensures that the PTPs are active only at deﬁned subcellular sites; speciﬁcally, localization to focal adhesion complexes mediated by proline-rich sequences has been observed for some PTPs. The presence of PDZ domains in cytoplasmic PTPs also shows that these can be integrated into larger signaling complexes formed for example, at ligand-gated ion channels.

Questions 10.1. Describe the typical domain structure of RTKs. What are the functions of these domains, and which PTMs are found on these domains? 10.2. Name at least five different classes of RTKs and their representative ligands. 10.3. Describe the principal sequence of events leading from RTK ligand binding to activation of the first effector protein. 10.4. Describe the events that lead from activation of the insulin receptor to the activation of Akt kinase. 10.5. Which mechanisms for ligand-induced RTK dimerization have been identified? Describe these mechanisms. Give an example of RTK heterodimerization. Why may this property be advantageous for the cell? 10.6. Describe at least three mechanisms by which autoinhibition of the TK domain of RTKs may be released. 10.7. Which functions can be ascribed to the TK activity in RTK signaling? Name at least four different types of partner proteins (including their general function) that are immediately downstream of RTKs. How are these proteins recruited to the activated receptor? 10.8. Describe the major mechanisms by which SH2 domains are involved in signal protein activation. 10.9. Explain the general functions of adapter/scaffolding proteins on the example of the function and regulation of IRS protein. References 533

10.10. What are the functions of Grb2 and Gab adaptors? 10.11. What are the major mechanisms for the downregulation and inhibition of RTKs? 10.12. Describe the domain structure of non-RTKs. Which function can be ascribed to these domains? 10.13. What is the structural basis of autoinhibition of Src kinase, and how can autoinhibition be relieved? Compare the autoinhibition of Src kinase to that of Abl kinase. 10.14. Describe the structural and functional basis of oncogenic activation of Abl kinase in chronic myelogenic leukemia. 10.15. Which classes of PTPs are known? Describe the enzymatic mechanism of P-Tyr-mediated cleavage of the major class of PTPs. 10.16. Which functions can be ascribed to PTPs in cellular signaling? Give examples of positive and negative effects on signal transmission. 10.17. Which mechanisms are used for the regulation of PTP activity?

References

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11 Signal Transmission via Ras Proteins

11.1 The Ras Superfamily of Monomeric GTPases

Summary Intracellular signal transduction employs central switching stations that receive, modulate, and transmit signals further. Small regulatory GTPases, the Ras protein being a well-known example, make up membrane-associated switching stations of particular importance for growth, differentiation, morphogenesis, vesicular trafficking, and formation of the cytoskeleton. The founding member of this class of GTPases is the Ras protein, which has attracted great interest because of its identification as a human oncogene. Accordingly, the small GTPases have been classified also as the Ras superfamily of monomeric GTPases comprising 150 human members. The Ras superfamily is divided into five major subfamilies on the basis of sequence and functional similarity [1]: the Ras/Rap; the Rho/Rac; the Rab; the Ran; and the Arf subfamilies (see Table 11.1). These proteins are monomeric regulatory GTPases of molecular mass 20–40 kDa, which run through a GTPase cycle as already discussed in Chapter 7; by cycling between the inactive GDP-bound state and the active GTP-bound state, these proteins function as molecular switches. Transit through the GTPase cycle of the Ras superfamily members is controlled by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) which show specificity for a particular subfamily within the Ras superfamily. The GDIs are regulators that are specific for the Rho/Rac and Rab subfamilies.

The main structural features of the major Ras superfamily proteins are shown in Figure 11.1. Much like the Ga-subunits of the heterotrimeric GTPases (see Section 7.4), the Ras superfamily members carry a conserved core G-domain, also named the Ras domain, that is responsible for GDP/GTP binding, GTPase activity,

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 536 11 Signal Transmission via Ras Proteins

Table 11.1 GEFs and GAPs of Ras superfamily members. For details, see Refs [2---4].

Ras Examples of subfamilies or Examples of GEF, gene or Examples of GAP, gene or family family members protein name protein name

Ras H-Ras, N-Ras, K-RasA, RasGRP, mSos, NF1, Ras-Gap, p120-GAP K-RasB mCDC25 R-Ras, M-Ras RasGRF Rap1Gap, TSC2 Rap1A,1B,2A,2B RapGEF1-6 Ral1A, Ral RLF Reb Rho/ RhoBTB subfamily mSos, BCR, Tiam1, Vav, BCR, ARHGAPs, Rac RasGRF2, CENTD DLC1 Rho subfamily Rac subfamily CDC42 subfamily RhoT subfamily Rnd subfamily Rab > 50 different Rabs RIN1, RABIF USP6, TBC1D1 Ran Ran, TC4 RCC1 RANGAP1 Arf ARF1-6 PSD, Sec7 CENTD, SMAP1

and the cycling of Ras proteins between active GTP-states and inactive GDP-states. The critical switch I and switch II elements of the G-domain change in conformation during GDP–GTP cycling and contribute to a preferential effector binding to the GTP-state and the core effector domain. Rho proteins carry an insert within the G-domain, and hypervariable sequences are found at the C terminus of the five major subfamilies, characteristic of the distinct family members. An important biochemical feature of a majority of Ras superfamily members is their posttranslational modification (PTM) by lipids. Most biological functions of the members of the Ras superfamily are linked to the cytoplasmic side of the cell membrane or to intracellular membranes, where specific signals are received and transmitted further. The interaction of Ras superfamily members with distinct membrane compartments and subcellular locations is mediated by lipidation at the N terminus and at the C terminus (Section 2.9). The majority of Ras, Rho and Rab family proteins terminate with a C-terminal CAAX tetrapeptide that directs the attachment of farnesyl or geranyl- geranyl groups to the cysteine residue of the tetrapeptide motif. The palmitoylation of cysteine is another PTM frequently found on Ras superfamily members. The ARF family members carry an additional hypervariable region at the N terminus, and these proteins use N-terminal myristoylation as additional membrane anchor. The principal functions of regulatory GTPases have already been outlined in a general sense in Section 7.4. The members of the Ras superfamily share the switch properties of the G proteins: by cycling between the inactive GDP-bound state and the active GTP-bound state, these proteins can receive and transmit signals. Incoming 11.1 The Ras Superfamily of Monomeric GTPases 537

Figure 11.1 Structural organization of the Ras farnesyl or geranyl-geranyl isoprenoid addition superfamily of small GTPases. The 20 kDa (Section 2.9.3). Some are modified additionally core G domain (corresponding to Ras residues by a palmitate fatty acid to cysteine residues in 4---166) is conserved among all Ras the HV sequence that contributes to superfamily proteins and is involved in GTP membrane association. Rab proteins also binding and hydrolysis. This domain is contain a C-terminal HV region that terminates comprised of five conserved guanine with cysteine-containing motifs that are nucleotide consensus sequence elements (Ras modified by addition of geranyl-geranyl lipids, residue numbering) involved in binding with some undergoing carboxymethylation. Arf þ phosphate/Mg2 (PM) or the guanine base family proteins are characterized by an N- (G). The switch I and II regions change in terminal extension involved in membrane conformation during GDP---GTP cycling, and interaction, with some cotranslationally contribute to preferential effector binding to modified by addition of a myristate fatty acid. the GTP-bound state and the core effector Rho proteins are characterized by an up to 13- domain (E; Ras residues 32---40). Ras and Rho amino acid “Rho insert” sequence positioned family proteins have additional C-terminal between Ras residues 122 and 123 involved in hypervariable (HV) sequences that commonly effector regulation. terminate with a CAAX motif that signals for

signals activate the members of the Ras superfamily by inducing the exchange of GDP for GTP. As outlined in Chapter 7, there are three classes of proteins that regulate the transit through the GTPase cycle of the Ras superfamily members:

GTPase-activating proteins (GAPs) Guanine nucleotide exchange factors (GEFs) Guanine nucleotide dissociation inhibitors (GDIs).

Alternation between the active GTP-bound and inactive GDP-bound states of the small GTPase is controlled by GEFs, which stimulate the exchange of GDP for 538 11 Signal Transmission via Ras Proteins

GTP, and by GAPs, which terminate the active state by stimulating GTP hydrolysis. The Rho and Rab families possess a third class of regulatory proteins, GDIs, which will be discussed in Section 11.4. Members of the individual families are distinguished by specific structural insertions or deletions that specify distinct interactions with the cognate GEFs, GAPs, GDIs and effector proteins, which are highly variable in nature. Numerous structural studies on the Ras superfamily have shown that the basic mechanisms of nucleotide exchange and GTPase action are well conserved among the different superfamily members, and that the GTPase switching station represents a conserved module for signal transduction within a large signaling network. The activating signals are conveyed to the GTPases mostly via the GEFs that act on the GDP-bound state to promote GDP/GTP exchange. Due to their multidomain structure, the GEFs can receive and relay a multitude of different signals, allowing for multiple controls of GTPase signaling. In the GTP-bound state, the signal is passed on to downstream effectors. Typically, each small GTPase mediates its functions through association with multiple and functionally distinct effectors, the selection of which may depend on the identity of the activating GEF effectors. This may be achieved by each GEF causing a spatially distinct distribution of GTPase activation, and by the function of the GEF as a scaffold that facilitates effector activation. Overall, small GTPases act as signaling nodes, with multiple input signals converging on GEFs and GAPs and upon GTPase activation, which initiates multiple output signals. The GAPs and GEFs show specificity for a particular subfamily within the Ras superfamily, and a large number of different GAPs and GEFs are now known that act on distinct Ras superfamily members [2]. As an example, there are more than 70 predicted GAPs in mammals with specificity for distinct members of the family of Rho proteins. Whereas, each branch of the Ras superfamily is regulated by GAPs and GEFs, the GDIs are regulators specific for the Rho/Rac and Rab families. The main properties of the GAPs, GEFs and GDIs of the Ras superfamily are presented in the following subsections.

11.1.1 Crosstalk Among Ras Superfamily Members

The Ras proteins and most of the other monomeric GTPases form part of a signaling network where a given GTPase can be activated by a variety of signals and can activate a set of different effectors that, in turn, can relay the signal to another GTPase belonging to the same family or to a different branch of the Ras superfamily. It is now well established that an intensive interplay exists between the various members of the Ras superfamily allowing for functional interactions. A key biochemical mechanism conveying signals to GTPases and facilitating crosstalk involves the guanine nucleotide exchange factors (GEF)s. 11.2 GTPase-Activating Proteins (GAPs) of the Monomeric GTPases 539

11.2 GTPase-Activating Proteins (GAPs) of the Monomeric GTPases

Summary GAPs negatively regulate signal transmission of the corresponding GTPase by increasing the rate of GTP hydrolysis. All GAPs are composed of multiple domains. In addition to the G-domain, GAPs harbor interaction modules for membrane association, for binding to PTMs on partner proteins, and for protein–protein interactions.

The primary function of the GTPase-activating proteins (GAPs) is to negatively regulate signal transmission. The human genome encodes at least 160 genes that are predicted to encode proteins with GAP activity. This large number underscores the importance of GAP activity for controlling GTPase signaling. The GAP proteins stimulate GTPase activity of the corresponding GTPase by an active role in catalysis, and the function of negative regulation of GTPase signal transduction is generally attributed to them. These proteins control the intensity of signal transduction via GTPases by reducing the lifetime of the active state of the GTPase, in some cases by several orders of magnitude. This reduces the number of GTPase molecules available for interaction with the downstream effectors, leading to an attenuation or termination of downstream signaling. The GAPs for the different Ras superfamily members are not conserved, and they employ a variety of methods to stimulate GTPase activity (Section 11.5.6). For a detailed review of function and regulation of GAPs, see Ref. [3].

& GAP proteins: — Negatively regulate GTPases — Provide catalytic residues for GTPase reactions — Contain distinct signaling modules such as SH2, SH3, PH, C1 and C2 domains — Are regulated by diverse mechanisms.

All GAPs are multidomain proteins. In addition to the catalytic domain harboring the GTPase-activating function, GAPs contain other signaling modules that allow GAP activity to be influenced by many regulatory signals; this indicates that the function of GAPs goes beyond the mere negative regulation of GTPase activity. PH domains, SH2 and SH3 domains, and C1 and C2 domains (see also Section 2.4.4), as well as other protein–protein interaction modules, are found on GAPs, and these domains may be used to target GAPs to activated receptors, to specific membrane compartments or to other signaling partners, and also to direct the GAPs to specific intracellular locations. Furthermore, some GAPs contain domains that mediate interaction with GEFs or GAPs for different GTPases, and 540 11 Signal Transmission via Ras Proteins

Figure 11.2 Domain structure of selected GAP explanation of the domains, see Section 2.4.4. proteins. The functional domains of Ras- PIP3, phosphatidyl inositol-3,4,5- specific p120-GAP, Rho-specific BCR and Rho- trisphosphate; RA, Ras association domain; and Arf-specific CENTD are shown, together SAM, sterile alpha-motif. with their interacting targets. For an

this allows for crosstalk between different branches of the small GTPases. The multidomain structures of three different GAPs are shown in Figure 11.2. GAP activity is subject to multiple regulatory inﬂuences such as protein–protein or protein–lipid interactions, and the binding of second messengers and/or PTMs such as phosphorylation and ubiquitination. Regulated membrane association is an important aspect of GAP function, and this may be mediated by PH and C2 þ domains that bind PIP3 and Ca2 , respectively. Both second messengers are formed in response to distinct signals (Chapter 8), allowing for a signal-dependent membrane-association of the Ras superfamily members.

& GAPs are regulated by: — Phosphorylation/dephosphorylation — Phospholipid binding to PH domains — Targeted degradation.

These multiple inﬂuences place the GAPs into a signaling network centered around the signaling node of the GTPase. 11.3 Guanine Nucleotide Exchange Factors (GEFs) of the Monomeric GTPases 541

11.3 Guanine Nucleotide Exchange Factors (GEFs) of the Monomeric GTPases

Summary GEFs activate signaling through the small regulatory GTPases by catalyzing GDP/GTP exchange between the inactive GDP-bound state and the active GTP- bound state. The exchange activity of the GEFs is located on distinct catalytic domains, of which the CDC25 and Dbl homology (DH) domains stand out. Much like the GAPs, GEFs are multidomain proteins that are subjected to multiple PTMs and undergo multiple protein–protein interactions. GEFs transduce a variety of activating signals from RTKs, GPCRs and second messengers to the small GTPases, and these proteins are the major activators of small GTPase signaling.

The GEFs play an essential role in the function of the Ras superfamily members by relaying the incoming, activating signals to the GTPase [2,3]. They do this by catalyzing the dissociation of GDP from inactive GDP-state of the GTPase. GEFs modify the nucleotide-binding site such that the GDP is released and subsequently replaced by GTP.

& Catalytic domains for nucleotide exchange: — CDC25 domain — Dbl homology domain — Sec7 domain.

Members of the different branches of the Ras superfamily are regulated by GEFs with distinct catalytic domains, and a large list of different GEFs has been identiﬁed that show speciﬁcity for distinct small GTPases. There is a large diversity of GEFs, as illustrated by the observation that the 20 human Rho GTPases are regulated by 83 Rho-GEFs, and these can be grouped into different families according to the nature of the catalytic domain.

11.3.1 Catalytic Domains

The following types of domain are responsible for the catalytic activity of GDP/GTP exchange:

The CDC25 homology domain is found on the members of Ras branch of GTPases. The Dbl homology (DH) domain is characteristic for the GEFs for most members of the Rho family of small GTPases. Typically, the DH domain is found in tandem arrangement with a PH domain that assists exchange activity by a variety 542 11 Signal Transmission via Ras Proteins

of mechanisms, for example by promoting membrane association or contributing to GTPase binding. The DH domain has been named after the oncoprotein Dbl, which contains a domain of approximately 180 amino acids for which homologs were later found in a growing family of oncogenes. Proteins containing the tandem DH-PH domain cluster are now included in the DH protein family, which comprises more than 60 members. The Dock domain has been identiﬁed on members of the DOCK subfamily of RhoGEFs as being responsible for nucleotide exchange. The Sec7 domain is the catalytic domain of the GEFs for the Arf family GTPases.

11.3.1.1 Mechanism of Nucleotide Exchange The afﬁnity of most small GTPases for GDP/GTP is in the lower nanomolar to picomolar concentration range. As a direct consequence, the dissociation rate of nucleotides is very slow and requires the participation of GEFs that accelerate dissociation – and thus the exchange reaction – by several orders of magnitude. GEFs catalyze the dissociation of GDP by modifying the nucleotide-binding site such that the nucleotide afﬁnity is decreased; as a result, GDP is released and subsequently replaced by GTP that is present in a 10-fold higher concentration compared to GTP. The mechanistic basis of nucleotide exchange by GEFs can be inferred from high-resolution structures between GEFs and members of the Ras-GTPase superfamily. The results of structural studies have shown that the various GEFs, despite carrying different catalytic domains, employ similar principles to deform the nucleotide-binding site. GEF binding induces conformational changes in the P- þ loop as well as in switch I and/or switch II. The P-loop and Mg2 are displaced from binding to the phosphates by inserting GEF-residues into the nucleotide binding site, so as to sterically and electrostatically expel the nucleotide leading to an empty G-nucleotide binding site (for details, see Ref. [3]).

11.3.1.2 Multivalency of GEFs Studies performed on a large number of GEFs have shown that there is no universal mechanism by which GEFs are activated and integrated into GTPase signaling pathways. Key to the multiple functions of GEFs in Ras signaling networks is their complex domain structure that allows for numerous interactions during GEF action. In addition to the catalytic domain, GEFs contain signaling modules that mediate for example, membrane association and subcellular location, þ the binding of second messengers such as Ca2 , and interactions with other signaling proteins such as transmembrane receptors (Figure 11.3).

& GEFs transduce signals from: — RTKs — GPCRs þ — Second messengers, such as Ca2 . 11.3 Guanine Nucleotide Exchange Factors (GEFs) of the Monomeric GTPases 543

Figure 11.3 Domain structure of selected GEFs. For DH, Cdc25, see the text; RA, Ras association domain; REM, Ras exchange motif; RBD, Ras binding domain; PH, Pleckstrin homology domain; EF, C1, calcium binding motifs; for PDZ, see Section 2.4.4.

These modules allow GEFs to transduce signals from receptor tyrosine kinases, G protein-coupled receptors (GPCRs), adhesion molecules and second messengers, among others. Often, adapter proteins help GEFs to communicate with upstream signaling proteins. The mechanisms by which GEFs become activated include membrane recruitment and subcellular sequestration, relief of autoinhibition upon phosphorylation, allosteric regulation by binding of phospholipids and second messengers, and recruitment into multiprotein complexes.

11.3.1.3 GEFs Can Activate Two Different GTPases An important contribution to the diversiﬁcation of signaling by GTPases is provided by the presence of two nucleotide exchange domains in some GEFs. For example, the exchange protein mSos (Section 11.5.7) harbors two different exchange domains. The DH/PH domain of mSos serves for the activation of Rac 1, whereas the REM/CDC25 domain is responsible for the activation of Ras proteins. However, which of the two exchange domains is used appears to depend on the nature of the ligand that directs the membrane association of mSos.

11.3.1.4 GEFs as Effectors of GTPases: Linkage of and Crosstalk between GTPases GEFs are important downstream effectors of GTPase superfamily members, and this property is used to diversify GTPase signaling and to provide for an interplay between different GTPases. Many GEFs harbor a Ras-association domain that mediates binding to Ras-GTP, the active state of Ras. The binding activates the exchange activity of the GEF for its substrate GTPase and allows for transmission of the signal to this GTPase. 544 11 Signal Transmission via Ras Proteins

One particularly interesting example of the complexity of regulation and e effector function of GEFs is PLC- , an enzyme that hydrolyzes PtdInsP2 to þ generate InsP3/Ca2 -signals and diacylglycerol (DAG) signals (see Chapter 8). In addition to the catalytic domain for lipase activity, PLC-e also harbors a CDC25 domain and two Ras-association domains. The CDC25 domain can mediate nucleotide exchange on members of the Ras family, whereas the Ras- association domain of PLC-e interacts with activated Ras and Rap proteins, and this interaction promotes lipase activity.

11.4 Guanine Nucleotide Dissociation Inhibitors (GDIs)

The GTPases of the Rab and Rho/Rac families are subject to an additional level of regulation owing to their association with a class of proteins known as guanine nucleotide dissociation inhibitors (GDIs). These proteins bind the GDP or GTP form of the GTPase and prevent dissociation of the nucleotide. However, the GDIs also perform other functions [5]. Three distinct biochemical activities have been described for GDIs:

They inhibit the dissociation of GDP from the Rho/Rac or Rab GTPases, maintaining the GTPase in the inactive state and preventing GTPase activation by GEFs. GDIs also can bind to the GTP form of the GTPase, blocking both intrinsic and GAP-catalyzed GTPase activity and preventing interactions with downstream effectors. This property points to a crucial regulatory function of GDIs in GTPase signaling, although it remains unclear under what circumstances an interaction with the GTP-bound state of the GTPase might take place. GDIs modulate the cycling of the partner GTPases between cytosol and membranes. By forming high-afﬁnity complexes, the GDIs maintain the GTPases as soluble cytosolic proteins, preventing association with membranes. High-resolution structures of the complexes show that the C-terminal geranyl- geranyl lipid anchor of the GTPase is shielded from the solvent by its insertion into a hydrophobic pocket of the GDI. When the GTPase is released from the complex, it can associate with the lipid bilayer via its lipid anchor, allowing activation by membrane-bound GEFs and signaling to effector targets at the membrane.

& GDIs: — Act on Rho/Rac and Rab proteins — Inhibit the dissociation of GDP from the GTPase–GDP complex — Inhibit the dissociation of GTP from the GTPase–GTP complex — Keep the GTPase in the cytosol — Are regulated by phosphorylation and GDI dissociation factors. 11.5 The Ras Family of Monomeric GTPases 545

The ability of GDIs to prevent both GEF action and GTPase activation of partner GTPases identiﬁes the GDIs as crucial elements of Rho/Rac and Rab signaling. An important question to be answered in this context relates to the mechanisms that trigger a release of the GTPase from the inhibitory complex with its GDI. The cell appears to employ two mechanisms for the dissociation of GTPase–GDI complexes:

Proteins named GDI dissociation factors (GDFs) can trigger the dissociation of GDIs from the complex with the GTPase. Phosphorylation of the GTPase–GDI complex is another tool for the regulation of GDI function. Both, Rho-GDIs and the Rho-GTPase have been shown to be phosphorylated by distinct protein kinases, and this phosphorylation may affect GTPase–GDI interaction in either a positive or negative manner.

11.5 The Ras Family of Monomeric GTPases

Within the Ras superfamily of monomeric GTPases, the Ras family historically has attracted the most interest, in large part because of a critical role of some of its members in human oncogenesis. The Ras protein in its narrow sense is a prominent member of the Ras family that is frequently converted to an oncogene, and it is for this reason that the Ras protein is currently one of the best- characterized signaling proteins with respect to both structure and function. In many aspects, the Ras protein is seen as being exemplary for the monomeric GTPases. Many of the basic principles of the switch function of Ras can be transferred to the other members of the Ras family, and to the whole Ras superfamily. The cellular function of the Ras family members can be summarized as being signaling nodes that are activated in response to diverse extracellular stimuli. These proteins process growth-promoting signals received by receptor tyrosine kinases and by receptors with associated tyrosine kinase activity, and transmit these into the cell interior (Figure 11.4). Furthermore, Ras family members transduce signals from heterotrimeric G proteins and from other monomeric GTPases, and relay these signals to a large variety of different downstream effectors that regulate the cytoplasmic signaling networks involved in control of cell proliferation, differentiation, and survival. Most of these effects are mediated through Ras signaling- induced changes in transcription.

11.5.1 General Properties of the Ras Protein

The Ras family of monomeric GTPases (36 human members) comprises the Ras proteins in its narrow sense, and also other GTPases such as the Rap, R-ras, Ral, and Reb proteins. The Ras protein (known also as p21ras due to its size of 21 kDa), as 546 11 Signal Transmission via Ras Proteins

Figure 11.4 The Ras protein as a central tyrosine kinase activity, via G protein-coupled switching station of signaling pathways. A receptors, via other small regulatory GTPases main pathway for Ras activation is via receptor and via second messengers. For clarity, the tyrosine kinases, which pass the signal on via membrane association of the Ras protein is adapter proteins (Grb2, Shc, Gab; see Section not shown. In addition, not all signaling 10.2.1) and guanine nucleotide exchange pathways that contribute to activation of the factors (GEFs) to the Ras protein. Activation of Ras protein are shown, nor are all effector Ras protein can also be initiated via reactions. Gbc: bc-complex of the transmembrane receptors with associated heterotrimeric G proteins.

the founding member of the Ras family, and the whole Ras superfamily received their name when Ras genes were identiﬁed as the tumor-causing principle of retroviruses that trigger sarcoma-type tumors in rats (Ras ¼ rat sarcoma). The general importance of Ras proteins in growth regulation was ﬁrst fully appreciated during the early 1980s, when close to 30% of all solid tumors in humans were shown to carry a mutation in the Ras gene; thereafter the Ras protein became the prototype of an oncoprotein.

& Ras proteins in the narrow sense: — H-Ras — K-Kas — N-Ras.

Mammals have at least three different Ras genes – H(arvey)-ras; K(irsten)-ras or K-ras; and the N(euroblastoma)-ras gene – with the K-ras gene producing a major (K-Ras 4B) and a minor (K-Ras 4A) splice variant. For each of these genes, oncogenic mutations have been identified in human tumors. The most frequently 11.5 The Ras Family of Monomeric GTPases 547 mutated gene is K-ras, with 70–90% mutations in pancreatic cancer and 20–50% in lung cancer. Most of the structural and biochemical data are available for the H-Ras protein [6] which, in the following subsections, is referred to as “the Ras protein” for simplicity. The Ras protein undergoes the typical G protein cycle of activation and inactivation that is mainly driven by the rate of GDP/GTP exchange and the rate of GTP hydrolysis. Considered in isolation, the Ras protein is a very inefficient – not to say a “dead”–enzyme. On the one hand the intrinsic rate of GTP hydrolysis is very low, whilst on the other hand the complex of Ras protein and GDP is very stable and dissociates only very slowly. The rate constants of the two processes are in the region of 10 4 s 1. However, as both reactions may be accelerated by Ras- specific GEFs and GAPs during the process of signal transduction, these proteins are essential elements of the switch function of Ras proteins.

11.5.2 Structure of the GTP- and GDP-Bound Forms of Ras Protein

The structure of the GTP-bound form of the Ras protein is shown in Figure 11.5.

Figure 11.5 Structure of the GTP form of the L2 and L4 loops, which have a switch function Ras protein. Crystal structure of the Ras in GTP hydrolysis. The numbers give the protein in complex with the GTP analog sequence positions of amino acids in the bc-imino-GTP (GppNHp). The figure shows loops. the P loop, in which Gly12 is located, and the 548 11 Signal Transmission via Ras Proteins

The Ras protein shows the G-domain typical of the regulatory GTPases (see Figure 8.14) with the sequence motives (Section 8.5) involved in binding the þ nucleotide and Mg2 . Three structural elements are of particular importance for the switch function of Ras protein: the P-Loop; the switch I region; and the switch II region. All three structural elements contact the c-phosphate of GTP. Upon GTP hydrolysis, the switch I (residues 30–38) and switch II (residues 59–67) regions undergo signiﬁcant changes in conformation that can best be described as a “loaded spring” mechanism (see Figure 7.23). & Important structural elements of Ras: — P-Loop: contacts c-phosphates of GTP — Switch I: changes conformation upon activation, interacts with effectors — Switch II: changes conformation upon activation.

The L2 loop of the switch I region is also known as the effector loop, and it is part of the core effector domain (residues 32–40). In the active GTP state, the effector domain provides interaction surfaces to downstream effectors that are not available in the inactive GDP form. The effector domain is essential for all effector interactions and thus serves to transmit the signal to the immediate downstream components of the Ras signaling node. The switch II region contains the conserved Gly60 that forms an H-bridge to the c-phosphate. Switch II also harbors the catalytically essential Gln61 residue and is involved in the interaction with GTPase-activating proteins.

11.5.3 GTP Hydrolysis Mechanism and Stimulation by GAPs

The rate of GTP hydrolysis is very low for the Ras–GTP complex and would not be suitable for cellular signal transduction, which normally includes complete inactivation within minutes after GTPase activation. Therefore, termination of the active GTP state requires the participation of GAPs as an essential step, increasing the rate of GTP hydrolysis up to 105-fold. Structural and biophysical studies have shown that GTP hydrolysis proceeds via an in-line nucleophilic attack of a water molecule on the b-phosphate of the leaving group GDP. Effective catalysis of GTP hydrolysis requires an optimal positioning and polarization of the attacking water molecule, and stabilization of the transition state. These requirements are fulﬁlled by the various GAPs in different ways. The GAPs for the members of the Ras superfamily are not conserved and employ various mechanisms to enhance GTP catalysis (for details, see Ref. [3]). The mechanisms used by different GAPs to enhance GTP catalysis are illustrated in Figure 11.6. One interesting approach is used by those GAPs speciﬁc for Ras protein and for Rho family members. Structural determinations of the Ras–GAP complex have shown that the GAP protein actively participates in catalysis by making an Arg residue available, which helps to stabilize the transition state of GTP hydrolysis. 11.5 The Ras Family of Monomeric GTPases 549

Figure 11.6 Mechanism of GAP-induced GTP GTP is shown in the transition state of the hydrolysis. (a) Schematic representation of hydrolysis. All figures are based on the crystal GTP hydrolysis, assuming an in-line structure of the mentioned G protein in replacement reaction with an associative complex with the GAP and a slowly hydrolyzing transition state and inversion of GTP analog or GDP-aluminum fluoride with stereochemistry of the c-phosphate; (b) the exception of Rap and RapGAP, which is Different GAPs use different ways to stimulate only a model based of the structure of Rap- GTP hydrolysis. Residues of the G protein and GAP alone. For details of the heterotrimeric G the GAP that are directly involved in catalysis proteins, see Section 7.5.4. Bos 2007 [3], figure are shown in blue and red, respectively. The 2B. Reproduced with permission of Elsevier.

& GTP hydrolysis by Ras: — Active participation of GAP in GTP hydrolysis — GAPs accelerate GTP hydrolysis up to 105-fold.

A structural element of the GAP known as ‘Arg ﬁnger’ harbors an invariant Arg residue (R789) that interacts with the b- and c-phosphate during hydrolysis. Arg789 stabilizes the transition state by neutralizing the charge of the c-phosphate developed during hydrolysis. A central function in GTP hydrolysis is attributed to Gln61 of switch II, since it is located in an ideal position for exact alignment of the water molecule and for stabilization of the transition state of GTP hydrolysis (Figure 11.7). The observation that position 61 – after position 12 – is the second most frequent site of oncogenic mutations in solid tumors is in agreement with the central importance of Gln61 for GTP hydrolysis by Ras protein. 550 11 Signal Transmission via Ras Proteins

Figure 11.7 The Ras---GAP complex. Structural view of the active site, with the important elements of catalysis. Ras structural elements are shown in yellow, GAP elements are shown in red.

As shown schematically in Figure 11.6, the GAPs speciﬁc for Rab and Ran proteins use different residues to enhance GTP hydrolysis. The GTPase of the a-subunits of heterotrimeric G proteins also uses an Arg residue for stabilization of the transition state of hydrolysis. In contrast to the Ras protein, this is localized in the cis conﬁguration on the a-subunit itself and is found in the linker between the helical domain and the G-domain.

11.5.4 Structure and Biochemical Properties of Transforming Mutants of Ras Protein

The ras genes are the most frequently mutated oncogenes detected in human cancer [7]. A comparison of the biochemical properties of mutated Ras proteins with those of the wild-type Ras protein shows that oncogenic activity correlates with increased lifetime of the GTP form. Ras proteins can be converted to oncogenic, transforming forms by mutations, in particular at positions 12, 13, and 61.

& Oncogenic mutations of Ras: — Frequently at positions 12, 13, and 61 — Increase lifetime of GTP state — Interfere with GAP function.

Gly12 located in the P-loop is especially sensitive to amino acid substitutions, and mutations at this position are the most frequent Ras mutations found in human tumors. The replacement of Gly12 with any amino acid other than proline leads to 11.5 The Ras Family of Monomeric GTPases 551 oncogenic variants of Ras protein that are no longer susceptible to negative control by GAPs. In the presence of GAPs, the oncogenic variants of Ras protein spend a much longer period in the activated state than does the wild-type Ras protein and can transmit a dominant signal in the direction of cell proliferation, favoring tumor transformation. An explanation for the oncogenic activity of the Ras mutants was provided by the structure of the Ras–GAP complex. The G12 of the P-loop is located very close to the main chain of the Arg finger of the GAP protein and to the Gln61 of the Ras protein. The replacement of glycine by other amino acids would lead to van der Waals repulsion and thus to displacement of the Arg finger and of Gln61. In the oncogenic G12 mutant of the Ras protein, an active role of the Arg finger in GTP hydrolysis is, according to this model, no longer possible. Gln61 is another amino acid that is highly sensitive to oncogenic mutations. It has a central function in GTP hydrolysis in that it contacts and coordinates the hydrolytic water molecule and the O-atom of c-phosphate of GTP, and thus stabilizes the transition state. Amino acids with other side chains apparently cannot fulfill this function, as shown by the oncogenic effect of Gln61 mutants in which Gln61 is replaced by other amino acids (other than Glu).

11.5.5 Membrane Localization of Ras Protein

The function of the Ras protein in cellular signal transduction is inseparably bound up with the plasma membrane. The Ras proteins associate with the inner side of the cell membrane with the help of lipid anchors, such as farnesyl residues and palmitoyl residues (see Section 2.9). Farnesylation (Figure 11.8) of the Ras protein occurs at the C-terminal CAAX sequence (A: aliphatic amino acid, X: Ser or Thr). In addition, the Ras proteins have a palmitinic acid anchor at different Cys residues in the vicinity of the C terminus. The membrane localization of the K-Ras protein is also supported by a polybasic sequence close to the C terminus. Membrane anchoring via the C-terminal modiﬁcations is absolutely necessary for the function of the Ras proteins. The lipid anchors have no inﬂuence on the catalytic activity of Ras GTPase. Rather, the membrane anchoring of the Ras protein has the role of bringing the latter to the membrane inner side, into the neighborhood of its upstream and downstream signaling partners.

11.5.5.1 Ras Nanoclusters A novel aspect of Ras-membrane interaction has been revealed by the observation that activated Ras proteins can cluster in distinct membrane microdomains called Ras nanoclusters [8]. The H-Ras, K-Ras and N-Ras isoforms harbor different C- terminal lipid anchors (see Figure 11.8), and their GTP-bound states segregate into distinct nanoclusters in an isoform-speciﬁc manner. It is believed that effector activation is augmented in these nanoclusters. The underlying mechanism is not yet understood, but biophysical experiments have shown that the membrane 552 11 Signal Transmission via Ras Proteins

Figure 11.8 The processed hypervariable C-terminal farnesylation, carboxymethylation, regions (HVR) of H-, N-, and K-Ras. In the case and the palmitoyl modifications or the stretch of H-ras, the division of the linker region of the of basic residues. The minimal plasma HVR into HVR1 and 2 (blue hues) is shown. membrane targeting sequence of H-ras is The minimal plasma membrane targeting abbreviated tH. sequences are outlined in orange and include

orientation of the GTP-bound G-domain is different for the Ras isoforms, allowing for an isoform-selective access of downstream effectors such as Raf kinase and PI3 kinase [9]. Both, the nanodomain localization and membrane orientation appear to cooperate in deﬁning binding of different Ras isoforms to their effectors.

11.5.6 GAPs in Ras Signal Transduction

The GAPs speciﬁc for the Ras branch of monomeric GTPases are known as Ras- GAP proteins, of which 14 are predicted to be encoded in the human genome.

& Ras-GAPs: — p120-GAP: multidomain protein with SH2, SH3, and PH domain — Neurofibromin.

A well-characterized Ras-GAP is the protein p120-GAP, the domain structure of which is shown in Figure 11.2. p120-GAP has a hydrophobic amino terminus, two 11.5 The Ras Family of Monomeric GTPases 553

SH2 domains, an SH3 domain, a PH domain, and a domain that is homologous to the calcium-binding domain of phospholipase A2. The catalytic domain for GAP activity is found in a 250-amino acid section close to the C terminus; three other highly conserved sequence elements are also found in this region. The PH domain is thought to facilitate membrane association via binding to phosphatidylinositol phosphates, and the SH3 and SH2 domains are involved in protein–protein interactions. As an example of the latter function, the SH2 domains of p120-GAP mediate specific binding to P-Tyr771 of the b-type PDGF receptor (see also Figure 10.11). Another Ras-GAP, the protein neurofibromin, is implicated in the disease Recklinghausen neurofibromatosis type I. Deletions of the neurofibromin gene or mutations leading to loss of catalytic activity have been found in neurofibromatosis patients.

11.5.7 Guanine Nucleotide Exchange Factors (GEFs) in Ras Signal Transduction

The members of the Ras branch of monomeric GTPases receive, in particular, signals promoting growth and differentiation, starting from RTKs and other transmembrane receptors. A major link between activated receptors and Ras protein is provided by GEFs, often acting in cooperation with adapter proteins. Signaling from activated transmembrane receptors to Ras involves a family of Ras- speciﬁc nucleotide exchange factors of which three members are known: Sos (Sos: son of sevenless, because of the role of this protein in signal transduction of the sevenless gene in Drosophila); Ras-GRF; and Ras-GRP. Historically, the function of Sos in Ras signaling was characterized ﬁrst and will be discussed in the following subsection.

& Ras-GEFs: Sos: — A multidomain protein with two exchange domains: DH-PH domain for Rac proteins, CDC25 domain for Ras — Is linked to activated receptors via the adapter Grb2 — Is activated by membrane recruitment.

11.5.7.1 Structure and Activation The Ras-specific Sos protein of mammalia (mSos) is a large protein with a complex domain structure allowing for diverse regulatory inputs (Figure 11.9; see also Figure 11.3). mSos proteins (two subtypes mSos1 and mSos2) consist of an N- terminal histone-binding domain, followed by a DH-PH domain specific for Rac, a Ras exchange motif (REM) domain, a CDC25 domain specific for Ras, and a C- terminal Pro-rich domain. The latter domain mediates interaction with SH3 domains of adapter proteins such as Grb2. Sos proteins are subject to multiple allosteric regulations. The results of structural studies have suggested a complex autoinhibitory mechanism according 554 11 Signal Transmission via Ras Proteins

Figure 11.9 Regulation of Sos1 by receptor membrane association. Importantly, Sos1 tyrosine kinases. An activated receptor tyrosine carries two G-nucleotide exchange motifs, the kinase (RTK) transduces signals to the Dibble homology domain (DH) and the Cdc25 downstream effectors PI3 kinase (PI3K; see exchange domain. The DH domain is directed Section 8.4), to the adapter Grb2 and the towards the Rac proteins, whereas the Cdc25 adapter complex Abi1---Eps8. The adaptors domain is specific for G-nucleotide exchange then bind to Pro-rich motifs PXXP on Sos1. PI3 on Ras. The exchange activity of the Cdc25 kinase produces phosphatidyl inositol-3,4,5- domain is subject to intramolecular inhibition trisphosphate (PIP3; see Section 8.4) that by the DH domain. REM, Ras exchange motif; binds to the PH domain of Sos1 and mediates H2A, histone 2A homology.

to which both the C-terminal and N-terminal regions of mSos negatively regulate the exchange activity. How this blockage is relieved is not yet clear (for details, see Ref. [3]). A positive feedback control is exerted by RasGTP. The Ras-GEF activity of mSos is allosterically stimulated by RasGTP, which binds to an allosteric site constituted by parts of the REM and CDC25 domain, and this interaction stimulates CDC25 activity towards Ras.

11.5.7.2 Regulation A major regulation of mSos is by recruitment to the plasma membrane where Ras is located. When transmembrane receptors with intrinsic or associated tyrosine kinase become activated, a redistribution of mSos from the cytosol to the plasma membrane and the activated receptor is observed. This membrane recruitment is a critical step in mSos function because it brings mSos into the vicinity of its substrate, the membrane-bound Ras protein. Translocation of mSos is mediated by the adapter protein Grb2 that has a SH2–SH3–SH2 domain structure (see Section 10.2.1.2). In the cytosol, mSos is found in tight association with Grb2 and, upon activation and autophosphorylation of the receptor, Grb2 binds via its SH2 domains to phosphotyrosines of the receptor and thus brings the associated mSos 11.6 Raf Kinase as an Effector of Signal Transduction by Ras Proteins 555 to the membrane. Formation of the Grb2–mSos complex is mainly mediated by binding of the SH3 domain of Grb2 to a Pro-rich sequence on mSos. Following membrane recruitment of Grb2–Sos, Sos and Ras engage in an interaction that results in the expulsion of bound GDP and subsequent formation of the active Ras- GTP form. The identiﬁcation of two monomeric GTPases, Ras and Rac, as targets of mSos and the presence of several domains interacting allosterically indicates a complex and variable function of mSos in vivo. Sos is an interesting example of an exchanger protein that is used in two different branches of the Ras superfamily of GTPases that control both growth-regulating and cytoskeletal functions. How both activities are coordinated, and how the various functional elements collaborate in vivo is largely unknown at present. Historically, the activation of Ras by the Grb2–mSos complex has been ﬁrst described. There are several pathways by which the Grb2– mSos complex can participate in Ras signal transduction:

Direct binding of Grb2-Sos to activated receptor tyrosine kinases: In this pathway, the SH2 domain of Grb2 binds to the phosphotyrosine of the activated receptor, whereby the Grb2–mSos complex is brought to the receptor and thus to the cell membrane (Figure 11.10). In the membrane-localized form, mSos protein interacts with Ras protein, which is also membrane-associated, and induces nucleotide exchange in the latter. Use of further adapters: The recruitment of GRb2–mSos to the activated receptor may include further adapter proteins, as for example, the p66Shc protein or the Gab1 adapter (see Section 10.2).

The Shc protein speciﬁcally binds via its PTB domain to autophosphorylated receptors such as the PDGF receptor and the EGF receptor, and becomes phosphorylated itself in the process (Figure 11.10). The phosphotyrosine residues may then serve as attachment points for the SH2 domain of Grb2 protein, whereby the Grb2–Sos complex is attached to the membrane. By a similar mechanism, the Gab 1 adapter can also mediate recruitment of Grb2–Sos to the receptor.

11.6 Raf Kinase as an Effector of Signal Transduction by Ras Proteins

A multitude of downstream effectors of Ras proteins have been identiﬁed that show preferential binding to the GTP-state of Ras and become activated upon exchange of GDP for GTP [10]. The best-characterized and validated effector of Ras function is the Ser/Thr-speciﬁc protein kinase Raf that is at the top of the three- tiered MAP/ERK cascade (see Chapter 12). Three isoforms of Raf kinase are known – named A-Raf, B-Raf and C-Raf (formerly named Raf-1) – that are encoded by distinct genes and differ in the details of regulation. The Raf family members are activated by the GTP-form of the Ras protein and transmit a growth-promoting signal via the MAP/ERK pathway down to the transcriptional level. Ras-GTP 556 11 Signal Transmission via Ras Proteins

Figure 11.10 Model of the function of Grb2--- phosphorylation of Shc is performed by the mSos and the adapter protein Shc in the Ras activated receptor. The Grb2---mSos complex pathway. The figure shows a highly simplified binds to the newly created phosphotyrosine version of the two known pathways of residues and is drawn into the signal pathway. involvement of the Grb2---mSos complex in In case (b), the Grb2---mSos complex acts signal transduction via the Ras protein. directly between the receptor and the Ras Phosphotyrosine residues of an activated, protein. In both situations, the Grb2---mSos autophosphorylated receptor R may serve as complex is targeted to the membrane and attachment points for the PTB domains of the from there, it can catalyze nucleotide exchange Shc adapter protein (a) or for the SH2 domain at the Ras protein. of the Grb2---mSos complex (b). In case (a), Tyr

interacts in a speciﬁc manner with Raf kinase and thus mediates membrane localization and activation of Raf kinase. Upon stimulation of Raf kinase, the signal is transmitted to the downstream effectors of Raf, the MEK1 protein kinases

& Raf kinase isoforms: — A-Raf, B-Raf, and C-Raf — Activation by Ras-GTP — Transmit signals to MAPK/ERK pathway.

The importance of RAF kinase for the regulation of cell proliferation is underscored by the observation that members of the Raf kinase family are frequently mutated in tumors. For example, about two thirds of malignant 11.6 Raf Kinase as an Effector of Signal Transduction by Ras Proteins 557

Figure 11.11 The domain organization of B- positive effect) and activation loop Raf. Conserved regions (CRs) and conserved phosphorylation, are indicated. The size of B- phosphorylation sites, involved in Raf Raf is noted at the right. regulation via 14-3-3 binding (negative or

melanomas harbor a mutation in the gene coding for B-Raf. Furthermore, various oncogenic variants of Raf genes have been found in tumor-causing retroviruses.

11.6.1 Structure of Raf kinase

All three Raf kinases share a common structure comprising three conserved regions, CR1, CR2, and CR3 (Figure 11.11):

The CR1 domain harbors two Ras-binding sites, the Ras-binding domain (RBD) þ as well as a Cys-rich domain. The latter also binds Zn2 and phospholipids such as phosphatidylserine and phosphatidic acid, and it is assumed that this region mediates membrane association of Raf, in addition to Ras association. The CR2 domain contains Ser and Thr residues that serve as regulatory phosphorylation sites. Mutations in CR1 and CR2 regions are known that lead to constitutive activity of Raf kinase and its oncogenic activation. The protein kinase activity is located on the CR3 region. In the inactive state, the kinase activity is subject to autoinhibition by the CR1 region. Activation requires the relief of autoinhibition and phosphorylation of the activation loop on Ser/Thr residues.

11.6.2 Mechanism of Activation and Regulation of Raf Kinase

Raf kinase is speciﬁcally activated and recruited to the membrane by the GTP-form of all three Ras-isoforms. Structural and biochemical studies have shown that the Ras-binding domain of Raf and the switch I region of Ras are involved in complex formation between Raf and Ras-GTP (Figure 11.12). Overall, Ras-dependent 558 11 Signal Transmission via Ras Proteins

Figure 11.12 Model of regulation and Raf kinase then activates the MAPK kinase activation of Raf kinase-1. Raf kinase-1 exists in cascade (see Chapter 12). Removal of the inactive and active states. The inactive state is activating phosphorylations by PP2A and the stabilized by binding to 14-3-3 proteins to introduction of inhibiting Ser-phosphorylations inhibitory Ser-P sites. Activation of Raf kinase-1 (e.g., PKA, Akt kinase, ERK) reverts Raf kinase requires: (i) removal of the inhibitory into the inactive state. The figure does not phosphorylations by protein phosphatase 2A address Raf dimerization and interaction with (PP2A); (ii) Ras protein in the active Ras-GTP KSR (see Figure 11.13). For details, see the state; and (iii) activating phosphorylations by text. various protein kinases (e.g., PKC). Activated

activation and membrane recruitment of Raf is a multistep process, during which inactive Raf transits into an active state in a complicated series of events including distinct protein–protein, protein–lipid interactions and speciﬁc phosphorylations [11]:

Inactive state: In the cytosol, Raf exists in an autoinhibited, closed conformation with the N-terminal regulatory region bound to the kinase domain. This state is stabilized by the binding of 14-3-3 proteins (Section 2.4.4) to distinct P-Ser residues as for example, Ser365-P within CR2 of C-Raf. Two protein kinases have been implicated in the inhibitory phosphorylations, PKA and Akt kinase. Activation: Ras-GTP binding to CR1 transitions Raf from the closed to the open state by relieving the autoinhibitory interactions. Activation and recruitment to the membrane is associated with the dephosphorylation of Ser365 by, for example, protein phosphatase 2A, dissociation of 14-3-3 proteins from CR2, and stabilization of the open conformation. During these processes, the Cysteine-rich domain of Raf appears to become accessible for binding of phospholipids, which enforces membrane association and activation. 11.6 Raf Kinase as an Effector of Signal Transduction by Ras Proteins 559

Activating phosphorylations: Further activation of Raf requires phosphorylation at several sites, including Ser/Thr phosphorylation within the activation loop which is thought to occur by autoactivation within Raf dimers. Furthermore, Tyr- phosphorylation by Src-family kinases has been reported to enhance Raf activity.

11.6.2.1 Formation of Homodimers and Heterodimers Raf subtypes A, B and C form homodimers and heterodimers that are now considered as the activating units that transmit the signal to the downstream effectors. All possible homodimers and heterodimers may be formed, and each dimer may have slightly distinct regulatory properties [12]. Together with the possibility of becoming activated by each of the four Ras isoforms (see Figure 11.13), this opens up a large variation and branching in signaling from Ras to Raf. Further diversification may be produced by the ability of various dimers to be regulated in differential fashion and to interact with different downstream components (Section 12.3). The influence of dimerization on Raf activity can be explained by an allosteric interaction between two kinase domains in a specific side-to-side dimer arrangement. The dimer state is thought to position the C-helix of in the kinase domain in a productive conformation necessary for optimal catalytic activity. As shown for the heterodimer B-Raf/C-Raf, activation within the dimer is also possible when one of the partners contains a kinase domain which is inactive due to binding of a specific inhibitor. The same binding mode appears to be used upon Raf binding to the scaffolding protein KSR (Kinase suppressor of

Figure 11.13 (a) Multiplicity of interactions probably of C-Raf causes the dissociation of between Ras, Raf, MEK and ERK. Six of the Raf heterodimers and prevents the possible 24 interactions between the four Ras reassociation of Raf with Ras, limiting signal components and the six Raf components are duration. In the next tier, phosphorylation of indicated by arrows. Two of the 12 possible Raf MEK1 by ERK represses the activation of stable and MEK interactions and two of the four MEK1---MEK2 heterodimers. Finally, ERK possible MEK and ERK interactions are homodimers, which bridge scaffolds (KSR1) indicated by arrows; (b) Dimer formation in and substrates (phospholipase A2; cPLA2), are the Raf pathway. Ras promotes the required to maintain signal specificity at heterodimerization of B-Raf with the scaffold different subcellular locations. Blunt arrows KSR1, C-Raf, or A-Raf. These heterodimers denote inhibitory effects; normal arrows have high MEK kinase activity and probably indicate activation; double arrows indicate represent the MEK activating unit. ERK- dimer formation. mediated phosphorylation of B-Raf and 560 11 Signal Transmission via Ras Proteins

(b) Ras

KSR1

B-Raf C-Raf

A-Raf

P P P P MEK-1MEK-2

P P P P KSR1 ERK2 ERK2 P P P P ERK2 ERK2 cPLA2

Figure 11.13 (Continued)

Ras). KSR links Raf signaling to downstream substrates within the MAPK/ERK cascade (see Figure 11.13 and Chapter 12). How the dimerization of Raf is induced is not yet understood, but it is possible that an activation-induced dimerization of Ras-GTP or the inclusion of Ras-GTP into nanostructures is involved.

11.6.2.2 Negative Feedback by Inhibitory Phosphorylations A major control of Raf activity is effected by inhibitory Ser-phosphorylations introduced by ERK kinase. This kinase is at the bottom of the MAPK/ERK cascade, and transmits Ras-Raf signals to many downstream components. ERK regulates RAF in a negative feedback by phosphorylating Raf on ﬁve Ser residues; these phosphorylations interfere with Raf dimerization and thus have a negative impact on kinase activity (Figure 11.13b).

& Regulation of Raf kinase by: — Binding to Ras-GTP — Phosphorylation — Dephosphorylation — Oligomerization — Binding to 14-3-3 proteins — Raf kinase inhibitory protein.

Protein–protein interactions are also implicated in Raf regulation. Inactive Raf is found in a multiprotein complex, and a large number of proteins have been reported to interact with Raf kinase. One widely expressed and highly conserved 11.7 Further Ras Family Members: R-Ras, Ral, and Rap 561 modulator of Ras function is the Raf kinase inhibitory protein that binds to Raf and inhibits the transduction of signals to downstream effectors of Raf. By this function, the Raf kinase inhibitory protein is considered to be another important control element of signaling through the MAPK/ERK pathway. A further important regulatory element within the Ras/Raf network is the E3 ligase IMP (impedes mitogenic signal propagation) which is an effector of Ras. IMP inhibits Raf signaling by inactivation the adapter KSR that couples activated Raf to its substrate MEK1 [13].

11.6.3 Oncogenic Activation of Raf

The pathway from growth factor receptors to Ras protein and – via Raf kinase – to the MAPK pathway is critical for growth regulation, and mutations of its central components are frequently associated with tumorigenesis. It is therefore not surprising that many mutants of Raf kinase are known that lead to constitutive Raf activity and to oncogenic activation. This is especially true for the B-Raf gene that has been identified as a human oncogene mutated in approximately 7% of human cancers, with a focus on melanomas. A large number of different missense mutations of B-Raf have been identified in human cancers, with the majority positioned in the kinase domain of B-Raf. Some 90% of these mutations correspond to a V600E substitution in the activation loop of B-Raf that leads to an enhanced kinase activity and can cause growth transformation of cultured human cells. The V600E mutation is thought to mimic phosphorylation in the activation loop by inserting a negatively charged residue close to an activating Ser-phosphorylation site, and it has been identified as a marker for aggressive subtypes of thyroid cancer [14]. The oncogenic activation of Raf kinases can also be achieved by deletions or mutations at the N-terminal conserved regions that relieve autoinhibition.

11.7 Further Ras Family Members: R-Ras, Ral, and Rap

Further monomeric GTPases are known that are closely related to Ras proteins, but perform distinct cellular functions and form part of the Ras signaling network. Of these, the R-Ras, Ral and Rap proteins have been well studied.

R-Ras: The R-Ras proteins are involved in the regulation of cell migration and in neuronal development. Members of the family of plexin transmembrane proteins have been identiﬁed as upstream regulators of R-Ras. The plexins are transmembrane receptors for axonal guidance molecules, known as semaphor- ins. The cytoplasmic domain of plexins harbors GAP activity for R-Ras and acts as a negative modulator of R-Ras during axon and dendrite remodeling. R-ras has a nearly identical effector-binding region to H-, N-, and K-Ras, and couples to a similar spectrum of Ras effectors. An important downstream effector is PI3 562 11 Signal Transmission via Ras Proteins

kinase (see Section 9.4) which becomes activated by R-Ras-GTP to produce the

second messenger PtdInsP3. Ral: The Ral GTPases have been implicated in the control of a variety of cellular functions, including the control of cell proliferation, cell motility, intracellular cell trafficking, and maintenance of cellular architecture. A large part of these functions is mediated by a linkage of the Ras signaling pathway to Ral function. GEFs with specificity for Ral have been identified as downstream effectors of Ras protein that interact specifically with the GTP-form of Ras. Furthermore, it has been shown that Ral activation can contribute to Ras-induced tumorigenicity, thus implicating Ral in the control of cell proliferation. Another major occupation of Ral is the regulation of a secretory machine, named the exocyst [15]. The exocyst is a large protein complex that participates in targeting and tethering vesicles to specific membrane sites. Protein components of the exocyst have been identified as effectors of Ral proteins. Rap: Rap GTPases, with Rap1 as its best-studied member, are implicated for example, in the control of integrin-mediated adhesion of cells to the extracellular matrix, and in cadherin-mediated cell–cell interactions. The function of Rap proteins in these processes is subject to multiple controls of multidomain GEFs and GAPs [16]. Of the upstream activators, the GEFs for Rap1 are of special interest because of their regulation by second messengers. The exchange protein þ CalDAG-GRF1 is activated by Ca2 , DAG and the GEF named Epac requires cAMP for its exchange function (Section 8.2). By these mechanisms, activation of Rap1 is linked to central signaling pathways of the cell (Figure 11.14).

& RAP GEFs: þ — RAPGRP: activated by Ca2 and DAG — Epac: activated by cAMP.

A large number of effector proteins have been identiﬁed for Rap proteins, as for example, protein kinase D (PKD), the adaptors Riam and Af-6, the Rac GEFs Tiam1 and Vav, and the Rho-GAPs RA-RhoGAP and Arap 3. These effectors link Rap activity to regulators of cell–cell interaction, cell adhesion and formation of the cytoskeleton. In particular, the linkage to Rho and Rac GTPases provides for a link to the major GTPases regulating junction formation and cell adhesion.

11.8 Reception and Transmission of Multiple Signals by Ras Protein

11.8.1 Multiple Input Signals of Ras Protein

The signals that activate Ras are delivered by the most part via the Ras-speciﬁc nucleotide exchange factors. As outlined above, the GEFs are multifunctional 11.8 Reception and Transmission of Multiple Signals by Ras Protein 563

Figure 11.14 Regulation of Rap GTPase. CalDAG---GEF1 is linked to phospholipase þ Major regulatory inputs to Rap GTPases are C (PLC) signaling via Ca2 and diacylglycerol delivered via GEFs. The exchange protein Epac (DAG) signals. A negative feedback between is activated by cAMP signals originating for Epac and RapGTP helps to dampen the example, from activated GPCRs. influence of cAMP on Rap signaling. proteins that can interact with a variety of signaling proteins and form parts of major signaling pathways. The main upstream signals of Ras activation originate from (Figure 11.15):

Binding of growth factors to receptor tyrosine kinases: This well-characterized pathway of Ras signal transmission was the ﬁrst to be discovered (see above) and involves adapter proteins (Grb2, Shc, Gab1) and GEFs (e.g., Sos). Binding of cytokines to receptors with associated tyrosine kinase activity (Chapter 13). Activation of integrins (Chapter 13). þ þ Ca2 and diacylglycerol signals: Changes in the concentration of DAG and Ca2 lead to activation of the Ras protein in brain. This effect is possibly mediated via 564 11 Signal Transmission via Ras Proteins

Figure 11.15 Summary of input signals of Ras. Input signals originate mostly from Ras-GEFs, Shc-Grb2---mSos complexes, and from G proteins. A negative regulation of Ras occurs by various GAPs.

þ specific GEFs. Ras-specific GEFs, which are regulated by Ca2 , are found in brain. Examples are the Ras guanyl nucleotide-releasing protein (RasGRP), þ which contains a Ca2 -binding motif, a DAG-binding motif, and the Ras guanyl þ nucleotide-releasing factor 1 (RasGRF1), which is activated by Ca2 /calmodulin. NO signals: Stimulation of N-methyl-D-aspartate (NMDA) receptors in the nervous system is linked to the activation of NO synthase and the creation of an intracellular NO signal. NO can directly activate Ras protein by redox-modification: the S-nitrosylation (see Section 8.10) of Cys118 of Ras has been shown to trigger GDP/GTP exchange by a complex mechanism and to convert Ras into the active GTP state.

& Input signals to Ras: — Receptor tyrosine kinases/growth factors — Receptors with associated tyrosine kinase/cytokines þ — Ca2 /diacylglycerol signals — NO

11.8.2 Multiple Effector Molecules of Ras Proteins

Operationally, Ras effector proteins are characterized by their preferential binding to the active GTP form of Ras as compared to the inactive GDP form. An intact Ras effector domain (residues 32–40 of Ras) is required for this interaction, and mutations within the Ras effector core impair binding of the effectors. Following 11.8 Reception and Transmission of Multiple Signals by Ras Protein 565

Table 11.2 Summary of Ras effector function.

Protein Function Substrate or target

Raf-1, A-Raf, B-Raf Serine/threonine kinase MEK1 and MEK2 serine/ tyrosine kinases p110a, p110b, p110d, Phosphoinositide 3-kinase Phosphatidylinositol (4,5)- p110c bisphosphate RalGDS Guanine-nucleotide-exchange RalA and/or RalB small factor GTPases RGL, RGL2, RGL3 Guanine-nucleotide-exchange RalA and/or RalB small factor GTPases Tiam1 Guanine-nucleotide-exchange Rac small GTPase factor AF-6 Adapter Profilin RIN1, RIN2, RIN3 Guanine-nucleotide-exchange Rab5 small GTPase factor NORE1 (also called Adapter MST1 serine/threonine RASSF5) kinase e PLC Lipase, Guanine-nucleotide- PtdIns(4,5)P2 exchange factor p120RasGap Ras GTPase-activating Ras, ras isoforms protein IMP (Impedes Mitogenic E3 ubiquitin ligase Kinase suppressor of Ras signal Propagation) this definition, a number of other signal proteins have been identified, in addition to Raf kinase, to which an effector function in Ras signal conduction has been attributed (Table 11.2). Which of the various effectors will be used by Ras appears to depend largely on the cell context. Verified Ras effectors are characterized by a Ras- binding domain of which three classes are known. The major downstream effectors are (Figure 11.16):

& Downstream effectors of Ras: — Raf kinase — MEK kinases — PI3 kinase — GEFs — PLCe

Raf kinase (see above): Most of the biological effects are mediated by the activation of Raf kinase, which is part of the MAPK/ERK signaling module (Chapter 12). MEK kinases: In addition to Raf kinase activation, Ras protein also mediates the stimulation of other protein kinases known as MEK kinases. These are signal proteins in another MAPK module, the JNK signaling pathway (Chapter 12), and transmit signals at the level of gene expression. 566 11 Signal Transmission via Ras Proteins

Figure 11.16 Targets of activated Ras. Only the major downstream effectors of Ras are depicted. Note that the activation of the GEFs such as Tiam, PLCe, Rin1 and RalGEfs links Ras signaling to other members of the Ras superfamily.

PI3-kinase: The GTP form of Ras protein speciﬁcally binds to and activates the catalytic 110 kDa subunit of PI3 kinase (Section 9.4). The activation of PI3 kinase leads to the formation of the membrane-localized messenger substance Ptd-Ins

(3,4,5)P3, which binds to the PH domains of signal proteins and can lead these to the membrane and activate them (Section 8.6). As a consequence, the Akt kinase pathway is activated which is a major pathway for the control of cell proliferation and apoptosis (Section 9.4). The linkage of Ras to the PI3 kinase/Akt pathway is therefore ascribed an important role in mediating the pro-survival and proliferation-promoting functions of Ras. GEFs: At least four different GEFs have been identiﬁed as Ras effectors (Figure 11.16). The signaling from Ras to GEFs that speciﬁcally activate other members of the Ras superfamily places Ras into the network of interacting GTPases, as discussed below. PLCe: The PLC isoform PLCe (Section 7.7.2) is a Ras effector that links Ras 2þ activation to formation of the second messengers DAG and InsP3/Ca that regulate a variety of key signaling enzymes, including protein kinase C isoforms. Furthermore, PLCe has exchange activity towards Rap proteins.

11.8.3 The Ras Signaling Network

The multiple inputs and outputs of Ras proteins place these regulators into the center of a signaling network functioning as a signaling node that controls major cellular processes. Currently, a wide spectrum of oncogenic mutations of Ras proteins is known, and the study of activating Ras mutations has revealed how 11.8 Reception and Transmission of Multiple Signals by Ras Protein 567 these proteins are involved in the regulation of central cellular functions (for details, see Ref. [7]). The central role of Ras proteins in tumorigenesis is illustrated by the ﬁndings that inappropriate Ras activation can lead to major disturbances in central cellular processes such as cell proliferation, apoptosis and cellular metabolism favoring tumor formation:

Cell proliferation: Ras proteins are major transducers of growth-promoting signals originating from growth factors and their receptors (Figure 11.17a). By activating key signaling proteins such as Raf kinases and PI3 kinase, Ras proteins promote transition through the cell cycle. The Raf kinases are at the top of the MAP/ERK cascade, and Raf activation ﬁnally triggers the transcription of many genes involved in cell-cycle progression, such as cyclin D and CDK6. Furthermore, MAP/ERK signaling regulates growth factor expression and the expression of antiproliferative proteins such as TGFb (see Section 13.1). Apoptosis: Ras proteins regulate both pro-apoptotic and anti-apoptotic proteins. The activation of PI3 kinase and the MAPK/ERK cascade promotes cell survival by decreasing the expression of pro-apoptotic Bak1 protein and increasing the expression of caspases, p53 and anti-apoptotic BCL-2 protein. Upon inappropriate Ras activation, these linkages enhance cell survival and suppress apoptosis.

Figure 11.17 Effects of oncogenic Ras on cell cyclin D1 and suppresses the cyclin-dependent proliferation. Oncogenic RAS (Ras) kinase inhibitor (CDKI) p27. The newly establishes independence from extracellular synthesized cyclin D1 associates with and growth factors and growth inhibitors, thereby activates the cyclin-dependent kinases CDK4 promoting exit from the G0 phase of the cell and CDK6, leading to the phosphorylation of cycle, progression through G1 and entry into Rb and transition into S-phase (for details, see S-phase. RAS induces the transcriptional Chapter 15). Aberrant activation of S-phase upregulation of growth factors and interferes entry by mutated Ras can lead to replicative with transforming growth factor-b (TGFb) stress and --- finally --- to genomic instability and signaling (Section 14.1) through inhibition of hyperproliferation of cells harboring mutated TGFb receptor expression or downstream Ras. For details, see Ref. [7]. signaling. RAS also upregulates the levels of 568 11 Signal Transmission via Ras Proteins

Energy metabolism: ERK and PI3 kinase signaling downstream of activated Ras have a profound inﬂuence on energy metabolism by increasing metabolites for the synthesis of major cellular macromolecules (Figure 11.17). These effects are mediated by the activation of mTor kinase and by increased levels of the transcription factor HIF1 (hypoxia-inducible factor). The upregulation of HIF1 augments multiple steps in glycolytic metabolism. For example, hexokinase, phosphofructokinase and lactate dehydrogenase become upregulated on the transcriptional level upon Ras activation. These effects allow tumor cells to shift their energy metabolism towards high levels of glycolysis, as observed in many tumors.

11.9 The Further Branches of the Ras Superfamily

Based on sequence and functional similarity, the superfamily of Ras proteins is grouped into ﬁve major branches: Ras, Rho, Rab, Arf, and Ran. Many of the basic properties and regulations of the Ras branch presented in the preceding sections can be transferred also to the other branches of the Ras superfamily. Therefore, only a short characterization of the other branches will be given in the following [1].

11.9.1 The Rho/Rac Family

The human genome harbors at least 20 different genes encoding proteins with a small GTPase domain of the Rho family consensus type. Within this family, the RhoA, Rac1 and CDC42 proteins are well-studied members (reviewed in Ref. [4]). The Rho proteins are key regulators of actin reorganization in response for example, to growth factors, stress conditions and cell–cell interactions. The regulation of cell polarity, cell movement, cell shape, cell–cell and cell–matrix interactions are major occupations of the Rho protein family members. As an example, RhoA promotes stress fiber formation and focal adhesion assembly, Rac 1 is involved in membrane ruffling, and CDC42 regulates filopodium formation. The RhoA, Rac1 and CDC42 proteins are each regulated by a diversity of GEFs and GAPs, and utilize a similarly diverse set of downstream effectors [3]. Another tool for the regulation of Rho GTPases are GDIs, which mask the prenyl lipid anchor and promote the cytosolic localization of these proteins. A major regulation of Rho/Rac proteins operates via changes in their subcellular distribution, which is dictated largely by the exact nature of the C-terminal lipid modification and variable sequences at the C terminus. For example, RhoA localizes predominantly to endomembranes when geranyl-geranylated, and to the plasma membrane when farnesylated. An essential requisite for the activation of Rho/Rac proteins is the dissociation of the RhoGDI which is regulated at different levels as for example, by RhoGDI displacement factors and by phosphorylation. 11.9 The Further Branches of the Ras Superfamily 569

L RTK H GPCR

R* G

Src PI3K PLCγ PLC

CamKII PDZRhoGEF RacGEFs Tiam1 p190RhoGAP

PI3K Rho

Cytoskeleton: stress fibers Membrane ruffling Lamellopodia

Figure 11.18 Major components of Rac and phosphorylation (see Section 9.6) for þ Rho activation. Receptor tyrosine kinase (RTK) activation. The Ca2 -signals may be initiated by and G protein-coupled receptors (GPCRs) RTKs and GPCRs that transmit signals to the activate Rac and Rho proteins via specific G- PCLb and PCLc. Rho is activated by a PDZ- nucleotide exchange factors (GEFs). The containing GEF and inhibited by the GTPase- RacGEFs are activated by PI3 kinase (see activating protein (GAP) p190Rho GAP. The Section 9.4), whereas the Tiam1 exchange latter also activates Rac. þ protein requires Ca2 signals and CamKII

The incoming signals to the Rho/Rac proteins are mostly delivered by the GEFs that are themselves regulated at multiple levels. The main ﬂow of signals used to regulate the Rho/Rac proteins is illustrated in Figure 11.18. More than 70 different downstream effectors for Rho/Rac proteins have been identiﬁed, and these are activated by a variety of mechanisms. As with Ras proteins, the tethering of effectors to the cell membrane by Rho/Rac proteins forms an essential part of the activation. Furthermore, Rho/Rac GTPases can activate effectors such as the protein kinase Pak by relieving them of autoinhibitory conformations. Overall, complicated crosstalk exists between the various members of the Rho/ Rac family and Ras family members (further details on this subject are available in the review of Bustelo et al. [4]).

11.9.2 The Rab Family

The Rab proteins comprise the largest branch of the Ras superfamily, with 61 members in human cells. Rab GTPases are regulators of intracellular vesicle transport and the trafﬁcking of proteins between donor and acceptor compartments 570 11 Signal Transmission via Ras Proteins

of the endocytic and secretory pathways [17]. The localization of Rab proteins to distinct intracellular compartments is dependent on C-terminal prenylation, and speciﬁcity is dictated by divergent C-terminal sequences. During its membrane targeting functions, Rab proteins cycle between the cytosol and the cell membrane, and this cycle is superimposed on a GDP/GTP cycle. The cytosolic pool of the Rab proteins is thereby maintained in the GDP-bound state by GDI proteins.

11.9.3 The Ran Family

The Ran (Ran: Ras-related nuclear) protein is the most abundant small GTPase in the cell, and is best known for its function in nucleocytoplasmic transport [18]. There is a single human Ran protein that is regulated by a nuclear Ran-speciﬁc GEF and cytoplasmic GAPs. During nucleocytoplasmic transport, the Ran protein interacts in a cyclical manner with various import and export receptors, thereby allowing the transport of cargo proteins in and out of the nucleus. One essential feature of the cyclical transport is the asymmetric distribution of the GDP- and GTP-bound forms of Ran between the nucleus and cytoplasm, which in turn is caused by the asymmetric distribution of GEFs and GAPs for Ran. In the nucleus, there is a prevalence of the GEF, and this results in a high nuclear concentration of Ran-GTP that interacts with importin to facilitate cargo release. Ran-GTP also interacts with exportin-complexed cargo to promote cargo export (for details, see Ref. [18]). RanGDP/RanGTP cycling is also involved in the control of DNA of replication and assembly of the mitotic spindle and nuclear envelope.

11.9.4 The Arf Family

The Arf proteins [19] are homologous proteins involved in the transport of speciﬁc types of vesicles, namely Cop-coated (Cop, coat protein) vesicles, between the endoplasmic reticulum and the Golgi apparatus. Similar to the Rab proteins, the Arf family members cycle between a cytosolic form and a membrane-associated form, regulated by the transition between the GDP- and GTP-bound states, which in turn is controlled by the action of GEFs and GAPs. Membrane translocation is in part controlled by a myristoyl switch (see Section 3.7.5), where GDP/GTP exchange by GEFs induces a conformational change that allows the myristoylated N-terminal helix of Arf proteins to interact with phospholipid bilayers, thereby promoting membrane insertion. Examples of effector proteins are the Cop-components of vesicles, among others.

Questions 11.1. Which are the major families within the Ras Superfamily of small regulatory GTPases? Name the structural and functional features that are common to these families. References 571

11.2. Describe at least two mechanisms by which GAPs activate GTP hydrolysis by regulatory GTPases. 11.3. Which catalytic domains are found on GEFs specific for Ras superfamily members? Which domains may be used by GEFs to associate with upstream and downstream effectors? 11.4. What functions can be ascribed to GDIs? 11.5. Which structural elements of Ras are most important for nucleotide binding, Ras activation, and effector binding? 11.6. What are the positions of H-Ras that are most frequently mutated in cancers? 11.7. Which modifications mediate the association of Ras proteins with the membrane? Describe the reactions involved in these modifications. 11.8. Which function has Raf-kinase? Which domains are found on Raf and what is the function of these domains? Which function is ascribed the V600E mutation in B-Raf? 11.9. Which reactions and protein-binding events are involved in Raf regulation? 11.10. Which major signaling paths mediate Ras activation? Describe the major reaction steps of these signaling paths. 11.11. Ras has many downstream effectors. How can it be verified whether these effectors are directly coupled to activated Ras? Name at least four different downstream effectors and describe the signaling events mediated by these effectors.

References

1 Wennerberg, K., Rossman, K.L., and Der, Rho/Rac family: regulation, effectors and C.J. (2005) The Ras superfamily at a glance. functions in vivo. BioEssays, 29 (4), 356–370. J. Cell Sci., 118 (Pt 5), 843–846. PubMed 5 DerMardirossian, C. and Bokoch, G.M. PMID: 15731001. (2005) GDIs: central regulatory molecules 2 Vigil, D., Cherﬁls, J., Rossman, K.L., and in Rho GTPase activation. Trends Cell Biol., Der, C.J. (2010) Ras superfamily GEFs and 15 (7), 356–363. PubMed PMID: 15921909. GAPs: validated and tractable targets for Epub 2005/06/01. eng. cancer therapy? Nat. Rev. Cancer, 10 (12), 6 Vetter, I.R. and Wittinghofer, A. (2001) The 842–857. PubMed PMID: 21102635. guanine nucleotide-binding switch in three Pubmed Central PMCID: 3124093. dimensions. Science, 294 (5545), 3 Bos, J.L., Rehmann, H., and Wittinghofer, 1299–1304. PubMed PMID: 11701921. A. (2007) GEFs and GAPs: critical elements Epub 2001/11/10. eng. in the control of small G proteins. Cell, 7 Pylayeva-Gupta, Y., Grabocka, E., 129 (5), 865–877. PubMed PMID: and Bar-Sagi, D. (2011) RAS oncogenes: 17540168. weaving a tumorigenic web. Nat. Rev. 4 Bustelo, X.R., Sauzeau, V., and Berenjeno, Cancer, 11 (11), 761–774. PubMed PMID: I.M. (2007) GTP-binding proteins of the 21993244. 572 11 Signal Transmission via Ras Proteins

8 Abankwa, D., Gorfe, A.A., and Hancock, 14 Nucera, C., Lawler, J., Hodin, R., and J.F. (2007) Ras nanoclusters: molecular Parangi, S. (2010) The BRAFV600E structure and assembly. Semin. Cell Dev. mutation: what is it really orchestrating in Biol., 18 (5), 599–607. PubMed PMID: thyroid cancer? Oncotarget, 1 (8), 751–756. 17897845. Pubmed Central PMCID: PubMed PMID: 21321384. Pubmed Central 2761225. PMCID: PMC3074562. Epub 2011/02/16. 9 Abankwa, D., Gorfe, A.A., Inder, K., and eng. Hancock, J.F. (2010) Ras membrane 15 Wu, H., Rossi, G., and Brennwald, P. (2008) orientation and nanodomain localization The ghost in the machine: small GTPases as generate isoform diversity. Proc. Natl Acad. spatial regulators of exocytosis. Trends Cell Sci. USA, 107 (3), 1130–1135. PubMed Biol., 18 (9), 397–404. PubMed PMID: PMID: 20080631. Pubmed Central PMCID: 18706813. 2824305. 16 Gloerich, M. and Bos, J.L. (2011) Regulating 10 Udell, C.M., Rajakulendran, T., Sicheri, F., Rap small G-proteins in time and space. and Therrien, M. (2011) Mechanistic Trends Cell Biol., 21 (10), 615–623. PubMed principles of RAF kinase signaling. Cell Mol. PMID: 21820312. Epub 2011/08/09. eng. Life Sci., 68 (4), 553–565. PubMed PMID: 17 Hutagalung, A.H. and Novick, P.J. (2011) 20820846. Role of Rab GTPases in membrane trafﬁc 11 Roskoski, R. Jr (2010) RAF protein-serine/ and cell physiology. Physiol. Rev., 91 (1), threonine kinases: structure and regulation. 119–149. PubMed PMID: 21248164. Epub Biochem. Biophys. Res. Commun., 399 (3), 2011/01/21. eng. 313–317. PubMed PMID: 20674547. 18 Rensen, W.M., Mangiacasale, R., Ciciarello, 12 Wimmer, R. and Baccarini, M. (2010) M., and Lavia, P. (2008) The GTPase Ran: Partner exchange: protein–protein regulation of cell life and potential roles in interactions in the Raf pathway. Trends cell transformation. Front. Biosci., 13, 4097– Biochem. Sci., 35 (12), 660–668. PubMed 4121. PubMed PMID: 18508502. Epub PMID: 20621483. 2008/05/30. eng. 13 Matheny, S.A. and White, M.A. (2009) 19 Donaldson, J.G. and Jackson, C.L. (2011) Signaling threshold regulation by the Ras ARF family G proteins and their regulators: effector IMP. J. Biol. Chem., 284 (17), roles in membrane transport, development 11007–11011. PubMed PMID: and disease. Nat. Rev. Mol. Cell. Biol., 12 (6), 19091743. Pubmed Central PMCID: 362–375. PubMed PMID: 21587297. 2670105. Pubmed Central PMCID: 3245550. 573

12 Intracellular Signal Transduction: The MAP Kinase Pathways

Summary The MAPK (¼ mitogen-activated protein kinase) or MAPK/ERK (ERK ¼ extracellular regulated kinase) pathways use a cascade of three protein kinases to transduce extracellular and intracellular signals down to the level of transcription. These pathways are of modular construction and act in a linear sequence involving sequential phosphorylations. The kinase at the top of the cascade receives a signal from an upstream activator, while the kinase at the terminal end of the cascade delivers a signal to transcription factors or other protein kinases. Often, the external signals are relayed to the MAPK pathways via growth factor receptors and GTPase switching stations at the cell membrane, such as Ras and Rac proteins. The various MAPK pathways are named according to the kinase at the lower end of the cascade. Well-characterized examples include the ERK, Jun, and p38 pathways.

Historically, the name MAPK referred to a protein kinase that is activated by mitogenic signals (as for example, insulin and growth factors) and stimulates the transcription of speciﬁc genes. Later, the kinase was dubbed ERK and the term MAPK/ERK evolved into the family name of a number of related kinases that not only form part of the distinct signaling pathways, the MAPK pathways, but also respond to a multitude of extracellular stimuli including growth factors and both chemical and physical stress. The MAPK pathways share a common organization, with three protein kinases being activated sequentially in a kinase cascade (Figure 12.1). The kinase at the top of the cascade receives a signal from an upstream activator, while the kinase at the terminal end of the cascade delivers a signal to transcription factors or other protein kinases. The various MAPK pathways are named according to the kinase at the lower end of the cascade, which is often collectively referred to as the MAPK enzyme. In mammals, 14 MAPK enzymes have been described that are categorized as either conventional or atypical MAPKs.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 574 12 Intracellular Signal Transduction: The MAP Kinase Pathways

Figure 12.1 Principle of signal transduction membrane by central switching stations such through intracellular protein kinase cascades. as the Ras protein or the Rac protein. In the The intracellular protein kinase cascades are case of the MAP kinase pathway, the cascade organized in modules composed in most includes at least three different protein cases of three protein kinases and a kinases. Specific regulatory processes may take scaffolding protein. The modules process effect at every level of the cascade; in addition, signals that are registered, integrated and signals may be passed from the different passed on at the inner side of the cell protein kinases to other signaling pathways.

The conventional MAPKs are:

the extracellular regulated kinases 1 and 2 (ERK1 and ERK2) the c-jun-terminal kinases 1, 2, and 3 (JNK1, JNK2, and JNK3) the p38 kinases the extracellular regulated kinase 5 (ERK5). 12.1 Organization and Components of MAPK Pathways 575

The atypical MAPKs comprise:

the extracellular regulated kinases 3, 4, and 7 (ERK 3, ERK 4, and ERK7) the Nemo-like kinases (NLK).

By far the most extensively studied group of mammalian MAPKs are the ERK1/2, JNK, and p38 isoforms. Signal transduction within the kinase cascade is coordinated with the help of scaffolding proteins that organize the kinases into a multiprotein complex. The signal is passed on by the last member in the phosphorylation cascade in the form of a phosphorylation of substrate proteins. In many cases, this process is linked to translocation of the protein kinase into the nucleus, where nuclear-localized substrates – particularly transcription factors – are phosphorylated. Other important substrates include protein kinases that transmit the signal further. In addition, the phosphorylation and activation of enzymes catalyzing key reactions of metabolism are observed.

& Major MAPK pathways in mammals: — ERK1/2 — p38 — JNK — ERK5.

The signals leading to the activation of MAPK pathways often originate from cell- surface receptors, and are relayed to the MAPK cascade via members of the superfamily of Ras proteins, or via other protein kinases. MAPK pathways also respond to both chemical and physical stress, thereby controlling cell survival and cell proliferation.

12.1 Organization and Components of MAPK Pathways

Summary The three protein kinases of typical MAPK pathways are organized as functional units, often with the aid of scaffold proteins. The protein kinases at the bottom of the cascade, the MAPKs or ERKs, are activated by the MEKs (MEK ¼ MAP/ ERK kinase), also named MAPK kinases (MAPKK). The MEKs are phosphorylated and activated by the kinases at the top of the cascade, the MEK kinases (MEKKs) or MAPKK kinases (MAPKKKs). Activation of MEKKs by the further upstream effectors involves membrane translocation, phosphorylation, oligomerization, and binding to scaffold proteins. 576 12 Intracellular Signal Transduction: The MAP Kinase Pathways

Incoming signals are received by the top (or uppermost) protein kinase of the core module and passed on to substrates by the terminal protein kinase. Signal transduction via sequential protein kinase reactions is a very flexible and efficient principle for the amplification, diversification and regulation of signals. Protein kinases (as noted in Chapter 9) are open to a range of regulatory influences, with either positive or negative regulation being possible at every level of a protein kinase cascade, such that the intensity of a signal can be modulated within broad boundaries. The protein kinase at the top of the kinase cascade (Figure 12.2) is often the entry point of signals, and is referred to as MEK kinase (MEKK), where MEK ¼ MAP/

Figure 12.2 Components and activation of residue, and thus are classified as dual- the ERK pathway. Ordering and specificity of specificity protein kinases. Phosphorylated protein kinases in the ERK pathway. MAPKs are inactivated by the action of dual- Extracellular signals are registered via receptor specificity protein phosphatases, the MAPK tyrosine kinases and passed on to the Ras phosphatases that remove the activating protein. Ras-GTP activates protein kinases phosphates on MAPKs. MAPK, mitogenic- belonging to the group of MAPKK kinases activated protein kinase; ERK, extracellularly (Raf kinases and MEKKs). The MAPKK kinases regulated kinase; MEK, MAP/ERK kinase; phosphorylate the downstream group of MAPKK, MAPK kinase; MAPKKK, MAPKK protein kinases, the MAPKKs at two Ser kinase; MEKK, MEK kinase; MKP, MAPK residues. The MAPKKs phosphorylate the phosphatase. MAPKs (ERK1 and ERK2) at a Tyr and a Thr 12.1 Organization and Components of MAPK Pathways 577

ERK kinase. This class of kinases are also known as MAPKK kinases (MAPKKK or MAP3K). The MEKKs transmit the signal further on to the MAP/ERK kinases (MEKs), also known as MAP kinase (MAPKK). At the bottom of the cascade are protein kinases named MAPKs or ERKs, which transmit the signal to downstream effectors.

12.1.1 MAPKs

One of the ﬁrst MAPK pathways to be characterized is the ERK pathway, which leads from mitogens, via the Ras protein, to activation of the terminal protein kinases known as ERK1 and ERK2. Other groups of terminal kinases include the JNK, p38, ERK3/4 and ERK5 kinases, and each of these have given their name to the pathways of which they form part. The terminal kinases are sometimes collectively referred to as MAP kinases (MAPKs).

& MAPKs: — Activated by MAPPKKs — Phosphorylation at TyrXSer — Mostly dimers — Often nuclear translocation following activation — Recognize substrates via docking sites.

The MAPK/ERK proteins are activated by further upstream kinases of the cascade, the MEKs. The latter phosphorylate the MAPK/ERK enzymes at a Tyr and a Thr residue of a Thr-X-Tyr motif located in the activation loop. This phosphorylation activates the MAPK/ERK enzymes by relieving a steric hindrance to substrate binding, and by a reorganization of the catalytic site. Furthermore, the conformational change induced by phosphorylation facilitates the formation of homodimers (see also Figure 9.4), which is required for the nuclear translocation of MAPK/ERK enzymes. Location of the substrates of the MAPKs can be either nuclear or cytoplasmic. The recognition and selection of substrate proteins occurs via speciﬁc docking sites on the substrates, which are bound by complementary binding domains on the MAPK [1]. These docking sites are located at some distance from the phosphorylation site.

12.1.2 MAPK Kinases (MAP2Ks, MEKs)

The MAPK/ERK proteins are phosphorylated by the preceding protein kinase, the MAP/ERK kinase (MEK) or MAP kinase (MAPKK). Each of the groups of MAPKs/ ERKs is activated by specific MEKs (Figure 12.3). The MEK proteins are dual- specificity protein kinases, as they have twofold specificity with respect to the nature 578 12 Intracellular Signal Transduction: The MAP Kinase Pathways

Figure 12.3 Summary of the three major the Rho family of small regulatory GTPases. MAPK pathways in mammals. The input TNFR, tumor necrosis factor receptor signals and the components of the Ras/Raf/ (Section 14.3); GCK, germinal center kinase; MEK/ERK pathway, the SAPK/JNK pathway and PAK, p21-activated kinase; MLK, mixed lineage the p38 pathway are shown. The figure also kinase; TAO, thousand-and-one-amino acid illustrates the multiple interactions between protein kinase. the three pathways. Rac-1 and Cdc42 belong to 12.2 Regulation of MAPK Pathways by Protein Phosphatases and Inhibitor Proteins 579 of the acceptor amino acid at the phosphorylation site of their substrate MAPK/ ERK.

& MEKs, MAPKKs: — Activated by MEKKs (MAPKKKs) by dual Ser-phosphorylation — MAPKs as substrates.

The MEK proteins are phosphorylated and activated by the MEK kinases (MEKKs, MAPKKKs or MAP3Ks). These phosphorylate the MEK proteins at two Ser residues, which are separated by three other amino acids. All known MEK proteins have a similar phosphorylation site in the conserved sequence LID/NSXANS/T (X: any amino acid).

12.1.3 MEK Kinases (MAPKK Kinases, MAP3Ks)

The MEKKs are Ser/Thr-speciﬁc protein kinases and form the entry point for signal transduction in a MAPK module. The best-characterized representative, Raf-1 kinase, is activated by Ras protein in its GTP-bound form. Other representatives of the MEK kinase group are Mos kinase and the protein kinases MEKK1-3.

& MAPKKKs: — Are activated by upstream kinases or monomeric GTPases — Have MAPKKs as substrates.

Signaling proteins that deliver signals to the MAPKKKs comprise mostly small GTPases, such as members of the Ras and Rho/Rac family or other protein kinases. In the latter case, the MAP3Ks are activated by upstream protein kinases, which are then also referred to as MAP4Ks. In general, the activation of MEKKs is a complex process that requires the steps of membrane translocation, phosphorylation, oligomerization, and binding to scaffold proteins. The mechanistic details of MEKK activation are not yet available.

12.2 Regulation of MAPK Pathways by Protein Phosphatases and Inhibitor Proteins

Summary The MAPK pathways are regulated by multiple mechanisms, including dephosphorylation by phosphatases, the binding of inhibitory proteins, and posttranslational modifications (PTMs) such as ubiquitination, phosphorylation, and acetylation. The various regulatory mechanisms create feedback loops that regulate the signal flow through the three-tiered cascade in both a positive and a negative manner. 580 12 Intracellular Signal Transduction: The MAP Kinase Pathways

12.2.1 Feedback Loops in MAPK Regulation

The following regulatory loops are responsible for speciﬁc properties of MAPK signaling, namely the ampliﬁcation of signal sensitivity, robustness of signaling, and oscillatory behavior. The following feedback loops have been shown to shape the dynamic behavior of the MAPK cascade (Figure 12.4):

Negative feedback from ERK to Sos, the GEF for Ras: activated ERK can phosphorylate Sos, leading to its inactivation as GEF for Ras. Negative feedback from MAPK/ERK enzymes to MAPK phosphatases: an enhanced MAPK signaling leads to an enhanced expression of MAPK phosphatases which deactivate MAPK/ERK enzymes

Figure 12.4 Feedback regulation of MAPK negative feedback loop exists where the signaling by inhibitors (I) and by MAPK inhibitor gene or the MKP gene are activated phosphatases (MKP). Downregulation of via transcription factors that are substrates of signaling by MAPKs can be due to an the MAPKs. Further negative regulatory loops enhanced expression of inhibitor proteins use phosphorylation and inactivation of the directed against the MEKK proteins, and by GEF mSos and phosphorylation and an enhanced expression of MKPs that stabilization of MKPs. dephosphorylate the MAPKs. In both cases, a 12.2 Regulation of MAPK Pathways by Protein Phosphatases and Inhibitor Proteins 581

Positive feedback from ERK to Raf inhibitor, favoring signaling through the MAPK cascade: activated ERK phosphorylates the Raf inhibitor, relieving its inhibitory action.

12.2.2 Regulation by MAPK Phosphatases

A major control of the MAPK pathways is exerted by protein phosphatases (Figure 12.5) that remove the activating phosphorylations of the kinase components of the MAPK pathways to negatively control signal flow through the kinase cascade. A variety of different phosphatases have been shown to remove the activating phosphate modifications. In mammals, members of the phosphatase family of dual-specificity phosphatases (DUSPs) are the primary phosphatases responsible for deactivation of MAPK signaling [2]. These phosphatases target primarily the MAPK/ERK enzymes of the kinase cascade, and are often referred to as MAPK phosphatases (MKPs). The MKPs inactivate MAPKs by removing the activating

Figure 12.5 The function of scaffolding upstream effectors; (C) binding of protein proteins in MAPK signaling. Scaffolding phosphatases (PPase) for dephosphorylation proteins organize the three protein kinases of and downregulation of MAPKs; and (D) the MAPK cascade into a functional unit. Other binding of substrates. For further explanations, functions of the scaffolding proteins may be: see the text. (A) binding to adapter proteins; (B) Binding to 582 12 Intracellular Signal Transduction: The MAP Kinase Pathways

phosphorylations within the signature sequence – pTXpY – located in the activation loop of the MAPKs. Two types of phosphatases within the DUSP family are involved in the deactivation of MAPK enzymes. The subfamily of typical MKPs (10 members) have varying substrate specificities and subcellular locations, while the subfamily of atypical DUSPs (16 members) shares certain characteristics with the MKPs, with some members having been shown also to regulate MAPKs. However, the subfamily of atypical DUSPs is less well-characterized and generally has different substrate specificities and physiological roles from the MKPs. The best-characterized MKP member is MKP-1 (DUSP1), which is localized predominantly to the nucleus; this confines its activity towards phosphorylated MAKP members which have undergone cytoplasmic–nuclear translocation. Other MKP members, such as DUSP6, exhibit a predominantly cytoplasmic location. Most MKPs dephosphorylate all three major groups of MAPKs – that is, the ERK, p38, and JNK groups. Much like the other components of the MAPK/ERK cascade, the MKPs are subject to regulation at multiple levels and they themselves form part of the regulatory networks that control MAPK signaling by either negative or positive feedbacks. A major regulation of MKPs is exerted by modulating MKP expression at the level of transcription or protein stability. Feedback inhibition of MAPK activity is achieved via the enhanced transcription of MKPs upon activation of MAPK pathways. Among the targets of the transcription factors activated by MAPKs are genes for MKPs, and an enhanced expression of the phosphatase genes promotes the dephosphorylation and downregulation of activated MAPKs. Another negative feedback regulation uses phosphorylation of MKP-1 by activated ERK, which leads to a prolonged half-life and an increased stability of the MPK. This is achieved via reduced ubiquitination and degradation due to Ser-phosphorylation of MKP-1 by its substrate, ERK1 or ERK2. Another mechanism of MKP control, as exemplified by regulation of the JNK module, employs a redox-regulation of the phosphatases (see Sections 9.7.2 and 10.4.3). The oxidation of DUSPs by reactive oxygen species (ROS) leads to their inactivation and serves to sustain signaling through the JNK module under conditions of oxidative stress. The mechanisms by which MKPs gain access to their substrates are not well characterized, though one major mechanism for substrate recruitment appears to involve the MAPK scaffolding proteins. As described in the following section, the phosphatases may be recruited to the kinase cascade via their binding to the scaffolds that organize the kinase cascade into distinct functional units. Being major regulators of MAPK signaling, the dysregulation of MKPs has been shown be involved in multiple diseases [3]. For example, MKP-1 has been implicated in controlling obesity in mice, while MKP-1, DUSP2 and DUSP10 appear to be crucial players in the regulation of immune responses. As might be expected from the central role of the MAPK/ERK pathway in the transduction of growth-promoting signals, MKPs (e.g., DUSP6) have been ascribed critical roles in development and cancer. 12.3 Scaffolding in MAPK Signaling 583

12.2.3 MAPK Regulation by Inhibitory Proteins

Another level of regulation of the MAPK/ERK pathways uses inhibitory proteins that interact with speciﬁc components of the pathway. Often, these inhibitors form part of a negative feedback loop where transcription of the inhibitors is induced by the MAPK cascade. However, these inhibitors may also be part of a positive feedback. One example of this type of regulation includes the Raf-1 kinase inhibitory protein (RKIP), which targets Raf-1 kinase and interrupts RAF-1 signaling by interfering with the phosphorylation of its substrate MEK [4]. RKIP is part of a positive feedback loop where it becomes phosphorylated by activated ERK, leading to a relief of Raf inhibition and an enhancement of signal transmission through the MAPK/ERK cascade.

12.3 Scaffolding in MAPK Signaling

The existence of distinct MAPK signaling modules with similar architectures raises the question how signaling is organized through these pathways. A large number of different upstream signals, such as activation of the RTK-Ras signaling pathway, the activation of G proteins, the activation of Toll-like receptors (TLRs) and integrin activation can trigger signal transduction through the three-tiered MAPK kinase cascade. Hence, it is important not only to identify the mechanisms that permit the activation of a particular module by an upstream input, but also to determine how other modules can be insulated from stimulation by that same input. The major mechanism employed to segregate the signaling pathways among the MAPK cascades involves using scaffold proteins that assemble the kinases into a multiprotein complex (Table 12.1; Figure 12.6). These MAPK-speciﬁc scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffolding components of the MAPK pathways has several advantages, as exempliﬁed by the scaffolding proteins:

Table 12.1 Examples of scaffolding proteins for MAPK pathways in higher eukaryotes.

Scaffold protein MAPK module

MP1, KSR, Morg1 ERK1, 2 JIP1, 2, 3, 4 JNK, p38 Paxillin ERK1, 2 Arrestin ERK1, 2 JLP JNK, p38 OSM p38 584 12 Intracellular Signal Transduction: The MAP Kinase Pathways

Figure 12.6 Scaffold proteins involved in specifically involved in linking GPCRs to the MAPK-signaling pathways. (a) The scaffold ERK1/2 module; (b) The scaffolding protein protein KSR links signaling from RTKs and b-arrestin directs the signaling outputs from GPCRs to ERK-signaling modules. MORG1 is ERK-module to specific cytosolic 12.3 Scaffolding in MAPK Signaling 585

They contribute to the specificity and selectivity of signaling by the assembly of distinct kinases into distinct MAPK modules. Different scaffold proteins can recruit a specific kinase module so that it can be localized to different regions of the cells to carry out different functional responses. They orient and allosterically activate the associated kinases, thereby increasing signaling efficiency. Due to these properties, scaffolds are important regulators of signal flow through the MAPK modules mediating the modulation of signal strength and duration. They may actively participate in signaling due to intrinsic kinase activity. Furthermore, components of the MAPK modules can themselves perform the scaffolding function. They prevent unwanted crosstalk between different MAPK modules. They mediate specific contacts to the upstream effector of the cascade and recruit these into the signaling cascade. By interacting specifically with upstream activators, the nature of the scaffold protein may determine which signal can activate a MAPK module. They may specifically interact with local adaptors or with the upstream effector to ensure spatial and temporal signal flow through the cascade. They may recruit other proteins such as protein phosphatases into the cascade, which allows for the modulation of signal flow through the cascade.

& MAPK scaffolding proteins: — Organize MAPK components in a multiprotein complex — Select upstream activators of MAPK cascade — Mediate linkage to distinct subcellular sites.

Several types of mammalian MAPK scaffolds have been identiﬁed (see Table 12.1 and Figure 12.6), and some function simply as adaptors while others have additional functions [5]. Many scaffolds are multifunctional and harbor binding sites for multiple binding partners, as illustrated in Figure 12.5 (part A) for the JNK scaffold. The coupling of important scaffolds to upstream signaling components is illustrated in Figure 12.5, using the examples of KSR, Morg-1, JIP2, and b-arrestin scaffolds. Examples of mammalian scaffold proteins that bind speciﬁcally to components of the ERK1/2 pathway include KSR (Section 11.6), MP1, and Morg1. Furthermore, the multifunctional cytoskeletal protein paxillin links ERK1/2

J compartments (Section 7.8); (c) Scaffolding components such as cell-surface receptors, roles of JNK-interacting proteins. The receptor-like proteins, upstream G proteins scaffolding proteins, namely JSAP1 and (JNK- and/or GEFs that can activate the respective associated leucine zipper protein/JIP4) have kinase module. Cdo, receptor-like been identified to provide scaffolding functions transmembrane protein; TLR, Toll-like receptor for JNK as well as p38MAPKsignaling modules. (Section 14.4); FAK, focal adhesion kinase In addition to their role in assembling the (Section 13.4). Dhanasekaran 2007 [5], three-tier kinase module, these proteins also Figure 2. Reproduced with permission of interact with the upstream signaling Nature Publishing Group. 586 12 Intracellular Signal Transduction: The MAP Kinase Pathways

signaling to focal adhesion kinases (Section 13.4). An important link of ERK1/2 signaling to G protein-coupled receptors (GPCRs) is provided by the multifunctional b-arrestin proteins that relay GPCR signals independent of G proteins (Section 7.8). Whilst detailed information on the mechanism of scaffolding is lacking for most MAPK scaffold proteins, an interesting mechanistic aspect has been discovered for the ERK1/2 scaffold KSR – that is, in order to scaffold ERK signaling, KSR must contain multiple binding sites that bring RAF, MEK and ERK together at the plasma membrane. The recruitment of RAF and MEK requires the kinase-like domain of KSR, which is highly homologous to typical protein kinases but lacks a catalytically crucial lysine residue in mammal; this categorizes KSR as a pseudokinase that is not active. Yet, surprisingly, KSR has been now ascribed an active role in ERK signaling by its ability to form heterodimers or trimers with B- Raf, C-Raf and MEK1, the component downstream of Raf isoforms (for original reports and a review, see Ref. [6]). By performing this function the “pseudo” kinase activity of KSR becomes activated and phosphorylates MEK1, which further illustrates the complexities of KSR–Raf–MEK interplay. In most cases, the scaffolding proteins mediate the localization of the MAPK module to specific subcellular sites by specifically interacting with membrane- localized targets. The scaffolding proteins direct MAPK modules to multiprotein complexes where the upstream effectors and substrates are localized. As a consequence of this colocalization, activation of the MAPK pathway is restricted to specific subcellular region and a temporal and spatial regulation of the MAPK cascade is thus possible. As an example, the scaffolding protein arrestin mediates the binding of a MAPK cascade to activated GPCRs (see Section 7.8).

12.4 The Major MAPK Pathways of Mammals

Of the mammalian pathways, the ERK, JNK and the p38 pathways predominate, and these are described in greater detail in the following subsections (Figure 12.3).

12.4.1 The ERK Pathway

The ERK pathway, which was the ﬁrst MAPK pathway to be identiﬁed [7], has long been known for its activation by Ras proteins, which recruit MAP3Ks of the Raf family (Raf1, A-RAF, B-RAF) to activate two MEKs – MEK 1 and MEK 2. These in turn activate two ERKs – ERK 1 and ERK 2 (Table 12.2).

12.4.1.1 Input Signals The following stimuli have been shown to activate and regulate the ERK pathway; some of these are active only in speciﬁc cells, while others operate in most cell types: 12.4 The Major MAPK Pathways of Mammals 587

Table 12.2 The ERK1/2 module.

Input signals: Mitogens: via Ras-GTP, Src kinase, PKC

MAPKKK; MEKK A-Raf, B-Raf, Raf-1, MEKK1/2/3, Mos kinase MAPKK, MEK MEK1, MEK2 MAPK ERK1, ERK2 MAPK substrates: Transcription factors: Elk, c-Fos Phospholipase A Protein kinases: RSK1-4; MSK, MNK, MK

1) Mitogenic signals originating from growth factor receptors (Chapter 10); via Ras. 2) Mitogenic signals originating from cytokine receptors (Section 13.2.1); via Ras or Src kinase, which has been shown to activate Raf kinase. 3) Signals from integrins (Section 13.4); via Ras and Src kinase. 4) Signals from Rho/Rac proteins. 5) Activation of GPCRs. There are many routes by which ligand-binding to GPCRs can transmit signals to the ERK pathway, and activation of the ERK pathway is frequently observed upon ligand binding to GPCRs. A main entry point is the Raf kinase, which can be activated by protein kinase C and inhibited by protein kinase A. As outlined in Sections 9.3 and 9.4, both enzyme families can be activated via G protein-signaling pathways by multiple mechanisms.

& The ERK1/2 pathway is activated via: — Growth factor pathways, Ras — Cytokine receptors, Ras, Src kinase — Integrins — Rho/Rac GTPases — GPCRs.

12.4.1.2 Substrates of ERKs ERKs deliver signals both to nuclear and cytoplasmic substrates. The activation of ERKs by MEK-mediated phosphorylation promotes their dimerization, after which the ERK dimers can translocate into the nucleus, where various transcription factors are phosphorylated and activated. An example of a nuclear substrate is the transcription factor Elk-1, which is positively regulated via the ERK pathway. Elk-1 is phosphorylated by ERK proteins speciﬁcally at the sites essential for transcription activation. Several signals meet at the level of Elk-1, as the activation of Elk-1 is mediated by different MAPK proteins, which in turn are activated by different MAP kinase pathways (see Figure 12.3). 588 12 Intracellular Signal Transduction: The MAP Kinase Pathways

& ERK substrates: — Transcription factors: Elk, c-Fos — RNA polymerase II — Phospholipase A2 — Many protein kinases.

Much of the mitogenic effects of ERK signals can be explained by the observation that ERK-induced expression of transcription factors stimulates the transcription of

D-type cyclins, which promotes the G1/S-phase transition in the cell cycle (Sec- tion 15.4.1). Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Section 4.2.7) has also been reported to be mediated by the ERK1/2 proteins.

Phospholipase A2 is an example of a cytoplasmic ERK substrate. The phosphorylation of a Ser residue of phospholipase A2 by ERK proteins leads to an activation of lipase activity, with a consequent increase in the release of arachidonic acid and of lysophospholipids. These can either act immediately as diffusible signal molecules or they may represent the ﬁrst stages in the formation of second messenger molecules. An important cytoplasmic substrate of ERK1/2 proteins is the ribosomal S6 kinase (RSK), also termed MAPK-activated protein kinase 1 (MAPKAP-K1; see also Section 5.2.1). RSK has an interesting modular structure in that it harbors two kinase domains that appear to be phosphorylated in the activation loops by distinct protein kinases, namely by ERK1/2 and by the protein kinase PDK1. A number of cellular functions have been proposed for RSK, including the phosphorylation of transcription factors such as CREB and NFkB, as well as the stimulation of protein biosynthesis by phosphorylation of the ribosomal protein S6.

12.4.2 The JNK and p38 MAPK Pathways

Two major MAPK pathways have been identiﬁed in mammals that are activated in response to environmental stress and inﬂammatory cytokines; these are the JNK and p38 pathways (Tables 12.3 and 12.4). Another, less well-characterized class of stress- activated pathways is the ERK5 pathway [also termed “Big MAP kinase 1” (BMP1)

Table 12.3 The JNK module.

Input signal: cytokines, UV, irradiation, DNA damage, protein kinase G, reactive oxygen species

MAPKKK, MEKK MEKK1-4, MLK2,3; TAO1, TAK1, ASK1 MAPKK, MEK MEK4, MEK7 MAPK JNK1, JNK2, JNK3 MAPK substrates transcription factors: c-Jun, ATF-2, Stat3 cytoplasmic proteins 12.4 The Major MAPK Pathways of Mammals 589

Table 12.4 The p38 module.

Input signal: cytokines, UV, irradiation, DNA damage, osmotic shock, cytokines (TNFa), reactive oxygen species

MAPKKK, MEKK MEKK4; TAO1, TAK1, ASK1, DLK MAPKK, MEK MKK3, MKK4, MKK6 MAPK p38a, p38b, p38c, p38d MAPK substrates: Transcription factors: Sap-1a; ATF-1,2,6; NF-AT; HBP1 Protein kinases: MK2, MNK1, MSK1 PLA-2;

pathway], with the ERK5 protein as terminal kinase. This pathway is activated in response to stress and growth factors. A multitude of input signals can activate the JNK and p38 pathways. Moreover, the substrates are very diverse with substantial overlap in the substrate spectrum that makes characterization of these pathways difﬁcult. In almost all instances, the stimuli that recruit the JNK pathway also recruit the p38 pathway. Together, the JNK and p38 pathways are characterized by an enormous complexity, such that only selected aspects of these pathways can be presented in the following subsections.

12.4.2.1 Input Signals and Signal Entry Points of the JNK and p38 Pathways External stimuli that activate the JNK/SAPK and p38 pathways include osmotic stress, exposure to bacterial toxins, and environmental perturbations such as heat, ultraviolet light, ionizing radiation, chemicals such as tunicamycin, and alkylating agents. These stresses lead to (among other things) the misfolding of proteins and their accumulation in the endoplasmic reticulum, which in turn induces activation of the JNK/SAPK and p38 pathways. A variety of MAPKKKs that act upstream of the stress-activated MAPKs have been described, reﬂecting the many different stimuli that recruit these pathways. The input kinases acting at the level of the MAPKKK proteins can be broadly divided into three families: the MEKK1–4 proteins; the mixed lineage kinases (MLKs); and the “thousand-and-one amino acid kinases” (TAOs). The latter family is quite speciﬁc in activating only the p38 pathway. The following signaling elements have been shown to recruit the JNK and p38 pathways by activating the MAP3Ks:

Activation via Rho/Rac proteins: Members of this family of small GTPases have members of the family of p21-activated protein kinases (PAKs) as effectors, which phosphorylate and activate MAP3K proteins. Furthermore, the MEKK1 protein has been identiﬁed as a direct effector of GTP-activated Rho/Rac proteins. Activation by tumor necrosis factor receptor (TNFR): The TNFR (Section 14.3) has associated adapter proteins that in turn interact with various protein kinases. 590 12 Intracellular Signal Transduction: The MAP Kinase Pathways

These include the germinal center kinases (GCKs) that function as MAP4Ks and activate kinases of the MAPKKK group, for example, MEKK1, by a complex mechanism. MAPKs and apoptosis: During speciﬁc processes of apoptosis, a controlled proteolysis of the MEKK1 polypeptide by caspases (see Chapter 17) is observed. The enzymatically active MEKK1 fragment is thereby released from the multiprotein complexes of the MAPK module, and it is thought that this freely diffusible form of MEKK1 is responsible for its pro-apoptotic properties.

12.4.2.2 Substrates of the JNK and p38 Pathways As with the ERK proteins, the JNK and p38 proteins phosphorylate and activate not only transcription factors but also other protein kinases (see Figure 12.7). Some of the protein kinase substrates – the MAPK-activated protein kinases MAPKAP-K2 and -K3 – are selectively recruited by stress-activated MAPKs, while others, such as the mitogen-activated protein kinase-interacting kinase (MNK), are activated by

Figure 12.7 Substrates of the major MAPK MNK, mitogen-activated protein kinase- pathways. The major substrates of activated interacting kinase; MSK, mitogen- and stress- MAPKs are transcription factors (bottom) and activated protein kinase; PRAK, p38-regulated/ protein kinases that phosphorylate and activated protein kinase; RSK, 90 kDa- regulate a multitude of substrates (top). ribosomal S6 kinase. MAPKAP-K, MAPK-activated protein kinase; 12.4 The Major MAPK Pathways of Mammals 591 both stress and mitogenic signals. The MAPKAP-K2 and -K3 polypeptides relay signals to the level of the cytoskeleton; the MNKs regulate the initiation of protein biosynthesis by phosphorylating the initiation factor eIF-4E (Section 5.2.6). The transcription factors regulated by the JNK/SAPK and p38 proteins include the Elk1, ATF2, and c-Jun proteins. Substrate recognition and selection of the JNK/ SAPK and p38 proteins (and also the ERK proteins) are mediated both by specific docking sites and by the nature of the amino acids surrounding the phosphoacceptor site. For the transcription factor substrates, specific docking domains have been identified that are located at a distance from the phosphorylation sites in the transactivation domain. These docking domains serve to increase the selectivity and specificity of phosphorylation, and they are used to recruit MAPK kinases into protein complexes at promoters, where they can phosphorylate other regulatory transcriptional proteins.

12.4.2.3 The JNK Module

The JNK module (Figure 12.3) contains the c-Jun NH2-terminal kinases (JNK) as a terminal kinase [8]. These kinases have been named alternatively, because of the activation by stress signals, as stress-activated protein kinases (SAPKs). Mammals express three JNKs – JNK1, JNK2 and JNK3 – of which several splicing isoforms are known. As indicated by their name, the c-Jun terminal protein kinases phosphorylate the transcriptional activator c-Jun at residues Ser63 and Ser73. The phosphorylation sites are located within the transactivation domain of c-Jun, and their phosphorylation correlates with enhanced trans-activating activity. Several other transcription factors have been shown to be phosphorylated by the JNKs, such as ATF-2, NF-ATc, and Stat3. Originally identified by their ability to phosphorylate c-Jun in response to UV-irradiation, the JNKs now are recognized as critical regulators of various aspects of mammalian physiology, including cell proliferation, cell survival, cell death, DNA repair, and metabolism. Moreover, a diverse set of cellular functions – including processes of programmed cell death, T-cell differentiation, inflammatory responses, negative regulation of insulin signaling, control of fat deposition and epithelial sheet migration – have been shown to be regulated by the JNK pathway. The spectrum of activating signals of the JNK pathway is very similar to the p38 pathway, and both pathways share a number of MAPKKKs that receive the upstream signals. In contrast to the p38 pathway, only a few protein cytoplasmic proteins have been identified as downstream substrates.

12.4.2.4 The p38 Module The terminal kinase of the p38 pathway comprises four proteins – the kinases p38a, p38b, p38c and p38d – derived from a single gene by alternative splicing. Activation of the p38 isoforms has been shown to occur in response to extracellular stimuli such as UV light, heat, osmotic shock, inﬂammatory cytokines (TNFa, IL-1) and growth factors. There is a large list of MAPKKKs that feed these signals into the p38 module [9]. 592 12 Intracellular Signal Transduction: The MAP Kinase Pathways

Questions 12.1. Name the major components of the MAPK signaling pathways and describe their general function in these pathways. What may be the advantage of the cascade organization of MAPK signaling? 12.2. Describe the activation steps involved in the transfer of a signal from the top kinase to the kinase at the bottom of the cascade. 12.3. What role is ascribed to KSR in signaling through the MAPK cascade? 12.4. How is signaling through the MAPK/ERK pathway regulated? Which feedback controls ensure proper signal transfer? 12.5. Describe the function and properties of scaffold proteins involved in MAPK/ ERK signaling. 12.6. 12.6. How may GPCRs couple to signaling through MAPK pathways? 12.7. Which signals may active the ERK1/2 pathway? What steps are required for the activation of transcription factors? Name at least one transcription factor activated by ERK1/2. 12.8. Give examples of entry signals, entry points and ﬁnal substrates of the JNK and p38 pathways.

References

1 Cargnello, M. and Roux, P.P. (2011) suppressor. Cell Res., 18 (4), 452–457. Activation and function of the MAPKs and PubMed PMID: 18379591. their substrates, the MAPK-activated protein 5 Dhanasekaran, D.N., Kashef, K., Lee, C.M., kinases. Microbiol. Mol. Biol. Rev., 75 (1), Xu, H., and Reddy, E.P. (2007) Scaffold 50–83. PubMed PMID: 21372320. Pubmed proteins of MAP-kinase modules. Oncogene, Central PMCID: 3063353. 26 (22), 3185–3202. PubMed PMID: 2 Bermudez, O., Pages, G., and Gimond, C. 17496915. (2010) The dual-speciﬁcity MAP kinase 6 Shi, F. and Lemmon, M.A. (2011) KSR plays phosphatases: critical roles in development CRAF-ty. Science, 332 (6033), 1043–1044. and cancer. Am. J. Physiol. Cell Physiol., PubMed PMID: 21617065. 299 (2), C189–C192. PubMed PMID: 7 Rubinfeld, H. and Seger, R. (2005) The ERK 20463170. Epub 2010/05/14. cascade: a prototype of MAPK signaling. Mol. eng. Biotechnol., 31 (2), 151–174. 3 Wancket, L.M., Frazier, W.J., and Liu, Y. 8 Bogoyevitch, M.A., Ngoei, K.R., Zhao, T.T., (2012) Mitogen-activated protein kinase Yeap, Y.Y., and Ng, D.C. (2010) c-Jun phosphatase (MKP)-1 in immunology, N-terminal kinase (JNK) signaling: recent physiology, and disease. Life Sci., 90 (7–8), advances and challenges. Biochim. Biophys. 237–248. PubMed PMID: 22197448. Acta, 1804 (3), 463–475. PubMed PMID: Pubmed Central PMCID: PMC3465723. 19900593. Epub 2011/12/27. eng. 9 Cuadrado, A. and Nebreda, A.R. (2010) 4 Granovsky, A.E. and Rosner, M.R. (2008) Raf Mechanisms and functions of p38 MAPK kinase inhibitory protein: a signal signalling. Biochem. J., 429 (3), 403–417. transduction modulator and metastasis PubMed PMID: 20626350. 593

13 Membrane Receptors with Associated Tyrosine Kinase Activity

Summary Coupling of extracellular signals to tyrosine phosphorylation in the intracellular region may occur by two mechanisms, and involves two different receptor types. First, the receptor tyrosine kinases (RTKs), which harbor an intrinsic tyrosine kinase activity in the cytoplasmic receptor domain (see Section 10.1). The ligand- binding site and the tyrosine kinase are part of one and the same protein, and ligand binding stimulates the tyrosine kinase activity on the cytoplasmic side of the receptor. The second receptor type has tyrosine kinase associated on its cytoplasmic side, and in this case the tyrosine kinase and the receptor are not localized on the same protein. The associated tyrosine kinases belong to the group of Non-RTKs (Section 10.3). In most cases, the Non-RTK is permanently associated with the receptor and is activated as a consequence of ligand binding. Stimulation of the associated tyrosine kinase is then the starting point for transduction of the signal into the interior of the cell. In many cases, mechanisms described in previous chapters are used for further signal transmission. Four members of this receptor type will be presented in the following, namely the cytokine receptors type I and II, the immunoreceptors, and the integrins.

13.1 Cytokines and Cytokine Receptors

Summary Cytokines are a group of secreted signaling proteins that control the proliferation, differentiation and function of cells of the immune and hematopoietic systems, and they are also involved in the processes of inflammation. Often, signaling through cytokines is coupled to cell–cell interactions. Medically important cytokines include the interleukins (IL), erythropoietin (EPO), growth hormone (GH) and the interferons (IFNs). The receptors of cytokines are

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 594 13 Membrane Receptors with Associated Tyrosine Kinase Activity

single-pass transmembrane proteins with distinct ligand-binding modules on the extracellular domain. The cytoplasmic domain has non-RTKs associated that become activated upon ligand binding and function as immediate downstream effectors. All cytokine receptors function as dimers or higher oligomers, and bind one or more ligands via multivalent interactions on the extracellular domain. Most cytokine receptors are composed of different types of subunit that have specialized functions in ligand binding and tyrosine kinase activation. Ligand binding to the cytokine receptor triggers an activation of the associated tyrosine kinase via a structural reorganization of the receptor oligomer. Transmission of the signal to the downstream effectors includes autophosphorylation of the associated tyrosine kinase, Tyr-phosphorylation of the receptor subunits, and the binding and phosphorylation of effector proteins.

13.1.1 Cytokines

Cytokines are secreted proteins that serve as signal molecules in cell proliferation and cell–cell communication and, as such, perform a central and very diverse function in the growth and differentiation of an organism.

& Cytokines: — Extracellullar signaling proteins — Examples: Interleukins Erythropoietin Interferons Growth hormone Tumor necrosis factor (TNF).

Structural analysis has allowed the grouping of cytokines into different structural classes:

Helical cytokines Trimeric TNF family Cystine knot growth factors b-trifoil growth factors.

Classiﬁcation has been also made according to the type of receptor that they engage. In the present section, only cytokines of the helical type will be discussed, together with their receptors. Well-known cytokines include the interleukins (ILs), erythropoietin (EPO), growth hormone (GH), interferons (IFNs), and TNF (see Table 10.1). For a review on the structural aspects of cytokines and cytokine receptors, see Ref. [1]. 13.1 Cytokines and Cytokine Receptors 595

Figure 13.1 Structures of interleukin-6 and leukocyte inhibitory factor (LIF).

13.1.1.1 Cytokine Structure The ligands of the cytokine receptors share a common fold, named also the cytokine fold. As illustrated in Figure 13.1 for the examples of IL-6 and leukocyte inhibitory factor (LIF), the cytokine fold comprises a bundle of four antiparallel a-helices that adopt an up–up–down–down motif.

& Cytokine structure: — Bundle of antiparallel helices

The various cytokines differ in the length and straightness of the helices, and these structural differences dictate the speciﬁcity of receptor binding. Based on the length of the helices, the cytokines are grouped into three classes: (i) the (most common) long-chain or type I cytokines (10–20 residues) that include human growth hormone, erythropoietin and the gp130 cytokines; (ii) short-chain cytokines with helical lengths of eight to ten residues that include IL-2, -3, and -4; and (iii) cytokines formed by tandem four-helix bundle motifs to generate an eight-helix bundle architecture comprising IL-5 and IFN-c.

13.1.1.2 General Functions of Cytokines The cytokines are of considerable medical importance because of their essential function in controlling the immune system, in defense reactions, and for processes of inﬂammation. Many of the cytokines have the character of autocrine or paracrine hormones; that is, they only act locally and their targets are often cells of the same or similar type as the cytokine-producing cell. One characteristic that signiﬁcantly differentiates some of the cytokines from other hormones is the coupling of their activity to cell–cell interactions. The functions of some cytokines, such as IL-4, -5, -6 and -10 are, for example, closely associated with the interactions between B and T lymphocytes. 596 13 Membrane Receptors with Associated Tyrosine Kinase Activity

13.1.2 Structure and Activation of Cytokine Receptors

The cytokine receptors are trans-membrane polypeptides with distinct extracellular ligand-binding motifs and no known enzymatic activity in their cytoplasmic domains. The formation of dimers or higher oligomers is a characteristic feature of these receptors.

13.1.2.1 Classification and General Features Based on sequence homology, cytokine receptors are classiﬁed into types I to IV. The types I and II receptors use associated tyrosine kinase activity for signaling, while type III includes the receptors for TNF and for CD40 and Fas protein (Section 14.3), which are found on T lymphocytes. Type IV cytokine receptors comprise the Toll/IL-1 receptors (Section 14.4). Neither type III nor type IV receptors employ any associated tyrosine kinase activity for further signal transduction. Rather, adapter proteins function as immediate downstream effectors in these receptor systems, and receptor activation triggers the assembly of further signaling proteins on the adapter.

& Classification of cytokine receptors: — Type I: Single-chain family gp130 family bc-Family cc-Family. — Type II: Interferon receptors IL-10 receptor. — Type III: TNF receptors (Section 14.3) Have associated adaptors. — Type IV: Toll-like receptors (TLRs; Section 14.4) Have associated adaptors.

The domain structures of important members of types I and II cytokine receptors are shown in Figure 13.2. Type I receptors can be divided into four families: the single-chain family; the gp130 family; the common c chain (cc) family; and the common b chain (bc) family. Cytokine receptors of type II include receptors for the IFN-a, IFN-b and IFN-c, and also the receptors for IL-10 and related cytokines. At this point, attention will be focused on type I cytokine receptors. 13.1 Cytokines and Cytokine Receptors 597

Figure 13.2 Domain structure of class I and receptor; GHR, growth hormone receptor; class II cytokine receptors. WSXWS, conserved LIF-R, leukemia inhibitory factor receptor; GM- WSXWS sequence (W: tryptophan; S: serine; X: CSFR, granulocyte colony-stimulating factor nonconserved amino acid); CCCC, cysteine- receptor; PRLR, prolactin receptor; IFNR, rich motif; IL, interleukin; EpoR, receptor for interferon receptor. erythropoietin; TMPR, thrombopoietin

13.1.2.2 Structural Domains With the exception of ciliary neurotrophic receptor, which is membrane-anchored via a GPI anchor (see Section 2.9.7), receptor subunits are single-pass transmembrane proteins of N-terminal extracellular and C-terminal intracellular orientation. The extracellular region is of a modular structure and speciﬁes the particular receptor type. Cytokine binding is mediated by an about 200 residue-long cytokine- binding homology region (CHR) consisting of two ﬁbronectin-type III-like domains; one or more of this module are found on every type I cytokine receptor. Furthermore, immunoglobulin-like domains, four conserved cysteine residues and a WSXWS motif are found in the extracellular domain. 598 13 Membrane Receptors with Associated Tyrosine Kinase Activity

The cytoplasmic part of the receptors does not harbor catalytic activities as do the receptor tyrosine kinases. Rather, the cytoplasmic domains of cytokine receptors have non-RTKs associated that become activated upon ligand binding and function as immediate downstream effectors. Upon cytokine binding, receptor oligomerization is induced and this leads to the juxtaposition of the intracellular domains of the signaling subunits with their associated non-RTK. Activation of the non-RTK ensues, and the signal is conducted further without the activated receptor actually performing any enzyme function. Key to the activation are conformational changes within the receptor triggered by extracellular ligand binding. As a consequence, large signaling complexes assemble at the cytoplasmic part of the activated receptor. This situation is similar to the activation of the receptor tyrosine kinases (see Chapter 10).

& Cytokine receptors: — Extracellular ligand binding — Single-pass transmembrane domain — Form homo-oligomers or hetero-oligomers.

& Signaling by cytokine receptors: — Ligand binding activates associated tyrosine kinase — Tyrosine kinase phosphorylates receptor subunits — Tyr-P serve as binding sites for downstream effectors.

& Downstream effectors of activated cytokine receptors: — Non-receptor tyrosine kinases — Adapter proteins.

13.1.2.3 Oligomeric Structure Functionally active forms of cytokine receptors are composed of homodimers, heterodimers, trimers,orhigher oligomers. Only a minority of cytokine receptors, such as growth hormone receptors (GHRs) and erythropoietin receptors (EPORs), exist as preformed homodimers. Rather, the vast majority of cytokine receptors are heterooligomers, where one subunit is shared among different receptors. This structural feature allows cytokine receptors to recognize and respond to more than one cytokine, a property which forms the basis for a high cross-reactivity and redundancy in cytokine signaling. The subunits of heteromeric receptors can be divided into two classes:

Signaling subunits, which serve as signal-transducing receptor components within their receptor family. A long cytoplasmic tail with a permanently associated non-RTK is a characteristic structural feature of signaling subunits 13.1 Cytokines and Cytokine Receptors 599

that allows them to transduce the signal into the cell interior. Three types of signaling subunits are known that are shared among class I cytokine receptors, namely the gp130 subunit (or gp130 receptor), the common c-subunits (cc), and the common b-subunits (bc). The shared subunits have both functions in ligand recognition and signaling to the cell interior. Upon association with other signaling subunits or with non-signaling subunits, the shared subunits form the fully functional receptor complex that provides a high-affinity binding site for the cytokine. In doing this, the shared subunits must be able to interact with a large number of different cytokine ligands. Cytokine-specific non-signaling subunits (e.g., a-subunits) that provide a binding site for the ligand but lack a long cytoplasmic tail. These receptor components do not participate directly in signaling to the cell interior; rather, they carry major determinants for cytokine recognition. However, strong and specific cytokine binding involves binding sites on both signaling and non-signaling receptor components.

& Signaling subunits: — gp130 — Common c-subunits — Common b-subunits. Non-signaling subunits: — For example, a-subunits — Involved in ligand binding.

The subunit structure of the cytokine receptors is very variable, and the combination of shared subunits, other signaling subunits and non-signaling subunits leads to the formation of multiple functional receptors, as illustrated in Figure 13.3). For some cytokine receptors, including the growth hormone receptor and several interleukin receptors, soluble isoforms have been described that may arise by alternative splicing. These truncated receptors comprise all or part of the extracellular domain and may be able to bind the extracellular ligands. By association with other subunits of heterooligomeric receptors, such as the gp130 subunit, these soluble isoforms can function as either agonists or antagonists.

& Soluble receptor isoforms: — Only extracellular — Function as agonists or antagonists.

13.1.2.4 Cytokine Binding and Activation Structural analyses of cytokines in complex with the extracellular domain of the receptor have shown that the cytokines contact the receptor subunits via two to 600 13 Membrane Receptors with Associated Tyrosine Kinase Activity 13.1 Cytokines and Cytokine Receptors 601

three distinct contact points. Most cytokines activate the receptor by inducing the assembly of a heterooligomeric complex composed of shared, signaling, or non- signaling subunits. Only a minority of the cytokines bind to and activate preformed receptor oligomers. Examples are the GH receptor and the EPO receptor, both of which have a dimeric structure in solution; ligand binding activates these dimeric receptors by inducing a conformational change in the transmembrane domain and the cytoplasmic domains of the dimer. Although structural details on the conformational changes of the dimeric receptors are not yet available, subunit rotation and a closer apposition of the non-RTKs bound to the cytoplasmic tails have been implicated in the activation of kinase activity and further signal transduction. When in the inactive ground state, most cytokine receptors exist as monomers, and it is generally assumed that the non-RTKs bound to unliganded receptors are inactive. However, on ligand-binding-induced oligomerization a change occurs in the mutual orientation of the cytoplasmic part of the oligomeric receptor, which then undergoes an allosteric transition into the active state. This triggers an activation of the associated kinase activity (see Section 13.2.1). Structural information on these transitions is lacking. Once activated, the non-RTKs phosphorylate Tyr-residues on the cytoplasmic tails that serve as attachment points for further signaling proteins. This situation is very similar to the activation of the receptor tyrosine kinases (see Chapter 10). Further details of receptors containing the shared gp130 subunits, the b- and c-common subunits and cytokine type II receptors are presented in the following subsections.

& EPO-R, GH-R: — Homodimer — Activated by ligand-induced conformational change.

13.1.2.5 Gp130 Receptors The shared subunit gp130 is the founding member of the tall cytokine receptors, and is the common signal transducing component for the gp130 (or IL-6/IL-2) family of cytokine that comprise at least eight different cytokines. The signaling functions of gp130 cytokines are mediated through a set of receptor complexes that are formed by combining gp130 with other receptor components (see Figure 13.3b). The association of gp130 with cytokine-speciﬁc, non-signaling receptor IL-6Ra or IL-11Ra executes the activities of IL-6 or IL-11, J Figure 13.3 Diversity of shared receptors with shared cytokine receptors. TCCR, receptor---receptor interactions. Shared cytokine T-cell cytokine receptor; TSLPR, thymic stromal- receptors bc (a), gp130 (b), and cc derived lymphopoietin receptor. Adapted from (c) and their various receptor partners are Wang X, Lupardus P, Laporte SL, Garcia KC. depicted. These complexes are formed by the Structural biology of shared cytokine receptors. combination of ligand-specific a and/or b Adapted from Wang et al. 2009 [1]. 602 13 Membrane Receptors with Associated Tyrosine Kinase Activity

respectively. Other signaling receptors such as the LIF receptor (LIFR) and the OSM receptor (OSMR) can also participate in signaling complexes with gp130. Although high-resolution structures of complete gp130 receptors are not yet available, crystal structures of the extracellular domains of gp130 and the a-receptor in complex with gp130 cytokines have revealed important insights into the nature of cytokine binding and discrimination. The structure of the extracellular domains of the IL-6 receptor in complex with IL-6 shows a hexamer comprised of two cytokine molecules, two gp130 subunits, and two a-subunits. A membrane-distal headpiece and membrane-proximal “legs” can be differentiated in the receptor– ligand assembly (Figure 13.4). The cytokines use distinct sites (sites I, II, and III) to engage the receptor subunits. For example, sites I and II of IL-6 bind to the a-receptor and gp130, respectively, while site III in the cytokine engages the Ig domain of gp130 so that each gp130 contacts two different cytokines in an antiparallel fashion. The basic principle of a three-site contact to the cytokine also seems applicable to other gp130 cytokines and their receptors. Variations in the site III interactions are thought to dictate the final specificity of the receptor–cytokine interactions. Cytokine binding to the receptor occurs in several steps. IL-6 first binds specifically to the IL-6Ra, and this complex then triggers dimerization of the gp130 polypeptide, which has the tyrosine kinase permanently in association. Within the oligomeric complex thus formed the tyrosine kinase is activated and the signal is transduced further.

Figure 13.4 Assembly of the full-length the top. IgD, Ig-like domain; CHR, cytokine- gp130/IL-6/IL-6Ra complex. Components of binding homology region; FnIII, Fibronectin- the gp130/IL-6/IL-6Ra complex in pre- and type III domain. Lupardus et al. 2011 [2], post-assembly state. A surface rendering of the figure 1A. Reproduced with permission of structure of the signaling hexamer is shown at Elsevier. 13.1 Cytokines and Cytokine Receptors 603

& IL-6R: — Belongs to common gp130 family — IL-6Ra subunit and gp130 subunit form a heterotetramer — gp130 subunit becomes Tyr-phosphorylated by associated Tyr-kinase and activates downstream effectors.

13.1.2.6 Shared cc-Chain Receptors The cc-chain serves as a shared signaling receptor for IL-2, -4, -7, -9, -15 and -21, of which the IL-2 system is the best-characterized (Figure 13.3c). Cytokine IL-2 is mainly produced by antigen-activated T cells and promotes the proliferation, differentiation and survival of mature T and B cells, as well as the cytolytic activity of natural killer (NK) cells in the innate immune defense system. The biological importance of cc is illustrated by the fact that mutations in either cc or the associated JAK3 kinase can abolish the function of all cc-dependent cytokines and cause X-linked severe combined immunodeficiency disease (X-SCID). A specific feature of IL-2R (and also of IL-15R) complexes is their composition of three different subunits, namely the IL-2Ra, IL-2Rb and cc subunits. This is in contrast to the other receptors of this class, which heterodimerize cytokine-specific a-subunits and cc. The IL-2Ra-subunit has primarily the function of an affinity modulator and is not itself involved in signal transduction. For activated T cells, however, the presence of IL-2Ra is required for high-affinity binding of IL-2 by the other two subunits, which bind IL-2 in the absence of IL-2Ra only with intermediate affinity.

& IL-2R: — Belongs to cc-subfamily — a, b, c subunits — b-subunit carries signal further on.

In some cells such as macrophages, IL-2Rb and IL-2Rcc together are necessary and sufficient for efficient signaling, and both connect ligand binding to the activation of intracellular signaling components. The structure of the extracellular domains of IL-2Ra, IL-2Rb, and IL-2Rcc in complex with IL-2 shows that the cytokine contacts all three subunits in the high affinity complex formed (Figure 13.5). Despite an absolute requirement of IL-2Rcc for signaling, the majority of downstream-signaling pathways link through the IL-2Rb subunit to the activated receptor. Nearly all signals that are generated upon IL-2R activation can be mapped to the cytoplasmic tail of the IL-2Rb subunit.

13.1.2.7 Shared b-Chain Receptors The signaling chain bc serves as a shared subunit for the receptors of IL-3, IL-5, and GM-GCF (Figure 13.3a). The activated receptor complex consists of the cytokine ligand, the bc chain and a cytokine-speciﬁc a-chain. The results of structural studies have suggested that the actively signaling subunit is a higher 604 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Figure 13.5 Structure of IL-2R (IL-2/IL-2Ra/IL-2Rb/cc) with bound cytokine IL-2. Reproduced from [1].

oligomer composed of up to four copies of each of the signaling components. Downstream signaling is mediated primarily by the bc chain which has Jak2 as the signal-transducing kinase.

13.1.3 Activation of Cytoplasmic Tyrosine Kinases

As a consequence of ligand binding to cytokine receptors type I and type II, the activation of a tyrosine kinase activity is observed that is not part of the receptor protein. In most cases this tyrosine kinase is permanently associated with one of the subunits of the receptor, and ligand-induced restructuring or hetero-oligomerization of the receptor induces the activation of this tyrosine kinase. The tyrosine kinases most frequently associated with cytokine receptor subunits belong to the family of Janus kinases. However, protein tyrosine kinases of the Src family such as Lck and Fyn have also been shown to be direct downstream components of the cytokine receptors. & Associated tyrosine kinases of cytokine receptors: — Janus kinases — Src family kinases, such as Lck, Fyn, Tyk.

The following steps are involved in cytokine receptor signaling (Figure 13.6): Activation of the associated tyrosine kinase by autophosphorylation: This step occurs immediately after ligand binding, and is thought to function via 13.1 Cytokines and Cytokine Receptors 605

Figure 13.6 Steps involved in cytokine signaling. protein kinase also catalyzes Tyr phosphorylation Binding of a cytokine to the extracellular ligand- of the cytoplasmic domain of the receptor. The binding domain of the cytokine receptor activates phosphotyrosine residues serve as attachment the tyrosine kinase activity of the associated points for adapter proteins or other effector tyrosine kinase (e.g., Jak1). Tyrosine proteins containing phosphotyrosine-binding phosphorylation occurs in trans between motifs (PTB or SH2). The signal is then neighboring kinase molecules. The activated transmitted further into the cytoplasm.

autophosphorylation in trans between neighboring tyrosine kinases associated with the oligomeric receptor. This activation involves phosphorylation in the activation loop of the tyrosine kinase. Phosphorylation of receptor subunits: The activated tyrosine kinase then phosphorylates tyrosine residues in the cytoplasmic region of the receptor subunits. The phosphotyrosine residues serve as attachment points for the recruitment of other signaling proteins. 606 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Binding of other signaling proteins: Distinct phosphotyrosine residues are used for the attachment of signaling proteins that carry phosphotyrosine-binding domains such as SH2 or PTB domains. In this way, adapter proteins such as Shc or IRS are recruited to the cytokine receptor. As a consequence, signals are passed on to a variety of intracellular signaling pathways. Phosphorylation of substrates: Signaling proteins that have been recruited to the activated receptor are often substrates for phosphorylation by the activated tyrosine kinase. Examples are the Stat proteins (see below), the Shc adapter protein, and PI3 kinase. IL-2Rb activates PI3 kinase by inducing phosphorylation on tyrosine residues of the p80 regulatory subunit and recruiting PI3 kinase to the cell membrane.

The principles of cytokine receptor signaling are illustrated in Figure 13.7 for the EPO receptor, and in Figure 13.8 for the b-subunit of the IL-2 receptor.

Figure 13.7 Erythropoietin-mediated signaling actions in hematopoietic cells. For PI3K, Akt, pathways. The figure includes the stimulation PDK1 see Section 9.4. For Ras, Raf MEK, ERK, and downregulation of EPO-mediated see Chapters 11 and 12. For PTP1 and SHP1, signaling pathways, derived from EPO’s known see Section 10.4. 13.1 Cytokines and Cytokine Receptors 607

Figure 13.8 Signaling by the b-subunit of the Stat5 transcription factor can bind to all three IL-2 receptor. The cytoplasmic portion of the phosphorylation sites, is phosphorylated by b-subunit is shown with tyrosine Jak1, and then translocates to the nucleus to phosphorylation sites that mediate further activate transcription of target genes. Jak1 also downstream signaling events. Jak1 is phosphorylates and activates the non-RTK Syk constitutively associated with the Box 1 and which has the transcription factor Myc as a Box 2 regions. Following ligand binding, Jak1 substrate. Inhibition and attenuation of IL-2Rb becomes activated and phosphorylates distinct signaling is exerted by the SOCS proteins tyrosine residues on the b-subunit which serve which can inhibit Jak1 or interfere with Stat as attachment points for SH2 domains of activation, among others. further signaling proteins, as indicated. The

IL-2R is composed of the IL-2Ra, IL-2Rb and IL-2Rcc subunits, of which only the IL-2Rb and IL-2Rcc subunits conduct the signal further into the cytoplasm.

& Reactions triggered by activated cytokine receptors: — Autophosphorylation of associated tyrosine kinase — Receptor subunit phosphorylation — Docking of effectors to Tyr-P of receptor subunits — Binding of adaptors. 608 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Three non-RTKs – the Src family kinases Lck and Lyn, and the Jak family member Jak1 – have been shown to be associated with the IL-2Rb polypeptide. Most specific signals are mediated by Jak1, while the in vivo function of the Lck and Lyn proteins in IL-2R signaling is not yet fully established. Another member of the Jak family, Jak3,isfirmly associated with the IL-2Rcc subunit. It is not yet clear how the different kinases cooperate in IL-2R signaling. Most signaling specificity is imparted by Jak 1, which phosphorylates specific tyrosine residues on IL-2Rb. The phosphorylation of Tyr338 by Jak1 induces binding of the adapter Shc and, subsequently, recruitment of the Grb–mSos complex. As a result, the Ras protein is activated, a signal is delivered to the effector pathways of the Ras protein, and activation of the MAPK/ERK pathway has been observed many times as a consequence of IL-2R activation. The Jak1-mediated phosphorylation of tyrosine residues (e.g., Tyr392 and Tyr510) in the C-terminal half of the cytoplasmic tail creates binding sites for the Stat5 protein, which is then phosphorylated by Jak1 and activated for further signaling. Another effector is the PI3 kinase, which binds to the above-mentioned phosphotyrosine residues through the SH2 domain of its p85 regulatory subunit. Starting from the activated tyrosine kinase, the signal may be conducted along different signaling pathways, depending on the receptor type:

Jak-Stat pathway (see below) Ras pathway MAP kinase pathway Protein kinase C via phospholipase Cc PI3 kinase pathway.

13.2 The Jak-STAT Pathway

Summary The non-RTKs associated with cytokine receptors most often include the Jak kinases and Src family kinases such as Lck and Fyn. A well-characterized cytokine signaling pathway uses Jak/STAT signaling. In this pathway, Jak kinases phosphorylate the STAT proteins at Tyr-residues, which in turn triggers translocation into the nucleus. The nuclear STATs function as transcriptional regulators that bind to cognate DNA elements and cooperate with other transcription factors to stimulate the transcription of target genes. Signaling through cytokine receptors is regulated at various levels including PTMs, dephosphorylation by PTPs, and the binding of inhibitor proteins such as SOCS proteins and PIAS proteins. 13.2 The Jak-STAT Pathway 609

The Jak-Stat pathway is a signaling pathway, starting from cytokine receptors, that allows a very direct signal transduction from the membrane to the cell nucleus using only a few coupling elements. Many cytokines use this pathway to bring about a rapid change in the transcription activity of speciﬁc gene sequences [3].

13.2.1 The Janus Kinases

The non-RTKs most often involved in signal transduction via cytokines includes the non-RTK subfamily of Janus kinases (Jak kinases). The Jak family consists of four mammalian members – Jak1, Jak2, Jak3 and Tyk2 – that share seven homologous domains, named JH1 to JH7. A characteristic feature of the structure of Jak kinases is the occurrence of two tyrosine kinase domains (Figure 13.9): the JH1 and JH2 domains. The JH3 and JH4 domains share homology with SH2 domains. The amino-terminal domain (JH4–JH7) is also known as the FERM domain, which mediates binding to the cytokine receptor.

13.2.1.1 Kinase Regulation Of the two kinase domains, only JH1 possesses all of the structural features required for a canonical protein kinase activity, and this domain appears to be responsible for the phosphorylation of the receptor subunits and the Stat proteins. The function of JH2, which is also called a pseudokinase domain, has been elusive for a long time. Both, biochemical and clinical evidence have demonstrated an important autoregulatory function for JH2 in JAKs [4]. JH2 is predicted to adopt the canonical kinase fold but, as some of the key catalytic residues are missing, JH2 has been ascribed a negative regulatory role in Jak activity, without the participation of kinase activity, by autoinhibiting JH1 activity. The importance of this regulation is underscored by the observation that least 32 different mutations in JH2 of JAK2 have been shown to cause, or are linked to, hematological diseases such as myeloproliferative neoplasms. The most frequent somatic mutation, V617F, results

Figure 13.9 Schematic representation of the downregulates the activity of the kinase modular structure of Jak2. Jak2 associates with domain. The JH3---JH4 domain of Jaks shares the type II and most of the type I cytokine homology with Src homology 2 (SH2) receptors. Jaks have seven homologous domains. The amino-terminal domain domains (JH1---7), including the catalytic (JH4---JH7) is known as the FERM (short for domain (JH1) and the catalytically inactive 4.1 protein, ezrin, radixin and moesin) domain, pseudokinase domain (JH2), which putatively which engages with cytokine receptors. 610 13 Membrane Receptors with Associated Tyrosine Kinase Activity

in constitutively active JAK2. Surprisingly, the JH2 domain of JAK2 could be shown to harbor a dual-speciﬁcity kinase activity [5] that phosphorylates two negative regulatory sites in Jak2, Ser523 and Tyr570. The mechanism by which these phosphorylations suppress JH1 activity is not yet known. However, it is assumed that Ser523 and Tyr570 phosphorylations strengthen the JH1–JH2 autoinhibitory interactions. Most Jak kinases are constitutively associated with a cytoplasmic section of the receptor, which is in the vicinity of the membrane and contains two conserved sequence elements, Box 1 and Box 2.

& Janus kinases: — Jak1, Jak2, Jak3, Tyk2 — Carry JH1 and JH2 domains — Bind to box1, box 2 of activated receptor.

Cytokine-induced conformational changes of the receptor oligomer bring about a change in the juxtaposition of the associated JAKs, allowing the cross-phosphorylation of neighboring kinases in their activation loop, which leads to their activation (see Figure 13.6). Activation of the Jak kinases may take place in a homodimeric receptor complex, or it may also occur in heterooligomeric complexes. In addition to the activation loop, Jaks are phosphorylated on multiple sites, including the JH2 domain. These phosphorylation events may have either an activating or inhibiting inﬂuence on Jak signaling. In the absence of cytokine activation, Jak 2 is constitutively phosphorylated on Ser523; however, upon activation Jak2 becomes phosphorylated on as many as 20 Tyr sites.

13.2.1.2 Coupling to Other Receptor Types Jaks associate also with the RTKs and GPCRs, and therefore Jak kinases may be activated by many different extracellular stimuli.

13.2.1.3 Nuclear Functions of Jaks Novel functions of Jaks have been established that are distinct from their canonical roles in cytokine receptor or other receptor signaling. Jaks can shuttle between the cytoplasm and the nucleus in a similar manner as do the Stat proteins – their immediate downstream substrates – upon cytokine receptor activation. In the nucleus, Jak2 has been shown to phosphorylate histone H3 at Tyr 41 (H3Y41) [6], leading to gene derepression (Figure 13.10). H3Y41 phosphorylation disrupts the interaction of H3 with HP1, a transcriptional repressor involved in the maintenance of a suppressed chromatin state (see Section 4.5). By binding to H3K9 methyl marks, HP1 establishes a repression of nearby genes and this negative regulation of transcription can be relieved by H3Y41 phosphorylation. Constitutive Jak activation is frequently observed in leukemogenic malignancies, and it is thought that inappropriate Jak activity can lead to the removal of HP1 from genomic regions allowing aberrant gene activation during leukemogenesis. 13.2 The Jak-STAT Pathway 611

Figure 13.10 Jak2-mediated transcriptional target genes. Alternatively, the active Jak2 activation involves two separate pathways. In enters into the nucleus to phosphorylate the canonical Jak2---Stat5 pathway, Jak2 is histone H3 at Tyr41 (H3Y41). H3Y41 activated by autophosphorylation upon the phosphorylation disrupts the association of association of receptors and their ligands. The HP1a (Section 3.5.3.1) with chromatin, which active Jak2 further phosphorylates Stat5, which may lead to the activation of oncogenes. is translocated into the nucleus to activate its

13.2.2 The Stat Proteins

Starting from the activated Jak kinases, a signaling pathway leads directly to transcription factors that are phosphorylated by the Jak kinases on tyrosine residues and activated for stimulation of transcription. These transcription factors belong to a class of proteins known as Stat proteins (Stat ¼ signal transducer and activator of transcription). At least seven different Stat proteins are known in mammals, namely Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6.

& Stat proteins: — Transcription factors — Shuttle between cytosol and nucleus — Become Tyr-phosphorylated by Jaks. 612 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Figure 13.11 Domain structure of STATs. The C-terminal tyrosine (Tyr701 in STAT1) that becomes phosphorylated by Jak kinases is indicated. The ‘Y’ indicates the site of phosphorylation.

13.2.2.1 Structure of Stats The STAT proteins have an N-terminal oligomerization domain, a coiled- coiled region, an SH2 domain, a DNA-binding domain, and a C-terminal transactivation domain (Figure 13.11). The DNA-binding domain confers speciﬁcity for binding to cognate DNA elements, while the transactivation domain mediates interactions with the transcription complex and other transcriptional activators at cytokine- sensitive genes. The SH2 domain mediates binding to Tyr-phosphates of Stat dimers, Jak kinases, or interaction partners. In the unphosphorylated form, the Stat proteins exist as either monomers or dimers. Jak kinases catalyze the phosphorylation of Stats at a distinct Tyr-site (Tyr701 in Stat1), which leads to their activation and the formation of stable homodimers or heterodimers, or even higher oligomers.

& Stat structure and function: — SH2 domain — Oligomerization domain — Transactivation domain — Tyr phosphorylation sites — Dimerize upon phosphorylation — Bind to Stat elements in promoter regions of target genes.

In the activated state, the P-Tyr residue of one Stat protein binds to the SH2 domain of the partner, and vice versa, so that the phosphotyrosine–SH2 bonds function as a double clasp. Binding to DNA is in the form of a dimer with a parallel arrangement of the monomers.

13.2.2.2 Activation of Stats The signaling pathway where the STAT proteins are activated via Jak kinases is named the JAK/STAT pathway. On binding of the cytokine to the receptor and activation of the Jak kinase, the Stat proteins are recruited, via their SH2 domains, to P-Tyr residues of the receptor–kinase complex and are then phosphorylated by the Jak kinase on a conserved Tyr residue (Tyr701 for STAT1) at the C terminus. The activating phosphorylation can be catalyzed also by other protein tyrosine kinases such as the Src kinase, and it is indispensable for subsequent signaling events. The phosphorylated dimers of Stat proteins are transported as such into the nucleus (Figure 13.12), where they bind to corresponding DNA elements in the 13.2 The Jak-STAT Pathway 613

Figure 13.12 Model of activation of Stat to corresponding DNA elements, and activate proteins. The Stat proteins are phosphorylated the transcription of neighboring gene sections. (at Tyr701 for Stat1) as a consequence of In the figure, activation of Stat proteins is binding to the receptor---Jak complex, and Stat shown using the IL-6 receptor as an example. dimers are formed. The dimerization is Other Jak kinases and Stat proteins may also mediated by phosphotyrosine---SH2 take part in signal conduction via IL-6, in interactions. In the dimeric form, the Stat addition to the Jak kinases and Stat1 shown. proteins are transported into the nucleus, bind 614 13 Membrane Receptors with Associated Tyrosine Kinase Activity

promoter region of cytokine-responsive genes and activate the transcription of these genes. Stat-binding sites are often arranged in tandem on promoters, and STAT tetramers are then formed on the DNA. During the course of transcription activation, STAT proteins make contacts with the RNA polymerase II machinery via the transactivation domain. Furthermore, STAT proteins interact with and recruit histone acetylase complexes (see Section 4.5.2), and they often cooperate with other transcription factors such as glucocorticoid receptors and c-jun within enhanceosome complexes. The mechanisms of nuclear translocation differ for the various Stats. Both, Stat1 and Stat2 require Tyr-phosphorylation for interaction with the nuclear import factor importin and subsequent nuclear translocation. When dephosphorylated by PTPs, these STATs can redistribute to the cytosol. In contrast, Stat3 binds constitutively to the import machinery and its nuclear translocation appears to be independent of phosphorylation.

13.2.2.3 Signaling Function of STATs The Jak/STAT signal transduction is an example of a signaling pathway in which a signal is coupled, in the form of a tyrosine phosphorylation, directly to the activation of a transcription factor. In contrast to other signaling pathways that also regulate transcription processes, such as the Ras/MAPK pathway, the Jak/STAT pathway is impressive in its simple concept and the small number of components involved. STAT proteins transmit signals to the level of transcription also via other routes than the Jak/STAT pathway. Over 40 different proteins have been identiﬁed that induce the phosphorylation and activation of STAT proteins, and several signaling pathways converge at the level of the STAT proteins. STAT proteins can be activated via:

Janus kinases (as described) RTKs such as EGF-R and PDGF-R Nonreceptor tyrosine kinases (e.g., Src kinase and Abl kinase; see Section 10.3) GPCRs via indirect routes.

The biological outcomes of Stat signaling depend heavily on the nature of the cytokine receptor and STAT protein. There is a convergence of distinct cytokine and growth factor signaling pathways on an overlapping set of STATs, particularly STAT1 and STAT3. Both of these factors are targets for activation by distinct signals, particularly interferons, gp130-speciﬁc cytokines such as IL-6, and growth factors such as EGF. Despite the use of common STAT targets, these signals trigger distinct and often opposing effects on the target cells. Furthermore, the ﬁnal outcome of cytokine signaling varies with different cellular contexts. For example, STAT1 and STAT3 have opposing effects on cell survival and proliferation directing cells towards proliferation or apoptotic cell death. The interferon/STAT1 pathway negatively regulates cell survival by inducing pro-apoptotic and anti-proliferative genes, and by direct interaction with critical regulators such as p53 (Chapter 16) and tumor necrosis factor receptor (TNFR, Section 14.3). In contrast, STAT3 promotes proliferation through the induction of oncogenes and cell cycle regulatory genes [7]. 13.2 The Jak-STAT Pathway 615

13.2.2.4 Signaling by Unphosphorylated STATs A new twist in Stat signaling relates to the ability of unphosphorylated STATs (U- STATs) to translocate to the nucleus and perform distinct gene-regulatory functions [8]. The U-STATs can bind to DNA – albeit with a lower afﬁnity – and thus activate many of the genes that are not activated by the phosphorylated forms. After the initial cytokine stimulus, these genes are often activated for a prolonged time, leading to long-lived antiviral and immune responses. The stimulation of cells by IFN-c or by IL-6 has been shown to lead to substantial nuclear levels of U- STAT1 and U-STAT3, respectively. The mechanisms by which the U-STATs can activate genes in a manner that is different from their phosphorylated counterparts are not yet fully established. One speciﬁc approach to U-STAT signaling uses the transcription factor NFkB (for details, see Ref. [9]).

13.2.2.5 Acetylation of STATs In addition to phosphorylation at Tyr701, Stat functions are regulated by other PTMs, including Ser-phosphorylation, lysine acetylation, sumoylation, and ubiquitination. Of these modiﬁcations, Lys-acetylation by HATs appears to play an important regulatory role by negatively regulating cytokine signaling [10]. The acetylation of Lys-residues of the chromatin components is generally ascribed an activating function in gene transcription (Section 4.5.2). However, in gene induction by interferons, acetylation has been found to counteract interferon signaling. This inhibitory effect is ascribed to the acetylation of STAT1 by the histone acetylase CBP (or other acetylases) at two lysine residues (K410, K413) located within the DNA-binding domain. The Lys-acetylation of STATs appears to promote the binding of a highly active PTP (CTP45), which leads to the dephosphorylation of K701-P, a redistribution of Stat1, and the termination of interferon signaling. Such a switch, from an active Tyr-phosphorylated STAT1 to a suppressed Lys-acetylated STAT1 generates two different functional modes of STAT1.Via this mechanism, STAT1 target genes can be turned on rapidly and the cytokine signal can subsequently be turned off by CBP-mediated acetylation.

13.2.3 Regulation of Cytokine Receptor Signaling

Signaling through cytokine receptors and the Jak/Stat pathway is tightly controlled by a variety of mechanisms, including negative and positive controls. The importance of a negative control of cytokine signaling is illustrated by the observation that several hematological malignancies, inﬂammatory diseases and immune disorders are characterized by a constitutive activation of the cytokine and Jak/Stat signaling pathways.

& Cytokine signaling is controlled by: — Protein phosphatases — PIAS proteins 616 13 Membrane Receptors with Associated Tyrosine Kinase Activity

— SOCS proteins — E3 ligases — Soluble receptor forms.

The regulation of cytokine signaling occurs at multiple levels, including regulating the expression of the various components of the pathway, and controlling the activity and half-life of the main signaling components, namely the cytokine receptors, the Janus kinases, and the STAT proteins. Three types of proteins predominate as negative regulators of cytokine signaling (Figure 13.13):

Protein tyrosine phosphatases Suppressors of cytokine signaling (SOCS proteins) Protein inhibitors of activated STATs (PIAS proteins).

Another main mechanism for termination of signaling operates via the inhibitory acetylation of STAT proteins. Furthermore, ubiquitination and proteolysis of the central components of the cytokine signaling pathway serve to negatively regulate

Figure 13.13 Regulation of cytokine signaling. For an explanation, see the text. PPase, protein phosphatase; PIAS, protein inhibitors of activated STATs; SOCS, suppressors of cytokine signaling. 13.2 The Jak-STAT Pathway 617 cytokine signaling. In addition, soluble, truncated cytokine receptors may compete with fully functional receptors for binding of the cytokine ligand.

13.2.3.1 Protein Tyrosine Phosphatases The dephosphorylation of cytokine receptors and Jak kinases represents an efﬁcient means of modulating and terminating cytokine signaling. Members of the class of SH2-containing protein tyrosine phosphatases (SHP1, SHP2) have been shown to associate with phosphorylated cytokine receptors, such as the erythropoietin receptor, inducing their rapid dephosphorylation. Another major control point of Jak/STAT signaling is the dephosphorylation of nuclear localized STAT proteins, which is important for the recycling of these proteins (Section 13.2.2).

13.2.3.2 SOCS Proteins The family of “suppressors of cytokine signaling” (SOCS) proteins comprises eight members (SOCS1 to SOCS7 and CIS) that share a conserved sequence motif, the SOCS box, plus either an SH2 domain or other domains capable of mediating protein–protein interactions [11]. The SOCS box is believed to be involved in the degradation of proteins through the ubiquitin-dependent proteasomal pathway. The SH2 domain enables various SOCS proteins to bind to speciﬁc phosphotyrosines, and thus to inhibit molecules that are important for cytokine signaling, such as the cytokine receptors and the Janus kinases. Generally, the mRNAs for the SOCS proteins are present at low levels in unstimulated tissues, but are rapidly upregulated after stimulation with one or more of a broad spectrum of different cytokines; moreover, a negative regulation of cytokine signaling by the SOCS family members has been clearly demonstrated. Thereby, SOCS proteins can act in a classical negative feedback loop, whereby they inhibit the signaling pathway that stimulates their own production. As an example, the transcription of the socs1 gene is enhanced by the activated STAT transcription factors. The mechanisms by which SOCS family members inhibit cytokine receptor signaling are diverse, and depend on the nature of the SOCS protein and the receptor involved. The SOCS1 protein, for example, has been shown to associate with a phosphotyrosine residue of the activation loop of the Jak1 tyrosine kinase, leading to an inhibition of the kinase activity (see Figure 13.7). In addition, SOCS1 is part of an E3 ubiquitin ligase that serves to target activated Jaks to degradation, thereby terminating cytokine signaling. Another means by which SOCS proteins may inhibit cytokine signaling is via competition with STAT proteins for phosphotyrosine-binding sites on receptor subunits.

13.2.3.3 PIAS (Protein Inhibitors of Activated Stats) Members of the family of PIAS proteins inhibit Jak/Stat signaling by interfering with Stat protein functions. In addition to the Stat proteins, other transcription factors, such as p53, SMAD proteins, NFkB and the androgen receptor, are also regulated by PIAS proteins, and the PIAS proteins are now considered as general coregulators of transcription factors [12]. The mechanisms by which the PIAS 618 13 Membrane Receptors with Associated Tyrosine Kinase Activity

proteins interfere with the function of transcription factors are varied. As an example, PIAS1 binds to activated Stat1 dimers and inhibits their DNA binding. Another mechanism by which PIAS proteins regulate Stat proteins appears to involve the PTM of sumoylation (Section 2.8.6), and PIAS1 has been shown to serve as the E3 enzyme for the sumoylation of various target proteins. In addition, PIAS proteins can regulate transcription either positively or negatively by recruiting histone acetylases or deacetylases, respectively.

13.3 T- and B-Cell Receptors

Summary T- and B-cell receptors are multisubunit receptors, whereby the functions of ligand binding and conduction of the signal are localized on separate subunits. The signaling subunits, such as the TCRf chains, carry phosphorylation sites – the ITAMs – that serve to transmit the signal further. Following T-cell receptor stimulation, the tyrosine residues within the ITAMs become Tyr-phosphorylated by associated non-RTKs, a key early event in the TCR-signaling cascade. The non- RTKs Zap70 and Lck are involved in these phosphorylations. Further signal transmission employs adaptors such as LAT. Examples of signaling pathways stimulated by T- and B-cell receptors include the Grb2/mSos/Ras pathway, PLC- þ c signaling, Ca2 -signaling, and PI3K signaling.

At the surface of T and B lymphocytes, speciﬁc receptors are found that bind antigens and set intracellular signal chains in motion. These immunoreceptors mediate signals that lead to increased cell division, programmed cell death, or a functional recoining of lymphocytes. The receptors of the B lymphocytes recognize antigens in the form of foreign proteins, which exist in soluble, particle-bound or cell-bound forms. In contrast, the receptors of the T lymphocytes recognize antigens only during the course of a cell–cell interaction between the T lymphocyte and an antigen-presenting cell (APC). The APC presents the processed (i.e., proteolytically digested to form peptides) foreign protein as a peptide which, in turn, is bound to the major histocompatibility complex (MHC) of the APC and is recognized in this form by the receptor of the T lymphocyte.

13.3.1 Immunoreceptor Structure

13.3.1.1 T-Cell Receptor Structure The T-cell receptor (TCR) contains a minimum of eight polypeptides: the TCRa and TCRb chains, two copies of the TCRf chains that are linked by disulﬁde bridge, a heterodimer composed of CD3e and CD3c, and a heterodimer composed of 13.3 T- and B-Cell Receptors 619

CD3e and CD3d. Antigen binding takes place via the TCR-a and TCR-b subunits, which only have very short cytoplasmic structural portions and are not directly involved in conduction of the signal on the cytosolic side [13].

& T-cell receptor subunits: — TCRa, TCRb — CD3c, CD3e, CD3d — TCRf.

The function of signal conduction is performed by the CD3cde chains and the TCRf chain (Figure 13.14a), which contain sequence motifs on their cytoplasmic side that are critical for signal transduction at the cytoplasmic side. The sequence motifs include two pairs of Tyr and Leu residues in the consensus motif (D/E)

XXYXXL(X)6–8YXXL known as the immunoreceptor tyrosine activation motif (ITAM). There are 10 ITAM motifs on the intracellular part of the TCR: six on the TCR f dimer, and one each on the two CD3e and the CD3d and CD3c subunits. The ITAM motifs are the essential signaling modules of the TCR, and these motifs are also found on other immunoreceptors, such as the B-cell receptor.

& ITAM: — Signaling module — Found on T-cell receptors B-cell receptors Adapter proteins — Becomes Tyr-phosphorylated by Lck, Fyn, Nck, Zap70 kinases.

ITAM motifs are also found on adapter proteins with functions in immune signaling. Following TCR stimulation, the tyrosine residues within the ITAMs become Tyr-phosphorylated by associated non-RTKs, which is a key early event in the TCR-signaling cascade. Cooperation with other receptors subunits that help in synergistic fashion to trigger a signal is a particular feature of signal transduction via T- and B-cell antigen receptors. These other receptors are known as coreceptors, and examples are the CD4 and CD8 proteins which have Src family protein kinases associated. The coreceptors are essential for signal transduction by triggering the early steps of receptor activation. Furthermore, they have an amplifying effect on the sensitivity and speciﬁcity of antigen binding.

13.3.1.2 B-Cell Receptor Structure The B-cell receptor (BCR) complex is composed of the homodimeric BCR associated with an Iga and Igb heterodimer (Figure 13.14b). The extracellular immunoglobulin domains of BCR bind and recognize the antigen. Signal transduction to the cytosol is mediated by Iga and Igb that harbor ITAM motifs. 620 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Figure 13.14 Subunit structure of the T-cell motif; (b) Schematic structure of the B-cell receptors. (a) The different subunits of T-cell receptor (BCR). The BCR has Iga/Igb chains as receptors in a highly simplified representation. associated coreceptors. Following stimulation The stoichiometry of the subunits in the of BCR by the binding of antigen (Ag), the Tyr- complete receptor is not clear. The ab chains specific protein kinase Syk phosphorylates are also known as the Tiab complex; the ce ITAMs (yellow box) of Iga/Igb and and de chains together form the CD3 complex. downstream effectors. ITAM, immunoreceptor tyrosine activation

Following antigen binding to the BCR, an associated non-RTK (Lyn, Syp; see Section 10.3) becomes activated and phosphorylates the ITAM motifs, which in turn triggers a downstream signaling cascade involving non-RTKs, adaptors, and other signaling proteins. The main signaling pathways involved in BCR signaling þ are the PI3 kinase pathway and the PLC-c/Ca2 pathway (see Chapter 8). For details, see Ref. [14]. 13.3 T- and B-Cell Receptors 621

13.3.2 Activation and Signaling of the T-Cell Antigen Receptors

13.3.2.1 Initiation of Signaling TCR signaling involves a multiprotein complex and the action of many signaling proteins, which has made it difﬁcult to characterize the overall structure, the triggering mechanisms of activation, and the recruitment of the downstream components. Only the basic steps of the signaling process will be presented in the following. Activation of TCR occurs in the context of a T-cell–APC interaction where the antigen bound to the MHC is recognized by the TCRa and TCRb chains of the TCR complex. Antigen binding is thought to induce conformational changes within the TCR complex that trigger the activation of Lck kinase constitutively associated with the CD4 coreceptor on the cytoplasmic side. Lck is now brought into proximity of the CD3 subunits and phosphorylates the ITAM motifs on two Tyr sites. When doubly phosphorylated, the ITAMs recruit another non-RTK, Zap70 kinase (ZAP ¼ zeta-associated protein).

13.3.2.2 Zap70 This kinase has been shown to be an essential part of TCR signaling because its knockout leads to severe immunodeficiency. Zap70 belongs to the Syk family of non-RTKs, and contains two N-terminal SH2 domains and a C-terminal kinase domain. In unstimulated cells, Zap70 exists in an autoinhibited, inactive state. Following ITAM phosphorylation by Lck, Zap70 is recruited to the receptor via a high-affinity interaction involving the binding of Tyr-phosphates of ITAM to the tandem SH2 domains of Zap70. This binding event leads to an activation of Zap70 by release from its autoinhibited state. During this process, Zap70 becomes phosphorylated on five Tyr-sites, either by Lck or by Zap70, via a trans-mechanism. Two of the activating Tyr- phosphorylation are located in the activation loop of the kinase domain.

& TCR activation: — Antigen binding to TCRa, TCRb — Activation of associated Tyr kinase: Lck, Fyn, Nck, Zap70 — Phosphorylation of ITAM motif — Phosphorylation of LAT adapter — Activation of further effectors.

When activated, Zap70 acts on further signaling proteins without further regulation by other tyrosine kinases (Figure 13.15). In addition to Zap70 kinase, a notable number of other signaling proteins – including adaptors and other protein kinases – have been shown to associate speciﬁcally with the cytoplasmic parts of the TCRf receptor and the CD3 receptor. It appears that, upon TCR activation, variable and dynamic signaling complexes are formed at the cytoplasmic side. 622 13 Membrane Receptors with Associated Tyrosine Kinase Activity

Figure 13.15 An overview of signaling pass a signal to the Ras/MAPK pathway or to pathways associated with the activation of the nonreceptor tyrosine kinase Tec. lymphocytes. The triggering signal for the Furthermore, PLCc is recruited to LAT and a þ activation of T lymphocytes is generally antigen Ca2 /diacylglycerol signal is generated leading - binding to the T-cell receptor. The activated -- via calcineurin/NF-AT2 (see also Section receptor passes the signal on to associated 9.7.5; Figure 7.34; Figure 9.25) --- to tyrosine kinases such as Fyn, Lck, and Zap70. transcription of target genes and to the These phosphorylate the transmembrane activation of protein kinase C (PKC). DAG, protein LAT on cytoplasmic tyrosine residues. diacylglycerol; InsP3, inositol-1,4,5- The LAT phosphotyrosine residues are docking trisphosphate. sites for adaptors (Shc, Grb2) and GEFs which 13.4 Signal Transduction via Integrins 623

13.3.2.3 LAT (Linker for Activation of T Lymphocytes) A major substrate of activated Zap70 is the adapter protein LAT (see also Section 10.2.1) that belongs to a class of integral membrane proteins collectively named transmembrane adapter proteins (TRAPs) [15]. LAT has a short extracellular domain, a single membrane-spanning element, and a long cytoplasmic tail with nine conserved tyrosine residues, ﬁve of which have been shown to undergo phosphorylation. Furthermore, LAT harbors two palmitoylation sites that target LAT to the cell membrane. Once the TCR is engaged in antigen binding, LAT becomes multiply Tyr- phosphorylated by activated Zap70; the newly created Tyr-P sites then provide attachment sites for effector proteins containing SH2 domains. These LAT-binding proteins subsequently attract multiple protein partners, such that LAT functions as a crucial nucleation center for multiprotein complexes translating the immunoreceptor-mediated signals into the appropriate cellular responses. The signaling proteins recruited to phosphorylated LAT include PLC-c and adapter proteins such as Grb2, which assemble further proteins such as GEFs (Sos, Vav) and non-RTKs (Tec) into the TCR-signaling cascade. As illustrated in Figure 13.15, the following signaling pathways are activated following stimulation of TCRs:

Promotion of cell proliferation, via binding of Grb2-Sos, activation of Ras and MAPK cascades. þ Activation of PLC-c with subsequent release of intracellular Ca2 ,inﬂux of þ þ extracellular Ca2 , sustained intracellular Ca2 , and activation of the transcription factor NFAT (see also Chapter 8). Recruitment of further adaptors including SLP-76, leading to the activation of PI3K and a subsequent reorganization of the cytoskeleton, among others.

13.4 Signal Transduction via Integrins

Summary The integrins are transmembrane receptors that can transmit signals from “outside to inside,” as well as from “inside to outside.” Extracellular signaling partners may be other cells or the extracellular matrix. Outside-in signaling by integrins controls major cellular processes such as cytoskeletal organization, gene expression and the cell cycle. The integrins are heterodimers constructed from a- and b-chains, each of which have a single transmembrane element. Ligand binding to integrins induces conformational changes within the heterodimer and recruitment for the reorganization of associated scaffold proteins such as talin and kindlin. These scaffolds interact with further adapter proteins such as Grb2, with Ser/Thr-specific protein kinases, and with non-RTKs such as the FAK to carry the signal further. 624 13 Membrane Receptors with Associated Tyrosine Kinase Activity

The structure and function of the cell formations of higher organisms are highly dependent on adhesive interactions based on direct cell–cell contact and on the interactions of cells with the extracellular matrix. Surface receptors that can bind specifically to a neighboring cell or to the extracellular matrix serve as mediators of adhesion processes and, as a consequence, intracellular signaling pathways are activated. The protein family of the integrins are one such group of surface receptors which play a major role in regulating diverse cell adhesion migration events, both in stationary cells (such as fibroblasts) and in mobile cells (such as leukocytes) [16]. The integrins define attachment points for the extracellular matrix and for contact with neighboring cells, and they are involved in signal transduction into the cell interior (“outside-in”) as well as from the cell interior to the extracellular side (“inside-out”). With these functions, the integrins are involved in the regulation of embryonal growth, tumor formation, programmed cell death, tissue homeostasis, and many other processes within the cell.

& Integrins: — Transmembrane receptors, composed of a and b subunits, — Involved in Inside-out signaling Outside-in signaling.

Integrins participate in bidirectional signaling across the plasma membrane: “inside-out” signaling, whereby integrins become activated for extracellular ligand binding by intracellular signaling (Figure 13.16), and “outside-in” signaling, whereby integrins themselves signal into the cell after ligand binding. Outside-in signaling by integrins regulates many cellular processes including cytoskeletal reorganization, gene expression and the cell cycle. Integrin activation by inside-out signaling controls the binding of extracellular ligands (presumably by conformational changes) and the clustering of integrins in the cell membrane that is mediated by cytoskeletal interactions and membrane rafts. Overall, integrin signaling is a highly complicated process that involves a network of more than 150 components and over 600 interactions. Hence, only a very basic outline of integrin signaling is provided at this point. The integrins are composed of a- and b-chains, each of which has a single transmembrane element. In mammals, there are at least 24 different integrin heterodimers, formed from combinations of 18 different a-subunits and eight different b-subunits. Different activity states of the integrins have been described that are characterized by various binding afﬁnities for extracellular ligands. In the low-afﬁnity state, the extracellular parts of integrins adopt a bent conformation, while the transmembrane and intracellular parts exist in a twisted conformation. The binding of integrins to their extracellular ligands changes the conformation of the heterodimer and, because many of the ligands are multivalent, this contributes to integrin clustering. The conformational changes in the heterooligomer can be triggered during both outside-in signaling and inside-out signaling. In the latter 13.4 Signal Transduction via Integrins 625

Figure 13.16 The two directions of integrin clustering. The combination of these two signaling. During “inside-out” signaling, an events leads to intracellular signals that control intracellular activator, such as talin or kindlin, cell polarity, cytoskeletal structure, gene binds to the b-integrin tail, leading to expression and cell survival and proliferation. conformational changes that result in an Although these two processes are separated increased affinity for extracellular ligands conceptually they are often closely linked; for (integrin “activation”). Integrins also behave example, integrin activation can increase like traditional signaling receptors in ligand binding, resulting in outside-in transmitting information into cells by “outside- signaling. Conversely, ligand binding can in” signaling. The binding of integrins to their generate signals that cause inside-out extracellular ligands changes the conformation signaling. Shattil et al. 2010 [17], box 2. of the integrin and, because many of the Reproduced with permission of Nature ligands are multivalent, contributes to integrin Publishing Group. case, cytoplasmic signals promote a high-afﬁnity binding of external ligands (Figure 13.16).

13.4.1.1 Talin and Kindlin as Key Activators The trigger point for the integrin conformational changes and their activation are the cytoplasmic tails of the a- and b-chains that bind to two key cytoplasmic proteins, talin and kindlin [18]. Both of these proteins interact not only with integrin tails but also with a large number of other components of integrin signaling, inducing the formation of large cytoplasmic signaling complexes. The function of talin and kindlin can be described as a scaffold that binds in response to external cues to the b-chain tails and triggers a conformational change of the integrin heterodimer. As a consequence, the afﬁnity of integrins for their external ligands is modulated, a feature that is relevant during “outside-in” and “inside-out” signaling. Talin harbors a FERM domain that has PTB-like binding characteristics and mediates binding to conserved motifs on the integrin b-chain. This binding event induces an extended conformation of the b-chain tail and the transmembrane 626 13 Membrane Receptors with Associated Tyrosine Kinase Activity

domain, enforcing an altered membrane crossing-angle on the latter. In addition, talin binding is thought to interrupt interactions between the a- and b-chains, which leads to a relief of the closed conformation of the integrin extracellular domains. Mechanistic details of the sequence of events and the coupling of individual steps are not yet available. The kindlins are scaffolding proteins that are essential for integrin signaling and cooperate with talin function. The kindlin family of b-integrin binding proteins also carry a FERM domain, and cooperate with talin by binding to a site on the b-chain that is distinct from the talin site.

13.4.1.2 Downstream Signaling The recruitment of talin and kindlin to integrins is the starting point for the formation of a large adhesion complex. The following proteins have been shown to interact with integrins on the cytoplasmic side (Figure 13.17):

Microfilament or stress fiber components: a-actinin, filamin, talin, kindlin Adapter proteins: paxillin, grb2-Sos, Cas Membrane proteins: caveolin Ser/Thr-specific protein kinase: ILK, integrin-linked kinase non-RTKs: Syk, focal adhesion kinase (FAK).

As a consequence of ligand binding and clustering of integrins, the activation of different signaling pathways is initiated:

Activation of non-RTKs Activation of Ser/Thr-speciﬁc proteins kinases such as the integrin-linked kinase (ILK) þ Increase in Ca2 concentration Activation of the MAPK cascade Increased formation of Ptd-Ins messenger substances Activation of Rho/Rac GTPase signaling.

The integrin-mediated activation of MAPK pathways seems particularly important for integrin function, as this has an influence on transcription processes. Furthermore, the association of talin with microtubule components provides a rather rigid linkage between microfilament organization and the extracellular binding of other cells during cell–cell interactions. In this way, the integrin–talin system is thought to mediate the transfer of mechanical signals from across the cell membrane. It has to be assumed that the association of the large number of potential downstream effectors occurs in a fashion that is specific for each of the more than 20 different heterodimers. Each heterodimer may associate and activate a different set of effectors, with some effectors being more commonly used. Furthermore, the composition of the assembled signaling complexes may be dynamic and timely variable. Only the participation of protein kinases will be discussed in the following. 13.4 Signal Transduction via Integrins 627

Figure 13.17 Model of signal transduction via linked kinase (ILK) and the focal adhesion integrins. Upon “outside-in” signaling, kinase (FAK). Furthermore, proteins involved activated integrins transmit signals from the in reorganization of the cytoskeleton (paxillin, extracellular matrix to the cytoskeleton and cytohesin, endonexin) and in the formation of activate various intracellular signaling stress fibers associate with activated integrins. þ pathways. The diagram lists some of the signal Association of the Ca2 -binding protein proteins that have been shown to be involved calreticulin with integrins is thought to link þ in integrin signaling. The signal conduction integrin signaling with Ca2 -signaling. Signal involves --- among others --- the Ras/MAPK conduction via integrins occurs in multiprotein þ pathway and Ca2 -signaling pathways, setting complexes. It is therefore only possible to in motion a broad spectrum of subsequent show selected protein components of these reactions. At least two protein kinases complexes. participate in signal transduction, the integrin-

13.4.1.3 Focal Adhesion Kinase (FAK) Phosphorylation of the integrin tails has emerged as a dynamic mechanism that regulates the association of integrin signaling components. Most contacts to the downstream effectors of the integrins appear to be mediated by the integrin b-subunits that harbor many Ser/Thr and Tyr phosphorylation sites, and these direct the binding of adapter proteins, protein kinases, protein phosphatases and phospholipases. Furthermore, many of the integrin effector proteins are 628 13 Membrane Receptors with Associated Tyrosine Kinase Activity

phosphorylated during the process of integrin signaling, and their phosphotyrosine residues serve as attachment points for SH2-, SH3-, or PTB-containing signaling proteins

& Integrin signaling: — Mediated by phosphorylation of cytoplasmic tails of b-subunits. Downstream effectors of integrins: — Ser/Thr protein kinases — Tyr kinases — Adapter proteins — Stress fiber proteins. Protein kinases involved in integrin signaling: — Focal adhesion kinase — Integrin-linked kinase — Syk kinase — Src kinase.

Of the protein tyrosine kinases, the focal adhesion kinase (FAK; p125FAK) plays an important role in integrin signal transduction [19] by linking integrins to cell adhesion, Rho-GTPase activation, cell migration and crosstalk with growth factor signaling. In response to integrin clustering, the FAK protein undergoes autophosphorylation and the newly created phosphotyrosine residues serve as binding sites for SH2-containing signal molecules such as PI3 kinase, Src kinase, PLC-c and the adapter proteins Grb2, among others. The FAK protein also has a speciﬁc binding domain for binding to talin and another adapter protein, paxillin, which is found in adeﬁned complex together with FAK. Paxillin is a multifunctional docking protein that interacts with components of cytoskeleton structures (among others), so the recruitment of FAK to the cytoskeleton in the region of focal adhesion points seems possible via paxillin–FAK interaction.

Questions 13.1. Give examples of medically important cytokines. Which types of subunit are found in cytokine receptors type I and II? 13.2. Describe the functions of the subunits of the IL-2 receptor. 13.3. Which are the major tyrosine kinases associated with cytokine receptors? How may these kinases be activated by cytokine binding? 13.4. Activation of cytokine receptors can lead to transcription activation of target genes. How may cytokine receptors convey signals to the level of transcription activation? Describe the reactions and pathways involved. References 629

13.5. Which steps are involved in recruitment and activation of downstream effector proteins of cytokine receptors? Which pathways may be activated by cytokine receptors? 13.6. Describe the essential features of the canonical Jak/Stat pathway. How are the STAT proteins regulated? 13.7. How is cytokine signaling regulated? 13.8. Describe the principal structural organization of B- and T-cell receptors. 13.9. Which proteins are involved in the immediate signaling events following antigen binding? 13.10. Describe the structural organization of integrins and the function of the key downstream activators.

References

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19 (4), 414–422. PubMed PMID: 18708154. PMID: 20610546. Pubmed Central PMCID: Pubmed Central PMCID: 2597703. 2908767. 12 Shuai, K. (2006) Regulation of cytokine 16 Harburger, D.S. and Calderwood, D.A. signaling pathways by PIAS proteins. Cell (2009) Integrin signalling at a glance. J. Cell Res., 16 (2), 196–202. PubMed PMID: Sci., 122 (Pt 2), 159–163. PubMed PMID: 16474434. Epub 2006/02/14. eng. 19118207. Pubmed Central PMCID: 13 Wucherpfennig, K.W., Gagnon, E., Call, PMC2714413. Epub 2009/01/02. eng. M.J., Huseby, E.S., and Call, M.E. (2010) 17 Shattil, S.J., Kim, C., and Ginsberg, M.H. Structural biology of the T-cell receptor: (2010) The ﬁnal steps of integrin activation: insights into receptor assembly, ligand the end game. Nat. Rev. Mol. Cell Biol., recognition, and initiation of signaling. Cold 11 (4), 288 300. Spring Harb. Perspect. Biol., 2 (4), a005140. 18 Moser, M., Legate, K.R., Zent, R., and PubMed PMID: 20452950. Pubmed Central Fassler, R. (2009) The tail of integrins, talin, PMCID: 2845206. and kindlins. Science, 324 (5929), 895–899. 14 Kurosaki, T. (2011) Regulation of PubMed PMID: 19443776. BCR signaling. Mol. Immunol., 48 (11), 19 Arold, S.T. (2011) How focal adhesion 1287–1291. PubMed PMID: 21195477. kinase achieves regulation by linking ligand 15 Balagopalan, L., Coussens, N.P., Sherman, binding, localization and action. Curr. Opin. E., Samelson, L.E., and Sommers, C.L. Struct. Biol., 21 (6), 808–813. PubMed (2010) The LAT story: a tale of cooperativity, PMID: 22030387. Pubmed Central PMCID: coordination, and choreography. Cold Spring PMC3232291. Epub 2011/10/28. eng. Harb. Perspect. Biol., 2 (8), a005512. PubMed 631

14 Other Transmembrane Receptor Classes: Signaling by TGF-b Receptors, TNF Receptors, Toll Receptors, and Notch

Cells have evolved a variety of mechanisms by which activated transmembrane receptors create intracellular signals (Table 14.1). In the following, receptors with intrinsic Ser/Thr kinase activity (TGF-bR), and receptors undergoing intramembrane proteolysis (Notch) will be discussed. Furthermore, selected examples of receptors will be presented that use adapter proteins to assemble distinct signaling complexes at the cytoplasmic side of the receptor (TNFR, Toll receptors).

14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling

Summary The TGF-b receptors (TbRs) and their ligands, the family of TGF-b cytokines, are master regulators of cell proliferation, differentiation, migration and apoptosis. In most tissues, TGF-b signaling has a negative, suppressing effect on cell proliferation. The TbRs are transmembrane receptors that signal through intrinsic Ser/Thr kinase activity. TGF-b cytokine binding to TbR activates the intrinsic Ser/Thr kinase activity that leads to phosphorylation and nuclear translocation of transcriptional regulators, theSmadproteins,categorizedintoR-Smads,I-Smads,andSmad4proteins.These proteins cooperate with other transcription factors to influence the transcription of a large number of genes in either a positive or negative fashion. The TGF-b cytokines are dimeric proteins that are synthesized and stored as precursors that require proteolytic processing to produce the mature cytokine ligand. Active, cytokine-bound TbRs are tetramers, composed of two copies of receptor subunits TbRIandTbR II. The cytokine binds as a dimer to the receptor and contacts both the TbRIandTbRII receptors that cooperate in activating the intrinsic Ser/Thr kinase activity. The immediate downstream substrates of the kinase activity are the R-Smads that bind to Smad4 and then translocate into the nucleus, where the transcription of target genesismodulated.TGF-b signaling is controlled in several ways, including the inhibitory I-Smads, negative feedback control at the level of Smad transcription, dephosphorylation by phosphatases, and ubiquitination.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 632 14 Other Transmembrane Receptor Classes

Table 14.1 Classification of mammalian receptors by signaling activity. Extracellular ligand binding triggers the following activities on the cytoplasmic side of the receptors.

Signaling activity Location in book

Intrinsic tyrosine kinase activity Chapter 10 Associated tyrosine kinase activity Chapter 13 Intrinsic Ser/Thr kinase activity: This chapter TGF-b receptor Leucine-rich repeat receptor kinases of plants G protein-coupled receptors Chapter 7 Ligand-gated ion channels: NMDA receptor Intrinsic guanylyl cyclase activity Chapter 8 Intrinsic phosphatase activity Chapter 10 Intramembrane proteolysis: This chapter Notch GTPase-activating activity: Plexin-B1, Section 11.4 Robo receptors [1] Associated adapter proteins: This chapter Tumor necrosis factor receptor, TNF-R Toll-like receptors, TLRs Not discussed in this book: Glycosyl-phosphatidyl-inositol-linked receptors: Nogo [2] Histidine kinase activity, intrinsic or associated: “Two-component systems” in bacteria and yeast

In addition to receptors with intrinsic or associated tyrosine kinase activity, transmembrane receptors are also known that exert their signaling function through intrinsic or associated Ser/Thr kinase activity. A prominent example of receptors with intrinsic Ser/Thr kinase activity includes the receptors for the transforming growth factor-b (TGF-b) family of cytokines [3]. Other receptors with intrinsic Ser/Thr kinase activity are leucine-rich receptors, of which a large number is known in plants.

& TGF-b family of cytokines: — Activate receptors with intrinsic Ser/Thr kinase activity — Often suppress cell proliferation — Induce activation or repression of transcription. 14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling 633

Members of the TGF-b family of cytokines play a major role in the development of higher organisms. In particular, they regulate the establishment of the body plan through their effects on cell proliferation, differentiation, migration and apoptosis. In most tissues, TGF-b signaling has a negative, suppressing effect on cell proliferation, and it is not surprising therefore that inactivation of this pathway contributes to tumorigenesis, and that several components of the TGF-b signaling pathway have been identiﬁed as bona ﬁde tumor suppressors [4]. Overall, the signaling pathway of the TGF-b cytokines and their receptors employs, in most cases, a seemingly simple strategy. Binding of the cytokine to the TGF-b receptor triggers a Ser/Thr kinase activity in the receptor protein that leads to phosphorylation and activation of cytosolic proteins with the function of transcriptional regulators, the Smad proteins [3].

14.1.1 The Family of TGF-b Cytokines

The family of TGF-b cytokines consists of secreted peptides encoded by more than 40 open reading frames in humans. These proteins cluster in three major subfamilies: the TGF-b subfamily; the activin/inhibin subfamily; and the bone morphogenetic protein (BMP) subfamily, as deﬁned by sequence similarity and the signaling pathways that they activate.

& TGF-b family of cytokines comprises three subfamilies: — TGF-b/activin subfamily — BMP subfamily — Activin subfamily.

Although the diverse TGF-b family members elicit quite different cellular responses, they all share a set of common structural features. The active form of a TGF-b cytokine is a dimer that typically contains intersubunit disulfide bridges. Each monomer comprises several b-strands interlocked by conserved disulfide bonds, also known as the cystine knot motif. All members of the TGF-b subfamily are synthesized as precursor proteins that are secreted, stored and processed in larger complexes consisting of unprocessed and processed TGF-b. These complexes can associate with proteins of the extracellular matrix, and represent the latent stages of TGF-b ligands. The action of diverse proteases is then required for making the mature TGF-b ligands available for receptor binding. Unlike the TGF-b subfamily members, the BMP proteins are secreted in an active form and their activity is regulated by agonistic peptides that prevent interaction with the receptor. Binding of the dimeric cytokines to the extracellular domain of the receptors may be tightly regulated by two classes of proteins with opposing function. The first of these classes comprises a diverse set of proteins, collectively known as ligand traps, 634 14 Other Transmembrane Receptor Classes

that act by sequestering the ligand and barring its access to the receptor. The second class of proteins promote cytokine binding to the TGF-b receptors, are membrane-associated, and act as accessory receptors or coreceptors.

14.1.2 TGF-b Receptor

Active, ligand-bound TGF-b receptors are heterooligomers, composed of two types of receptor, TbRIand TbRII. The cytokine binds as a dimer to the receptor and contacts both the TbR I and TbR II receptors, which in turn leads to the formation of a receptor tetramer composed of two copies of TbR I and TbR II (Figure 14.1).

Figure 14.1 General mechanisms of TGF-b common Smad 4. Activated Smad complexes receptor activation. At the cell surface, the translocate into the nucleus, where they ligand binds a complex of type I and type II regulate the transcription of target genes, in transmembrane receptors and induces collaboration with coactivators such as CBP or transphosphorylation of the Gs segments (red) p300. Activation of R-Smads is inhibited by in the type I receptor by the type II receptor Smad 6 or Smad 7. Furthermore, TGF-b kinases. The consequently activated type I signaling is downregulated by the E3 ligases receptors phosphorylate selected Smads at C- Smurf1/2 that mediate ubiquitination and terminal serines, and these receptor-activated consequent degradation of R-Smads, Smad Smads (R-Smads) then form a complex with a 6/7, and of the type I receptor. 14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling 635

14.1.2.1 TGF-b Receptor Structure The five type II and seven type I TbRs that exist in humans and other mammals are characterized by a cytoplasmic kinase domain that has a strong serine/threonine kinase activity and a weaker tyrosine kinase activity; thus, they are classified as dual- specificity kinases. The type I receptors are also known as activin-receptor-like kinases (ALKs). Both types of receptor serine/threonine kinases are organized into an N-terminal extracellular ligand-binding domain, a single-pass transmembrane region, and a C- terminal protein kinase domain.

& TGF-b receptor is a tetramer, composed of two copies of: — TbRI — TbR II.

Type I, but not type II, receptors contain a characteristic SGSGSG sequence, termed the GS domain, located immediately N-terminal to the kinase domain. Activation of the type I receptor involves the phosphorylation of its GS domain by the type II receptor; hence, an active receptor signaling complex will comprise both types of receptor bound to the ligand. Within this complex, the type I receptor is responsible for signaling to the downstream effectors, the Smad proteins.

14.1.2.2 TGF-b Receptor Activation Two distinct modes of ligand–receptor interaction exist, one exemplified by members of the BMP subfamily, and the other represented by TGF-bs and activins. BMP ligands exhibit a high affinity for the extracellular ligand binding domains of the type I BMP receptors and a low affinity for the type II receptors. In contrast to the BMPs, TGF-b and activin display a high affinity for the type II receptors and do not interact with the isolated type I receptors. In this case, the ligand first binds tightly to the ectodomain of the type II receptor; this binding allows the subsequent incorporation of the type I receptor, forming a large ligand–receptor complex that incorporates a ligand dimer and four receptor molecules.

& TbR activation: — Binding of dimeric TGF-b ligand induces phosphorylation of TbR I on GS sequence

Binding to the extracellular domains of both types of receptor by the dimeric ligand induces a close proximity and a productive conformation for the intracellular kinase domains of the receptors, facilitating the phosphorylation and subsequent activation of the type I receptor. The type II receptor kinases are thought to be constitutively active and to phosphorylate multiple serine and threonine residues in the TTSGSGSG sequence of the cytoplasmic GS region of the type I receptor, leading to its activation. Because of its critical role in receptor activation, the GS region serves as an important regulatory domain for TGF-b signaling. 636 14 Other Transmembrane Receptor Classes

& Phospho-TbR I induces: — Phosphorylation of R-Smad — Activation of other signaling pathways such as ERK, JNK, PI3K, Ras, Rho.

The phosphorylated TbR I protein now provides a high-afﬁnity binding site for the signal protein next in sequence, the R-Smads; the latter are then phosphorylated by TbR I, allowing them to direct signals to the transcriptional level. The activation of TbRI/TbR II by TGF-b family members may be modulated by accessory proteins that function as coreceptors. TGF-b ligands can also interact with coreceptors such as endoglin to either facilitate or limit receptor kinase signaling.

14.1.2.3 Non-Smad Signaling Pathways Besides Smad-mediated transcription, TbR proteins activate other signaling cascades allowing for a high diversity of downstream signaling (for details, see Ref. [5]). The Smad-independent activities of TbR proteins are mediated by the association with activated TbR of a diverse set of signaling proteins including the Shc adapter and the E3 ligase TRAF (Section 14.4). The Tyr-Phosphorylation of TbR by Src kinase has been implicated in recruitment of Shc and subsequent activation of Ras/MAPK pathways. Furthermore, Rho signaling pathways and the PI3 kinase/Akt pathway have been shown to become activated by TbR proteins (Figure 14.2).

14.1.3 Smad Proteins

At least eight different Smad proteins have been identiﬁed in higher organisms [6], and these are divided into three functional classes: the receptor-regulated Smads (R- Smads 1–3, 5, and 8); the Co-Smad (Smad4); and the inhibitory Smads (I-Smads, Smad6, 7).

& Classes of Smads: — R-Smads: Smad 1–3, 5, 8 — Co-Smad: Smad4 — I-Smads: Smad6 and Smad7.

The domain structure of the Smads is shown in Figure 14.3. R-Smads and Smad4 contain a conserved MH1 domain and C-terminal MH2 domain, ﬂanking a divergent middle “linker” segment that harbors several regulatory phosphorylation sites. Inhibitory Smads lack a recognizable MH1 domain, but have an MH2 domain. Both, the MH1 and MH2 domains can interact with select sequence- speciﬁc transcription factors, whereas the C terminus of the R-Smads interacts with and recruits the related coactivators CREB-binding protein (CBP) or p300 (Section 4.5). With the exception of Smad2, the MH1 domains of Smads can bind

638 14 Other Transmembrane Receptor Classes

Figure 14.3 Domain structure and serine residues within the C-terminal SSXS phosphorylation sites in Smad2. R-Smads motif (X any amino acid). ERK, JNK (see (Smads 1---3 and Smad 5) and Smad 4 contain Chapter 12) and CDK4 (see Section 15.4.1) a N-terminal MH1 domain (MH, Mad alternatively phosphorylate Smad 2 at specific homology) and a C-terminal MH2 domain, sites in their middle linker regions, as the lines flanking a central “Linker” domain. indicate. A similar phosphorylation pattern is Catalytically, active TbR I phosphorylates two observed on Smad 3.

DNA, whereas the MH2 domains mediate Smad oligomerization and Smad– receptor interaction.

14.1.3.1 Smad Activation R-Smads, but not the other Smads, are directly phosphorylated by the activated type I receptors on a C-terminal SSXS motif. The phosphorylation of Smads by type I receptors is quite speciﬁc. R-Smads 2 and 3 are activated by TGF-b/activin receptors, whereas R-Smads 1, 3, and 5 are activated by the BMP receptors (Figure 14.4).

& Smad activation involves: — R-Smad binding to Phospho-TbRI — Help of adapter protein SARA — Phosphorylation of R-Smads — Formation of complex R-Smad–Smad4 — Translocation of R-Smad–Smad4 into the nucleus — Transcription regulation.

The recognition of R-Smads by the receptors may be facilitated by auxiliary proteins. For example, Smad2 and Smad3 can be specifically immobilized near the cell surface by the “Smad anchor for receptor activation” (SARA). This interaction allows a more efficient recruitment of Smad2 or Smad3 to the receptors for phosphorylation. In cooperation with Smad7, the SARA proteins also function as an anchor for the catalytic subunit of protein phosphatase PP2A, allowing the dephosphorylation and inactivation of TbRI. Phosphorylation of R-Smad induces the exposure of a nuclear import region on the Smad MH2 domain, leading to nuclear translocation. In addition, R-Smad phosphorylation augments its affinity for the Co-Smad, Smad4, and heterodimeric 14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling 639

Figure 14.4 Regulation of signaling by the R-Smads by the activated receptor. The TGF-b superfamily receptors. The extracellular inhibitory Smads are regulated at the signaling molecules TGFb, activin and bone transcriptional level via the TNFa/NFkB morphogenetic factor (BMP) each activate a (Chapter 14), EGF/MAPK (Chapters 10 and distinct receptor composed of type I and type II 12) and interferon-c/Jak/Stat (Chapter 13) subunits. TGF-b receptors and activin pathways, and by negative feedback via R- receptors activate the R-Smads Smad 2 or Smad signaling. R-Smad phosphorylation can Smad 3, while the BMP receptor uses Smad 1, be negatively regulated by protein kinase C 5 or 8 as R-Smads for further signal (PKC), CamKinase II and the extracellular transmission. Smad 6 and Smad 7 inhibit regulated kinase (ERK). Smad levels may be signaling by interfering with complex also downregulated by the action of the E3 formation between the R-Smads and the Co- ubiquitin ligase Smurf. Smad, or by inhibiting phosphorylation of the

or heterotrimeric complexes then form between phosphorylated R-Smad and Smad4. The association of these two proteins nucleates the assembly of transcriptional regulation complexes. In their basal state, R-Smads are predominantly localized in the cytoplasm, whereas the I-Smads tend to be nuclear. Smad4 is distributed in both the cytoplasm and the nucleus. After receptor activation, the phosphorylated R-Smads are translocated into the nucleus where they associate with Smad4 and bind to the 640 14 Other Transmembrane Receptor Classes

target DNA elements. Following dephosphorylation by protein phosphatases, the R- Smads are exported to the cytosol and can be activated again.

& R-Smads shuttle between cytosol and nucleus: — Phosphorylation promotes nuclear localization — Dephosphorylation promotes cytosolic localization.

Continuous shuttling of R-Smad with repeated cycles of receptor-mediated phosphorylation, and dephosphorylation appears to be a key event in TGF-b signaling, permitting a constant sensing of the activation status of the TGF-b receptor and ensuring an efﬁcient termination of signaling upon receptor inactivation.

14.1.3.2 DNA Binding and Transcriptional Regulation by Smads Smad4 and all R-Smads except for Smad2 bind to DNA in a sequence-specific manner, though with only low specificity. The minimal Smad binding element (SBE), initially identified as the optimal DNA binding sequence for Smad3 and Smad4, contains only four base-pairs, 50-AGAC-30. Because of the relatively low specificity of DNA binding by individual Smad proteins, Smads must cooperate among each other and with other DNA-binding proteins to elicit specific transcriptional responses.

& R-Smads: — Activate or repress transcription — Bind to DNA-elements — Cooperate with other transcription factors.

Smad access to target genes and the recruitment of transcriptional coactivators or corepressors to such genes is assumed to require the cooperation of speciﬁc partner proteins. Members from many different families of DNA-binding proteins – forkhead, homeobox, Jun/Fos, CREBP, and E2F – have been shown to function as Smad partners in this fashion. Many of these interactions are mediated by the MH2 domain that directs the binding of transcriptional coactivators as, for example, the histone acetylase CBP/p300 (Section 4.5).

14.1.4 Regulation of TGF-b and Smad Signaling

The TGF-b/Smad signaling pathway is controlled at various levels and is subjected to crosstalk with other signaling pathways [5], placing TbR signaling in a complicated regulatory network. The main regulatory inﬂuences are:

Binding of I-Smads PTM: ubiquitination, sumoylation, and phosphorylation 14.1 Receptors with Intrinsic Ser/Thr Kinase Activity: The TGF-b Receptor and Smad Protein Signaling 641

Activation of Smads via other signaling pathways Regulation of Smad expression.

14.1.4.1 Inhibition by I-Smads A main negative control of TGF-b/Smad signaling is exerted by the I-Smads, Smad6, and Smad7. Several mechanisms for this have been identiﬁed (Figure 14.4).

The I-Smads inhibit TGF-b family signaling through binding of their MH2 domains to the type I receptor, thus preventing the recruitment and phosphorylation of R-Smads 1–3. Smad6 also interferes with the heteromerization of BMP-activated Smads with Smad4, preventing the formation of an effector Smad complex.

& Regulation of TGF-b/Smad signaling: — By I-Smads — By phosphorylation — By ubiquitination via E3 ligase Smurf — By Smad expression.

14.1.4.2 Ubiquitination and Sumoylation Furthermore, the levels of TGF-b receptors are controlled by ubiquitin–proteasome-mediated degradation involving the I-Smads. I-Smads can recruit the HECT family E3 ubiquitin ligases Smurf 1 and Smurf 2 to the receptor, which induces ubiquitination and proteasomal degradation of the receptor complex. The sumoylation of TGF-b receptors is another regulatory modiﬁcation that appears to facilitate receptor activation.

14.1.4.3 Phosphorylation Besides the activating phosphorylations of TbR I and Smad 2/3, further phosphorylation events modulate TGF-b ligand signaling targeting both the receptors and the Smad proteins. For example, TbR I and TbR II also become Tyr- phosphorylated upon ligand binding. The Tyr-P sites on TbR II have been shown to recruit the Shc adapter (Section 10.2.1.2), providing a link to the Sos–Ras–MAPK signaling path. In this way, growth-promoting signals can be mediated by the TGF- b receptors. Furthermore, the Smad proteins are subject to regulatory phosphorylations by protein kinases other than the TbR kinases, and multiple phosphorylated isoforms of Smad proteins exist with distinct regulatory properties [6]. These phosphorylations are found mainly in the linker domain of the Smads, and are catalyzed by various protein kinases including MAPKs, CamK II (Section 9.6), and GRK (Section 7.3.3).

14.1.4.4 Activation of Smad Expression Importantly, the expression of the inhibitory Smads is highly regulated by extracellular signals. TGF-b cytokine ligands induce the expression of Smad6 and 642 14 Other Transmembrane Receptor Classes

Smad7, thus providing an autoinhibitory feedback mechanism for ligand-induced signaling. Other major receptor signaling pathways have also been shown to activate I-Smad expression (see Figure 14.4). Activation of the epidermal growth factor (EGF) receptor, interferon-c signaling through STAT proteins, and the activation of NFkB by tumor necrosis factor-a (TNF-a), induces Smad 7 expression, leading to an inhibition of TGF-b signaling.

14.1.4.5 Growth Regulatory Effects of TGF-b Signaling Depending on the cell type, TGF-b signaling can have either growth-promoting or growth-inhibiting effects. A multitude of genes are subject to transcriptional regulation of TGF-b signals, including genes that have proliferation-promoting effects and genes that have anti-mitogenic effects. Important genes regulated by TGF-b include the gene for the kinase inhibitor p15Ink4b (Section 15.2.3.2) that is activated by TGF-b in epithelial cells, and the gene for the transcription factor c- Myc that is repressed by TGF-b. Via these influences, TGF-b has a strong antiproliferative effect in these cells and a loss of TGF-b function by mutation promotes tumor formation. The proliferation-promoting effects of TGF-b signaling appear to be mediated by Ras-MAPK-mediated phosphorylation of Smad3 in the linker domain. In total, TGF-b–Smad signal conduction has distinct similarities to signal conduction in the Jak/STAT pathway (Section 13.2). In both pathways, cytosolic transcription factors are activated by phosphorylation and are translocated in oligomeric complexes to the nucleus and the DNA. Common to both pathways is the short distance from the extracellular signal to the transcription level. However, signaling through TGF-b/Smads is more complicated and versatile than it appears at first glance. Although there are considerably fewer receptors and Smads than there are ligands, a great versatility of signaling is possible. Combinatorial interactions of type I and type II receptors and Smads in oligomeric complexes allow substantial diversity, and are complemented by the many sequence-specific transcription factors with which Smads cooperate, resulting in context-dependent transcriptional regulation.

14.2 Receptor Regulation by Intramembrane Proteolysis: The Notch Receptor

Summary The Notch signaling pathway mediates short-range communication via cell–cell interactions in higher organisms. The Notch ligand on one cell binds to and activates the Notch receptor (or simply Notch) on a neighboring cell. Notch receptors are transmembrane proteins that undergo ligand-induced proteolytic processing to generate intracellular Notch signaling fragments that function as transcriptional regulators. Notch processing can be characterized as regulated intramembrane proteolysis, a process used for the processing and the 14.2 Receptor Regulation by Intramembrane Proteolysis: The Notch Receptor 643

maturation of many other transmembrane proteins. This processing includes a proteolytic shedding of the extracellular domain, proteolysis within the transmembrane domain, and the release of a cytosolic fragment that serves to transmit the signal further. In Notch signaling, the released fragment translocates into the nucleus where it cooperates with other transcription factors to regulate the transcription of target genes. In this way, Notch signaling promotes the development and/or proliferation of a variety of cell types and influences multiple developmental steps within a given lineage.

Notch receptors use a new concept of transmembrane signaling that is set apart from other conserved signaling pathways. Signaling by Notch receptors relies on the ability of a ligand to bring about receptor proteolysis, resulting in the intracellular release of a Notch fragment that carries the signal on. A second unusual feature of Notch signaling is that receptor activation is achieved by regulated intramembrane proteolysis (RIP).

14.2.1 Notch: General Function, Structure, and Ligands

The transmission of Notch signals requires physical contact between cells under most circumstances, and this signaling relies on the interaction between the Notch ligand presented on one cell and the Notch receptor (or simply Notch) on a neighboring cell. As with most signaling pathways, the effects of Notch signaling are exquisitely context and cell type-dependent, and can either promote or suppress cell proliferation, cell death, the acquisition of speciﬁc cell fates, or the activation of differentiation programs. Because Notch plays a critical role in many fundamental processes and in a wide range of tissues, an aberrant gain or loss of Notch signaling components has been directly linked to multiple human disorders, including many types of cancer. In this context, and depending on the cell, Notch can act as an oncogene or function as a tumor suppressor.

& Notch receptors: — Involved in cell–cell signaling — Activated by intramembrane proteolysis — Activate transcription of target genes.

14.2.1.1 Regulated Intramembrane Proteolysis A key event in Notch activation is its regulated intramembrane proteolysis (RIP), which is triggered by ligand binding. RIP is a an essential step in the maturation and processing of many transmembrane proteins serving to control the communication between cells and the extracellular environment, especially in the nervous system [7]. In the RIP process, a membrane protein typically undergoes two 644 14 Other Transmembrane Receptor Classes

consecutive cleavages. The ﬁrst cleavage results in the shedding of its ectodomain, while the second cleavage occurs within its transmembrane domain and results in the secretion of a small peptide and the release of the intracellular domain into the cytosol. Often, the released fragments function as transcriptional regulators, and speciﬁc changes in gene activity are observed as a consequence of activation of the RIP pathway. Examples of proteins processed by RIP include the Notch protein, the sterol regulatory element-binding protein (SREBP), the Alzheimer precursor protein (APP), the receptor tyrosine kinase ErbB4 (Section 10.1.3), and the cell adhesion molecule E-cadherin.

14.2.1.2 Domain Organization of Notch Notch receptors are large, single-pass transmembrane proteins with an extracellular N terminus and an intracellular C terminus. Mammals have four Notch paralogs (Notch 1–4) that display both redundant and unique functions. The domain organization of Notch 1 is shown in Figure 14.5a. The extracellular domain of all Notch proteins contains 29–36 tandem EGF-like repeats, of which repeats 11–12 are required for productive trans-interactions with ligands presented by neighboring cells. The EGF repeats are followed by a unique negative regulatory region (NRR), which is composed of three cysteine-rich Lin12-Notch repeats (LNR) and a heterodimerization domain. The NRR plays a critical role in Notch signaling by preventing receptor activation in the absence of ligands. The single-pass transmembrane part is followed by domains that mediate interaction with transcription factors and the transcription apparatus. Furthermore, nuclear localization signals and signals that regulate Notch internalization and degradation are contained within the intracellular region. Notch receptors are large glycoproteins: many of their EGF repeats can be modiﬁed by two forms of O-glycosylation, namely O-fucose, and O-glucose. This modiﬁcation in the Notch ligand-binding domain can determine which ligands can bind to and activate the receptor.

14.2.1.3 Notch Ligands Most Notch ligands are transmembrane proteins (Figure 14.5c) containing a small intracellular portion and a large extracellular portion composed of distinct domains that are used for the classiﬁcation of Notch ligands (for details, see Ref. [8]). The largest class of Notch ligands comprises the Delta, Jagged, and Serrate proteins. A critical role in the regulation of Notch ligand function has been ascribed to clustering of the ligand, to PTMs of the ligand, and to recycling of the ligand into speciﬁc membrane microdomains.

14.2.2 Processing and Activation of Notch

Notch activation is mediated by a sequence of proteolytic events (Figure 14.6). A functional Notch receptor is a heterodimer that is generated by cleavage at site S1 14.2 Receptor Regulation by Intramembrane Proteolysis: The Notch Receptor 645

Figure 14.5 Domain organization of Notch intracellular domain) heterodimer that is held and Notch ligands. (a) The mammalian Notch together by noncovalent interactions between paralog mNotch1. EGF repeats 11---12 (red) the N- and C-terminal halves of the and 24---29 (green) mediate ligand interactions. heterodimerization domain; (b) Details of the EGF repeats are followed by the negative mouse Notch1 transmembrane domain regulatory region (NRR), which is composed of (yellow box) and flanking residues showing the three cysteine-rich Lin12-Notch repeats (LNRs, Notch cleavage sites and corresponding green) and a heterodimerization domain cleavage products. After ligand binding, Notch (HD). Notch also contains a transmembrane is cleaved at site 2 by ADAM metalloproteases domain (TMD), nuclear localization sequences such as the Tace protease. c-Secretase can (NLSs), a seven-ankyrin repeats (Ank) domain, cleave multiple scissile bonds at site 3 and a transactivation domain (TAD) that (arrows). Cleavage then proceeds toward site 4 harbors conserved proline/glutamic acid/ until the short Nb peptides (most are 21 serine/threonine-rich motifs (PEST). amino acids) can escape the membrane lipid Mammalian Notch proteins are cleaved by bilayer; (c) The mammalian Jagged ligand. furin-like convertases at site 1 (S1), which Classical DSL ligands (DSL/DOS/EGF ligands) converts the Notch polypeptide into an contain the DSL (Delta/Serrate/LAG-2), DOS NECD---NTMIC (Notch extracellular (Delta and OSM-11-like proteins), and EGF domain---Notch transmembrane and (epidermal growth factor) motifs. 646 14 Other Transmembrane Receptor Classes

Figure 14.6 Overview of Notch activation and a neighboring cell. Endocytosis and membrane downstream signaling. The newly translated trafficking regulate ligand and receptor Notch receptor protein becomes glycosylated, availability at the cell surface. Ligand which is an essential step for the production of endocytosis is also thought to generate a fully functional receptor. The mature receptor mechanical force to promote a conformational is produced after proteolytic cleavage by Furin change in the bound Notch receptor. This protease at site 1 (S1). It is then targeted to the conformational change exposes site 2 (S2) in cell surface as a heterodimer that is held Notch for cleavage by ADAM together by noncovalent interactions. In cells metalloproteases. Juxtamembrane Notch expressing the glycosyltransferase Fringe, the cleavage at site 2 (see Figure 14.5b) generates O-fucose is extended by Fringe enzymatic the membrane-anchored Notch extracellular activity, thereby altering the ability of specific truncation (NEXT) fragment, a substrate for ligands to activate Notch. The Notch receptor the c-secretase complex. c-Secretase then is activated by binding to a ligand presented by cleaves the Notch transmembrane domain in 14.2 Receptor Regulation by Intramembrane Proteolysis: The Notch Receptor 647

from a large precursor protein. The cleavage occurs within the secretory pathway by the action of a protease named furin,andthecleavageproductis the heterodimeric Notch receptor that consists of a large ectodomain, noncovalently associated with the Notch transmembrane, and an intracellular domain. Ligand binding leads to the cleavage of Notch by Zn-metalloproteases of the ADAM family, such as the TACE protease at site 2 (S2), located 12 amino acids before the transmembrane domain and deeply buried within the negative regulatory region (Figure 14.5b). Shedding of the Notch ectodomain creates a membrane-tethered intermediate called Notch extracellular truncation (NEXT) that is a substrate for c-secretase, a multicomponent member of a growing family of intramembrane-cleaving proteases. c-Secretase cleaves NEXT progressively within the transmembrane domain, starting near the inner plasma membrane leaﬂet at site 3 (S3) and ending near the middle of the transmembrane domain at site 4 (S4) (Figure 14.5b). The product of c-secretase cleavage at site 3 is the Notch intracellular domain (NICD) that is now available for translocation to the nucleus. The protease responsible for the intramembrane proteolytic step is contained in the large c-secretase complex [10] that is composed of four polypeptides, namely presenilin, nicastrin, Aph-1, and Pen-2. The catalytic activity is located on presenilin, an aspartyl protease with nine transmembrane elements that undergoes endoproteolytic cleavage into two subunits upon formation of the active c-secretase complex. A key regulatory step in Notch activation is the ligand-induced cleavage at site 2 and a subsequent shedding of the ectodomain. The cleavage site S2 for ADAM metalloproteases resides within the negative regulatory region of Notch, which encompasses the LNR modules and the heterodimerization domain (see Figure 14.5a and b). In the absence of ligand, the negative regulatory region appears to exist in a closed state, thus preventing Notch proteolysis. Ligand-binding is thought to abrogate this control by inducing a transition into an open state in which proteolysis is possible. Activating or cancer-causing mutations in the negative regulatory region have been identiﬁed, and these mutations appear to shift the equilibrium from the “closed” to the “open” state. How ligand binding promotes the accessibility of site 2 for cleavage is not well understood. However, allosteric and mechano-transduction models have been proposed to explain the coupling between ligand binding and site 2 cleavage.

J NEXT progressively from site 3 (S3) to site 4 NICD binding, allosteric changes may occur in (S4) to release the Notch intracellular domain CSL that facilitate displacement of (NICD) and Nb peptide (see Figure 14.5b). transcriptional repressors. The transcriptional NICD then enters the nucleus where it coactivator Mastermind (MAM) then associates with the DNA-binding protein CSL. recognizes the NICD/CSL interface, and this In the absence of NICD, CSL may associate triprotein complex recruits additional with ubiquitous corepressor (Co-R) proteins coactivators (Co-A) to activate transcription. and histone deacetylases (HDACs) to repress From Ref. [9]. transcription of some target genes. Upon 648 14 Other Transmembrane Receptor Classes

& Ligand binding triggers Notch processing by the ADAM and c-secretase proteases: — Processing of Notch yields intracellular NICD fragment — NICD functions as a transcriptional coregulator.

14.2.2.1 Transcription Activation Once NICD is released by c-secretase, it translocates into the nucleus, although the processes and proteins that regulate such nuclear translocation remain unclear. In the nucleus, NICD is unable to bind DNA on its own, but rather acts to affect transcription with the help of its partner, a DNA-binding protein named CSL. In the absence of NICD, CSL represses transcription through interactions with a corepressor complex containing a histone deacetylase. On entering the nucleus, NICD displaces the corepressor complex from CSL and interacts with coactivators, the components of the mediator complex and the transcription apparatus. By employing this mechanism, NICD is able to provide a “transcriptional switch” to activate gene expression from the target promoter. The best-characterized Notch target genes include members of the Hes and Hey families of transcriptional repressors; other target genes include proteins involved in cell-cycle regulation such as p21Cip1, cyclin D1 (see Chapter 14), the transcription factors NFkB and Myc, and the inhibitor of apoptosis, survivin. Overall, a large number of Notch target genes have been identiﬁed, comprising both antiproliferative and proliferation-promoting members.

14.3 Tumor Necrosis Factor Receptor (TNFR) Superfamily

Summary Members of the tumor necrosis factor receptor (TNFR) superfamily play pivotal roles in many physiological processes including cell death, proliferation, differentiation, and the regulation of immunity. The ligands of TNFRs are the TNF cytokines that signal to their receptors as soluble, extracellular ligands or in membrane-bound form. In the latter case, the TNF ligands are single-pass transmembrane proteins presented on the surface of a partner cell during the course of cell–cell interactions, and the ligand–receptor assembly spans two interacting cells. TNFRs are single-pass transmembrane proteins that exist as preassembled oligomers composed of three receptor molecules. The TNF cytokines interact with the corresponding receptor in trimeric form via characteristic TNF homology domains to form the active, hexameric receptor complex. For downstream signaling, TNFRs engage two distinct classes of adapter protein, depending on the receptor type. TRAF signaling receptors engage TRAF proteins and trigger activation of the NFkB and MAPK pathways. Another type of TNFR uses distinct interaction domains, the Death domains 14.3 Tumor Necrosis Factor Receptor (TNFR) Superfamily 649

(DDs) for the recruitment of adaptors, and other signaling components. These receptors form part of the extrinsic apoptotic pathways (see Chapter 17).

The TNFR superfamily comprises 30 receptors (also classiﬁed as type III cytokine receptors; see Section 12.1) and 19 TNF cytokines [11]. The domain structure of selected TNFRs and their ligands are shown in Figure 14.7. In many cases, one

Figure 14.7 Ligands and receptors of the appear to lack function. By the intracellular TNFR superfamily. A schematic highlighting domain, TNFRs can be divided into two homology in the extracellular domains and the subsets: the apoptotic subset due to the extent of ligand overlap in the TNF/TNFR presence of a death domain (DD) and the superfamilies. The TNFR superfamily is TNFR-associated factor (TRAF) binding characterized by the presence of one or more receptors. Although most of the receptors are extracellular cysteine-rich domains (CRDs), membrane-bound, cleavage by which form the main structural framework of metalloproteases or alternative splicing can the extracellular domain. Most TNFRs are generate soluble forms, which play an transmembrane proteins with high homology important role in buffering and removing in the ECD, possibly as a consequence of gene excess ligand. duplication during evolution; some CRDs 650 14 Other Transmembrane Receptor Classes

ligand can bind to several receptors yet, conversely, different ligands often share the same receptor. This promiscuity in receptor–ligand interactions can lead to complicated and variable patterns of signaling responses. Although TNFRs are most often expressed at the cell surface, another layer of signaling modulation can occur through protein cleavage at the membrane by metalloproteases, or by alternative splicing to generate soluble forms of the extracellular domains (ECDs).

14.3.1 Biological Functions of TNFR Signaling

TNFRs and their ligands control a large variety of biological processes, including hematopoiesis, protection from bacterial infection, immune surveillance, and tumor regression. Furthermore, TNFR systems provide crucial signals for the morphogenesis of secondary lymphoid organs, and they also contribute to the function of cytotoxic effector cells in the recognition and destruction of viruses. These effects are primarily mediated by the activation of apoptotic pathways and by the transcriptional activation of a large number of target genes via the NFkB and MAPK pathways. In addition, TNFR members participate in another mode of cell death, necroptosis [12].

& TNFR ligand types: — TNF — Activin — BMP.

14.3.2 TNFR Structure

The extracellular domains of TNFRs are characterized by the presence of cysteine- rich domains (CRDs) that are pseudo-repeats and typically contain six cysteine residues engaged in the formation of three disulﬁde bonds. With few exceptions, the number of CRDs in a given receptor varies from one to four; however, the repeated and regular arrangement of CRDs confers an elongated shape on the receptors, and this is stabilized by a slightly twisted ladder of disulﬁde bridges. All TNFRs are single-pass transmembrane proteins with an extracellular N terminus and an intracellular C terminus [11]. The intracellular region is devoid of enzymatic activity, and a ligand-mediated association or clustering of diverse adapter proteins is used for further conduction of the signal. Two characteristic domains – the TRAF binding domain and the Death Domain (DD) – are used for the recruitment of distinct adaptors, allowing for a subdivision of the TNFR superfamily into the family of TRAF-signaling receptors (TRAF, TNFRa-associated factor) and the Death receptors. Another family of TNFRs, the decoy receptors, is devoid of an intracellular domain (Figures 14.7 and 14.8a). 14.3 Tumor Necrosis Factor Receptor (TNFR) Superfamily 651

Figure 14.8 Oligomer structure and ligand- membrane bound forms. The blue ovals binding of TNFR. (a) Schematic diagram of the represent the cysteine-rich domains (CRDs). structures of the three different subclasses of Receptors with four CRDs are shown for TNFRs. At point ‘a’, the death receptors are illustrative purposes only. TNFRs can have one characterized by the DDs in the signaling to six CRDs; (b) The pre-ligand assembly cytoplasmic tails. At point ‘b’, the majority of model. Upon ligand binding, the pre-ligand TNFRs signal through binding to one of six assembly domain (PLAD) interaction is TRAFs (TNFR associated factor). At point ‘c’, replaced by the more stable ligand---receptor the decoy receptors lack intact cytoplasmic interaction. A preassembled trimer is shown signaling tails and can exist as either soluble or for illustrative purposes.

14.3.3 TNF Cytokines

The TNF cytokines are single-pass transmembrane proteins with an intracellular N terminus and an extracellular C terminus, characterized by a conserved C-terminal domain of about 150 amino acids, the “TNF homology domain” (THD). The THDs, which are responsible for receptor binding, mediate an association of the cytokines to form a trimer, which is the active signaling state of the cytokine. While most TNF family members are synthesized as membrane-bound proteins, soluble forms can also be generated by a limited proteolysis (see Figure 14.7) catalyzed by distinct proteases; in fact, this solubilization is essential for the physiological function of some ligands. By contrast, some ligands function during cell–cell interactions, and proteolytic shedding of the ligand inhibits their function.

& TNFRs: — Signal in trimeric form — Use two types of adaptor for downstream signaling: Death domain-containing adaptors TRAF adaptors — Activate NFkB and MAPK pathways. 652 14 Other Transmembrane Receptor Classes

14.3.4 Receptor Activation and Downstream Signaling

Ligand–receptor assemblies are composed of the trimeric TNF cytokine and three receptor subunits (Figure 14.8b). This mode of interaction results in ligand–receptor assemblies with 3 : 3 stoichiometries that span two interacting cells. Previously, TNFRs were thought to signal through a ligand-induced trimerization of receptor monomers, but much evidence now indicates that many members within the TNFR family exist as preassembled oligomers prior to ligand stimulation [13]. This self- assembly depends on the pre-ligation assembly domain (PLAD), which includes the ﬁrst CRD and is not directly involved in ligand binding. (see Figure 14.8b). On ligand binding, the loose PLAD interactions are thought to be replaced by the more stable ligand–receptor interaction, a process which will change the orientation and spacing of the receptor subunits and bring about a closer spatial relationship of the intracellular parts. As a consequence, the binding and clustering of adapter proteins is triggered on the cytosolic side, allowing further signal transduction.

14.3.4.1 Downstream Signaling Based on the sequence and function of the intracellular region, the TNFR superfamily can be categorized into three overlapping classes: TRAF-signaling receptors; death receptors; and decoy receptors:

TRAF signaling receptors: These receptors (e.g., CD40) signal through the TRAF proteins and mediate activation of the transcription factor NFkB and the MAPK pathways. Death receptors: This receptor type is responsible for triggering apoptotic processes. Death receptors such as TNFR1 and FAS contain an 80-amino acid Death domain (DD) in their cytoplasmic domain. (Details of signaling by these receptors are presented in Chapter 16.) Decoy receptors: The decoy receptors, such as DCR1, lack a transmembrane domain. These are secreted receptors that bind to and neutralize distinct TNF ligands such as FasL. Decoy receptors have been shown to inhibit signaling by these ligands by causing their sequestration.

14.3.4.2 Recruitment of Adapter Proteins and Activation of NFkB Pathways The major targets of activated TNFRs are the MAPK signaling cascade and the transcription factor NFkB. As the steps leading from TNFR to NFkB have been discussed already in Section 2.8.5.3 and in Figure 2.21, only a short summary will be provided at this point. On the binding of TNF to the TNF receptor, the ﬁrst steps in signaling are recruitment of the adapter molecules TRADD (TNF-associated death domain) and RIP1 (receptor-interacting protein 1). The adaptors TRADD and RIP1 then serve as docking sites for the E3 ligases TRAF2/5 and the inhibitor of apoptosis (IAP). The E3 ligases catalyze the K63-linked polyubiquitination of RIP1 and TRFAF2/5, and the poly-Ub chains thus formed then serve as attachment sites for further proteins, 14.4 Toll-Like Receptor Signaling 653 including NEMO and TAK1. NEMO functions as an adapter and activator for IkB kinase 2 (IKK2) that, upon activation, phosphorylates the inhibitor if NFkB(IkB) leading to IkB ubiquitination, IkB degradation, and the release and nuclear translocation of NFkB, composed of p50 and p65 subunits. The protein kinase TAK1 (TNF-activated kinase 1) also becomes activated upon the polyubiquitination of RIP1. TAK1 is classiﬁed as a MAP3K, and engages the p38 and JNK pathways via the phosphorylation of various MAP2Ks (see Chapter 10). As a result, the transcription of many target genes is activated. In another mode of TNFR signaling, the non-canonical NFkB pathway is used (for details, see Ref. [14]). This pathway involves the activation of IKK1 and production of the RelB members of NFkB signaling. The non-canonical NFkB pathway is activated, for example, by ligand binding to the CD40 receptor and the lymphotoxin-beta receptor (LTbR).

14.4 Toll-Like Receptor Signaling

Summary The Toll-like receptors (TLRs) are germline-encoded transmembrane receptors that are essential for innate immune defense against infection by microbial agents. TLRs have been selected on an evolutionary basis to recognize either pathogen-derived compounds that are essential for pathogen survival, or endogenous molecules released by the host in response to infection. The pathogen-specific signature molecules are also known as pathogen-associated molecular patterns (PAMPs), that are sensed by a receptor class known as pattern recognition receptors, of which the TLRs constitute a particularly important group. The PAMPs comprise microbial lipids, carbohydrates, proteins, and nucleic acids. The binding of PAMPs to TLRs leads to TLR activation and the triggering of downstream signaling cascades to defend against microbial attack. The TLRs are single-pass transmembrane receptors composed of an N-terminal ligand-recognition domain, the ectodomain, and a C-terminal cytoplasmic signaling domain. The TLR ectodomains are found either on the extracellular surface or in the endosomes, where they encounter and recognize ligands that have been released by invading pathogens. Ligand-binding induces the dimerization of two receptor molecules to form a horse-shoe-shaped structure. Downstream signaling is mediated by the Toll IL-1 receptor (TIR) domain located on the cytoplasmic side of the receptor. Corresponding TIR domains have been found on a series of adapter proteins, and homotypic TIR–TIR interactions serve to recruit these adaptors into the signaling complex. Many of these adaptors also carry Death domains that mediate interactions with Death domains on signaling complexes further downstream. The final outcome is an enhanced activation of inflammatory pathways, for example, via the NFkB and MAPK pathways. 654 14 Other Transmembrane Receptor Classes

14.4.1 TLR Structure

In humans, 10 TLRs are found that respond to a variety of PAMPs in a receptor- speciﬁc fashion. For example, pathogen-derived lipopolysaccharides are recognized by TLR4, lipopeptides by TLR2 (associated with TLR1 or TLR6), bacterial ﬂagellin by TLR5, viral dsRNA by TLR3, viral or bacterial ssRNA by TLR7 and TLR8, and CpG-rich unmethylated DNA by TLR9 (Figure 14.9). The TLRs are single-span transmembrane receptors with an N-terminal ligand recognition domain, the ectodomain, and a C-terminal cytoplasmic signaling domain [15]. All TLR ectodomains are constructed from tandem copies of a motif known as the leucine-rich repeat (LRR), which is typically 22–29 residues in length and contains hydrophobic residues spaced at distinctive intervals. The TLR ectodomains contain 19–25 LRRs that form horse-shoe-shaped structures (Fig- ure 14.10). In most LRR proteins, ligand binding occurs on the concave surface. Typically, the ectodomains harbor N-linked glycosylations. The signaling domains of TLRs are known as TIR domains because they share homology with the signaling domains of IL-1R family members.

Figure 14.9 Extracellular and intracellular TLRs. TLRs can be subdivided into extracellular and intracellular TLRs. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 recognize their ligands on the cell surface. In contrast, TLR3, TLR7, TLR8, and TLR9 are intracellularly localized. Yamamoto and Takeda 2010 [16], figure 1. 14.4 Toll-Like Receptor Signaling 655

Figure 14.10 Crystal structures of TLR2---TLR1---Pam3CSK4 complex. The TLR2---TLR1/6 heterodimers induced by structures of the TLRs are dissected to reveal binding of triacyl and diacyl lipopeptides. (a) the shape of the lipid pocket; (c) A summary of Structure of the TLR1---TLR2---Pam3CSK4 the lipopeptide patterns recognized by the complex. TLR1, TLR2, and Pam3CSK4 are TLR1---TLR2 heterodimers. Adapted from colored light green, light blue and red, Ref. [15]. respectively; (b) Lipid-binding pocket in the

14.4.1.1 Ligand Binding and Activation Structural studies of ectodomain–ligand complexes have revealed diverse mechanisms of recognition for a wide variety of ligands [15,17]. One common feature of all TLR–ligand complexes resolved is the formation of an m-shaped TLR dimer, in which the ectodomain N termini extend to the opposite ends and the ectodomain C termini interact in the middle. The structure of TLR1/TLR2 in complex with a model lipopeptide is shown in Figure 14.10. It is now generally assumed that binding of the ligand induces dimerization of the ectodomains; this juxtaposes the intracellular TIR domains of the dimer in the correct orientation and/or spacing. Once correctly oriented, the TIR domains are thought to undergo homotypic and/ or heterotypic TIR–TIR interactions. In this way, agonist-binding to the receptor enables the association of TLR–TIR domains with TIR domains of adapter proteins, which in turn leads to the formation of a large cytoplasmic signaling complex. One very interesting mechanistic feature of TLR ligand recognition is the ability of the TLR3, TLR6 and TLR9 proteins to respond to foreign nucleic acids such as ssRNA, dsRNA and unmethylated CpG-rich DNA that encounter their receptors in 656 14 Other Transmembrane Receptor Classes

Figure 14.11 Structural model of the full- structure (2J67). The transmembrane length TLR3/dsRNA signaling complex. The portions have been modeled as a-helices. model is based on the mTLR3/dsRNA Bots 2011 [17], figure 7A. Reproduced with structure (3CIY) and a TLR3 TIR domain permission of Elsevier. homology model based on the TLR10 TIR

the lumen of the endosomes. The nucleic acid ligands contact two TLR ectodomains, as illustrated in Figure 14.11; however, the receptors fail to recognize the nucleic acid ligand in a sequence-specific fashion, and general structural features are rather used to discriminate between different nucleic acid types. For example, TLR3 interacts mainly with the sugar-phosphate backbone of the RNA substrate that must have a minimum length of 40 bp. The binding of DNA would not be possible because the B-helix of DNA would not be compatible with the dimensions of the binding sites of the TLR3 ectodomain dimer. 14.4.1.2 TLR Adaptors and Further Signaling The crucial signaling feature of the intracellular region of TLRs is the TIR domain, which is found not only on the receptors but also on a series of intracellular adaptors that serve to transmit the signal further. The adaptors MyD88, TRIF, TIRAP, TRAM and SARM all contain TIR domains, and these interact in a heterotypic fashion with the TIR domains of the receptor. Individual adaptors are selectively recruited to specific receptors via heterotypic TIR–TIR interactions. Because each adapter recruits further signaling proteins in a specific manner, signaling specificity is generated for each TLR. MyD88 is a master adapter that is used, in addition to all IL-1 receptors, by almost all TLRs, with the exception of TLR3. In addition to an N-terminal TIR domain, MyD88 also harbors a C-terminal DD, the presence of which allows interactions 14.4 Toll-Like Receptor Signaling 657 with other DD-containing proteins, notably the IL-1R-associated kinases (IRAKs). Structural analyses of MyD88–IRAK complexes have suggested the formation of a large signaling complex, named the Myddosome, containing multiple copies of MyD88 and IRAK [18], in a helical arrangement. Myddosome formation on the cytosolic side is proposed to promote the clustering of receptors, leading to the formation of higher-order receptor aggregates and enabling synergistic responses to microbial stimuli.

14.4.1.3 Adapter-Mediated Downstream Signaling The ligand-induced recruitment of cytosolic adaptors often involves TIR–TIR interactions between several adapter molecules, in either in a homotypic or a heterotypic manner. Furthermore, the presence of DDs on adaptors (notably MyD88) allows the recruitment of further DD-containing signaling molecules. Much like the TIR domains, the DDs can undergo homotypic and heterotypic interactions, leading to the formation of large signaling complexes. This function is well established for the receptor-mediated signaling pathways leading to apoptosis. For example, the Death Receptors – which belong to the TNFR superfamily – transmit apoptosis-inducing signals via interactions of their DD with other DD- containing signaling molecules (see Chapter 17). Of all TLRs, the signaling pathways involving the adapter MyDD88 are the best- characterized. An overview of the major TLR signaling pathways is provided in Figure 14.12. The ﬁnal outcome of these pathways is the enhanced expression of proinﬂamma- tory cytokines, as for example, TNF and IL-18 mediated by the NFkB and MAPK pathways. The production of interferon is also stimulated. Many of these downstream steps are also used in TNFR signaling pathways (see Sections 14.3, Section 2.8.5.3, and Figure 2.21), and the two pathways may share the same adaptors and signaling enzymes, leading to similar outcomes. The following steps have been shown to be involved in TLR4-MyD88 signaling:

MyDD88 is recruited to activated TLRs via TIR–TIR interactions, and may assemble further TIR-containing adaptors such as TIRAP. The DD of MyD88 serves to recruit members of the IL-1R-associated kinases (IRAKs) to the activated TLR via a heterotypic DD–DD interaction. IRAKs harbor a DD and are activated upon MyD88 binding. Activated IRAKs phosphorylate and activate the E3-ligase TRAF6. TRAF6 catalyzes the K-63-linked polyubiquitination of the Tak1 protein kinase complex, leading to its activation. The TAK1 complex has multiple regulatory functions, including activation of the p38 and JNK pathways (Chapter 12), and activation of NFkB. The control of transcription factor NFkB is mediated by TAK1 phosphorylation of the IkB kinase (IKK) complex that becomes activated. The IkB kinase complex then phosphorylates nuclear inhibitors of NFkB, the IkB proteins. These inhibitors sequester NFkB proteins in a silent state in the cytosol or in the nucleus. When phosphorylated, the IkB proteins are susceptible 658 14 Other Transmembrane Receptor Classes

Figure 14.12 TLR signaling pathways. TLRs 2.8.5.3 and Figure 2.21. Abbreviations: TIR, activate proinflammatory responses via NFkB Toll IL-1 receptor domain; IRAK, IL-1R- and MAPK pathways. Furthermore, interferon associated kinase; TRAF, Toll receptor- production is stimulated via the activation of associated factor; TAK1, transforming growth interferon regulatory factors (IRFs). MyD88, factor b-activated kinase 1. For simplicity, the TIRAP, TRAM and TRIF are TIR-containing oligomer structure of activated TLRs is not adapter proteins in the TLR signaling shown. pathways. For IKKs and NEMO, see Section

to Ub-dependent degradation and NFkB is released for transcription activation of target genes.

Questions 14.1. Describe the domain organization and functions of TbRs in TGF-b signaling. 14.2. Which types of Smad proteins do you know, and what is their function in TGF-b signaling? 14.3. How is TGF-b/Smad signaling regulated? Name at least three mechanisms. 14.4. Explain the reactions involved in intramembrane proteolysis. Give at least three examples of this type of protein processing. References 659

14.5. Describe the events that follow ligand-binding to Notch and lead to transcription activation. 14.6. Describe the domain structure of TNF cytokines and TNFRs. 14.7. Which types of TNFR do you know, and how do these receptors signal to downstream effectors? 14.8. Describe the events that are triggered by TNFR activation and lead to the activation of NFkB. 14.9. Name the classes of ligands that bind to and activate TLRs. Describe the domain organization of TLRs and the overall activation mechanism for these receptors. 14.10. How do TLRs signal to downstream effectors, and which pathways become activated upon TLR activation?

References

1 Ypsilanti, A.R., Zagar, Y., and Chedotal, A. 7 Lichtenthaler, S.F., Haass, C., and Steiner, (2010) Moving away from the midline: new H. (2011) Regulated intramembrane developments for Slit and Robo. proteolysis – lessons from amyloid Development, 137 (12), 1939–1952. PubMed precursor protein processing. J. Neurochem., PMID: 20501589. 117 (5), 779–796. PubMed PMID: 2 McDonald, C.L., Steinbach, K., Kern, F., 21413990. Epub 2011/03/19. eng. Schweigreiter, R., Martin, R., Bandtlow, C.E. 8 D’Souza, B., Meloty-Kapella, L., and et al. (2011) Nogo receptor is involved in the Weinmaster, G. (2010) Canonical and non- adhesion of dendritic cells to myelin. canonical Notch ligands. Curr. Top. Dev. J. Neuroinﬂamm., 8, 113. PubMed PMID: Biol., 92,73–129. PubMed PMID: 21906273. Pubmed Central PMCID: 20816393. Epub 2010/09/08. eng. PMC3177896. Epub 2011/09/13. eng. 9 Kopan, R. and Ilagan, M.X. (2009) The 3 Santibanez, J.F., Quintanilla, M., and canonical Notch signaling pathway: Bernabeu, C. (2011) TGF-beta/TGF-beta unfolding the activation mechanism. Cell, receptor system and its role in physiological 137 (2), 216–233. and pathological conditions. Clin. Sci. 10 Jorissen, E. and DeStrooper, B. (2010) (Lond.), 121 (6), 233–251. PubMed PMID: Gamma-secretase and the intramembrane 21615335. proteolysis of Notch. Curr. Top. Dev. Biol., 4 Meulmeester, E. and TenDijke, P. (2011) 92, 201–230. PubMed PMID: 20816396. The dynamic roles of TGF-beta in cancer. Epub 2010/09/08. eng. J. Pathol., 223 (2), 205–218. PubMed PMID: 11 Bodmer, J.L., Schneider, P., and Tschopp, J. 20957627. (2002) The molecular architecture of the 5 Moustakas, A. and Heldin, C.H. (2009) The TNF superfamily. Trends Biochem. Sci., regulation of TGFbeta signal transduction. 27 (1), 19–26. PubMed PMID: 11796220. Development, 136 (22), 3699–3714. PubMed Epub 2002/02/14. eng. PMID: 19855013. 12 Vandenabeele, P., Galluzzi, L., Van 6 Matsuzaki, K. (2011) Smad phosphoisoform denBerghe, T., and Kroemer, G. (2010) signaling speciﬁcity: the right place at the Molecular mechanisms of necroptosis: an right time. Carcinogenesis, 32 (11), 1578– ordered cellular explosion. Nat. Rev. Mol. 1588. PubMed PMID: 21798854. Pubmed Cell. Biol., 11 (10), 700–714. PubMed PMID: Central PMCID: 3204345. 20823910. 660 14 Other Transmembrane Receptor Classes

13 Chan, F.K. (2007) Three is better than one: 16 Yamamoto, M. and Takeda, K. (2010) pre-ligand receptor assembly in the Current views of Toll-like receptor signaling regulation of TNF receptor signaling. pathways. Gastroenterol. Res. Pract., 2010, Cytokine, 37 (2), 101–107. PubMed PMID: 240365. PubMed PMID: 21197425. PubMed 17449269. Pubmed Central PMCID: Central PMCID: 3010626. 1965282. 17 Botos, I., Segal, D.M., and Davies, D.R. 14 Schrofelbauer,€ B. and Hoffmann, A. (2011) (2011) The structural biology of Toll-like How do pleiotropic kinase hubs mediate receptors. Structure, 19 (4), 447–459. speciﬁc signaling by TNFR superfamily PubMed PMID: 21481769. Pubmed Central members? Immunol. Rev., 244 (1), 29–43. PMCID: 3075535. 15 Kang, J.Y. and Lee, J.O. (2011) Structural 18 Lin, S.C., Lo, Y.C., and Wu, H. (2010) biology of the Toll-like receptor family. Helical assembly in the MyD88-IRAK4- Annu. Rev. Biochem., 80, 917–941. PubMed IRAK2 complex in TLR/IL-1R signalling. PMID: 21548780. Nature, 465 (7300), 885–890. 661

15 Cell-Cycle Control by External Signaling Pathways

Summary Eukaryotic cells execute their reproduction in a cyclic process, in which at least two phases, S phase and M phase, can be differentiated on the basis of biochemical and morphological features. The biochemical characteristic of S (synthesis) phase is the replication of nuclear DNA and thus doubling of the genetic information. In M (mitosis) phase, division of the chromosomes between the daughter cells is prepared and carried out. In most cell types, two further phases can be distinguished, G1 and G2 phase.

G1 phase covers the period between M phase and S phase, while G2 phase covers the period between S phase and M phase (Figure 15.1). From G1 phase, G the cell may transfer into a quiescent state known as 0 phase. Appropriate signals (e.g., the addition of growth factors) can induce the cell to return from G0 into G1 phase and to proceed with the cell cycle.

The cyclical sequence of G1,S,G2 and M phases describes a standard cell cycle. Rapidly dividing cells in mammals require between 12 and 24 hours to complete a cell cycle. In some cell types, such as early embryonic cells, the period between the

S and the M phases is reduced to the extent that discrete G1 and G2 phases cannot be identiﬁed; the duration of the cell cycle is then only between 8 and 60 minutes.

15.1 Principles of Cell-Cycle Control

The different phases of the cell cycle include a number of highly ordered processes that ultimately lead to duplication of the cell. The various cell-cycle events are highly coordinated to occur in a deﬁned order and with an exact timing, requiring precise control mechanisms.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 662 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.1 The four phases of a typical cell cycle of a eukaryotic cell.

A biochemical system is at the center of the cell cycle, of which the most important players are Ser/Thr-specific protein kinases and regulatory proteins associated with these. The activity of this central cell-cycle “engine” regulates processes downstream that help to carry out the many phase-specific biochemical reactions of the cell cycle in a defined order. Furthermore, the system has built-in feedback mechanisms and allows for linkages between events that are not immediately consecutive in the cycle. In this way, it is ensured that the phases of the cell cycle are executed completely and in the correct sequence.

& Intrinsic controls of the cell cycle: — Completion of phases — Correct order of phases — Adequate cell size

Three aspects of the cell cycle are central to its function and control:

Cell growth: Cells must accumulate enough cell mass and organelles to establish two daughter cells. Survival: Cells must receive or produce survival signals that help to prevent programmed cell death (apoptosis). As an example, a balance of cell death and compensatory proliferation maintains the epithelia in a state of constant renewal. Proliferation: Cells must be instructed by the environment to divide at a given place and a given time. Proliferation-promoting signals are received by cells

almost exclusively during G1 phase. 15.1 Principles of Cell-Cycle Control 663

& External control of the cell cycle uses: — Anti-apoptotic signals — Proliferation-promoting signals

Progression of the cell cycle is subject to both internal and external controls.

15.1.1 Intrinsic Control Mechanisms

Intrinsic control mechanisms ensure that the cycle is executed completely so that, following cell division, both daughter cells are equipped with the same genetic information as far as possible. The following intrinsic control mechanisms are essential to cell-cycle progression: (Figure 15.2):

Coupling of mitosis to a completed S phase: Mitosis is only initiated when the DNA has been completely replicated during S phase. Coupling of S phase and mitosis: Entry into S phase is only possible if preceded by mitosis. Coupling of cell size and progress in G phase: 1 The daughter cells produced by cell division must reach a critical size during the course of G1 phase before S phase can commence.

Figure 15.2 Control points of the cell cycle: mitosis (metaphase/anaphase transition), and external and internal control mechanisms. in G1 phase. Internal controls are shown as Important controls of the cell cycle operate at broken arrows, and external controls as solid the end of G2 phase (G2/M transition), in arrows. 664 15 Cell-Cycle Control by External Signaling Pathways

15.1.2 External Cell Cycle Control

In addition to the internal control mechanisms, the cell is also subject to a number of external controls, which ensure that cell division occurs in balance with the overall development of the organism and with external growth conditions. This is a form of social control of cell division that regulates the progress of the cell cycle, with the help of circulating signal molecules or via cell–cell interactions. A set of mitogenic (i.e., proliferation-promoting) and antimitogenic (i.e., proliferation- inhibiting) signals serves to adjust cell division activity to the demands the development and function of the organism.

& External factors controlling cell-cycle progression: — Mitogenic signals Growth factors Cytokines Cell–cell interaction — Nutrient supply — Absence of proapoptotic signals.

15.1.2.1 Mitogenic Signals

A key event in cell cycle progression is the transition from G1 phase into S phase. G1 phase has a special regulatory function in the cell cycle; here, the decision is made to enter S phase and thus a new round of cell division, or to enter a resting,

quiescent state, G0. ﬁ ! The following requirements must be ful lled for G1 S transition in normal noncancerous cells:

Cells must receive mitogenic signals that allow cell division in the frame of the development of the organism. Cells must be large enough for division, and growth-promoting signals are required to this end. Cell death signals must be allowed to become active under conditions where the cell number is to be controlled and where accidents such as DNA damage call for cell death.

Entry into S phase is an important commitment for mammalian cells, with normal cells requiring external mitogenic signals to enter S phase and allow cell division. Once DNA replication has begun, however, the cell cycle must progress until cell division is complete, and this progression is no longer dependent on external signals. The “point of no return” for cell-cycle entry has often been termed the “restriction point”. Within the bounds of intercellular communication, the mitogenic signals are produced in the form of growth factors or cytokines. These proteins bind to speciﬁc receptors on the target cell and initiate signal chains that promote progress of the 15.1 Principles of Cell-Cycle Control 665

Figure 15.3 Mitogenic and antimitogenic extracellular signal is registered by signals in control of the cell cycle. The cellular transmembrane receptors and passed on to environment may emit either mitogenic or the cell cycle apparatus via an intracellular antimitogenic signals. Mitogenic signals (e.g., signal chain. TGFb, transforming growth factor growth factors) promote passage through the b; CDK, cyclin-dependent protein kinase; CKI, cell cycle; antimitogenic signals (e.g., TGFb) inhibitor of CDK. lead to a halt in the cell cycle. In both cases, the cell cycle (Figure 15.3) by affecting the expression, degradation or activity of central components of the cell-cycle apparatus. Mitogenic signals can be also created during the course of cell–cell interactions, as exempliﬁed by integrin signaling (Section 13.4). One of the hallmarks of cancer cells is the escape from the social control of mitogenic signaling. Often, cancer cells become independent of external mitogenic signals by producing their own mitogenic signals (see Chapter 16).

15.1.2.2 Antimitogenic Signals during Cell---Cell Communication In addition to growth-promoting signals, growth-inhibiting antimitogenic signals may also take effect in the organism. These lead to a halt in the cell cycle and may lead to transition of the cell into G0 phase. A lack of mitogenic signals can have the same effect on the progress of the cell cycle.

15.1.3 Cell-Cycle Checkpoints

Control mechanisms exist that are not active in every cell cycle; these are only induced when defects are detected in central cell cycle events. Such control mechanisms are referred to as checkpoints. 666 15 Cell-Cycle Control by External Signaling Pathways

The term “cell-cycle checkpoint” refers to mechanisms by which the cell actively halts progression through the cell cycle until it can ensure that an earlier process, such as DNA replication or mitosis, is complete. One checkpoint of central importance is that involving DNA damage and DNA replication; this biochemical pathway responds to damaged DNA or stalled DNA replication and creates a signal

that slows cell cycle progression or arrests cells in the G1,S,orG2 phase (Section 15.6). Another major checkpoint is the spindle or metaphase checkpoint of M phase, which assures the correct segregation of chromosomes during mitosis.

15.2 Key Elements of the Cell-Cycle Apparatus

Summary The cell cycle is driven by the activity of its core engine that has the following components as main tools: (i) cyclin-dependent protein kinases (CDKs); (ii) cyclins, the activating subunits of CDKs; and (iii) inhibitors of cyclin-dependent protein kinases (CKIs). CDKs associate with cyclins and/or CKIs to form an oscillating system that initiates downstream biochemical processes and thus determines the individual phases of the cycle. CDK protein levels do not change significantly during cell- cycle progression; rather, the kinase activity of CDKs is regulated in a dynamic manner through the association with cyclins and/or CKIs, both temporally and spatially. Furthermore, phosphorylation/dephosphorylation is a major tool for the control of CDK kinase activity. CDKs, cyclins and CKIs represent the main attack points for intrinsic and external control mechanisms. External controls are mediated by signal transduction pathways that regulate the activity of CDKs, cyclins and CKIs at the following levels: (i) signal-directed changes of cyclin and CKI expression serve to stimulate (resp. inhibit) the activity of the CDKs at distinct stages of the cycle; (ii) PTMs, mainly phosphorylation, are used to activate or inhibit the activity of CDK/cyclin complexes; the phosphorylation of cyclins and CKIs also regulates their stability and subcellular distribution; and (iii) the Ub-mediated proteolysis of cyclins and CKIs serves to reset the cell-cycle clock and ensures unidirectionality of the cycle.

15.2.1 Cyclin-Dependent Protein Kinases (CDKs)

The CDKs are proteins of 34–40 kDa with Ser/Thr-specific protein kinase activity.An oscillating change in CDK activity is observed during the cell cycle, and these changes are due to the phase-specific association of CDKs with their activating subunit (the cyclins), or with their inhibitory partners (the CKIs). In the absence of the cyclin, the CDKs show only very low basal activity, and the CDKs must associate with the corresponding cyclin (or cyclin-like proteins) to be active. Active CDKs are 15.2 Key Elements of the Cell-Cycle Apparatus 667 thus heterodimers in which the CDK subunit carries the catalytic activity and the other subunit, the cyclin, performs an activating and specificity-determining function. In addition to association of the cyclin, most CDKs require phosphorylation in the activation segment (T loop; Section 9.2.1) for full activation.

& CDKs: — Ser/Thr-specific protein kinases — Cyclins as activating subunit — Regulated by phosphorylation.

In mammals, 11 cyclin-dependent kinases (CDK1–CDK11; Table 15.1) are currently known, of which only CDKs1, 2, 3, 4, and 6 intervene directly in the cell cycle, while CDK7 plays only an indirect role as an activator of these CDKs. Furthermore, CDKs7, 8, 9, 10, and 11 perform regulatory functions in transcription.

& Mammalian CDKs involved in cell-cycle regulation: — CDK1 (CDC2) — CDK2, CDK3 — CDK4 — CDK6.

15.2.1.1 CDKs Involved in Cell-Cycle Regulation The CDKs of the cell cycle associate with distinct cyclins to yield preferred CDK–cyclin combinations. The canonical view of the mammalian cell cycle depicts the many CDK– cyclin complexes as fulﬁlling unique and essential steps that dictate the sequential order of cell-cycle events. CDK1 controls G2/M transition, while CDK2 (also known as CDC2), CDK3, CDK4 and CDK6 are implicated at G1/S transition. Nevertheless, the genetic ablation in mice of different combinations of cell-cycle cyclins and CDKs has shown that most of them are dispensable for mouse development, unveiling partly overlapping functions and unexpected compensatory mechanisms. Surprisingly, the activity of CDK1 alone sufﬁces to drive the mammalian cell cycles associated with early embryonic development in all cell lineages.

15.2.1.2 Other CDKs The CDKs 5 and 7–11, together with the corresponding cyclins, are not directly involved in cell-cycle control and perform other speciﬁc tasks, for example, in transcription regulation (see Table 15.1).

15.2.2 Cyclins

Cyclins function as allosteric regulatory subunits for the CDKs. There is speciﬁcity in the interaction of cyclins with CDKs, and distinct cyclin–CDK complexes become 668 15 Cell-Cycle Control by External Signaling Pathways

Table 15.1 Mammalian CDKs, cyclins and CKIs.

Cyclin CDK CKI Phase/function

Mammals A1 CDK1, CDK2 p21, p27, p57 Meiosis

A2 CDK1, CDK2 p21, p27, p57 S, G2,M B1, B2, B3 CDK1 ? M

C CDK3 G0/G1 transition C CDK8 ? Mediator, Transcription repression

D1, D2, D3 CDK2, 4, 5, 6 p15, p16, p18, p19, p21, p27 G1, Restriction point

E CDK2 p21, p27, p57 G1/S

F? ? G2, F-box protein H CDK7 _ CDC2 phosphorylation CTD phosphorylation T CDK9 ? CTD phosphorylation, HIV-TAT target p35, p39 CDK5 Phosphorylation of structural proteins CDK10 Phosphorylation of transcription factors L CDK11 Transcription, RNA splicing,

active during the cell cycle (Table 15.1). The cyclins were originally deﬁned as proteins that show cyclic concentration variations during the cell cycle (Figure 15.4) and thereby activate CDKs differentially during the cell cycle. A classifying feature of the cyclins today is the cyclin box, a conserved domain of about 100 amino acids that interacts with the corresponding CDK.

& Cyclins: — Activating subunits of CDKs — Bind CDK via cyclin box.

Figure 15.4 Concentration changes in cyclins figure only depicts the course of concentration during the cell cycle. Cyclins of types D, A, E changes in the cell cycle, and does not indicate and B show characteristic concentration the relative cyclin concentrations. changes during the course of the cell cycle. The 15.2 Key Elements of the Cell-Cycle Apparatus 669

The cyclins of the mammalian cell cycle can be roughly divided according to their activity in the different phases of the cycle:

Cyclins of type D are synthesized during early G1 and are present at a fairly constant level during S and G2 phases until their level falls during M phase. The level of E-type cyclins increases at late G1 and decreases sharply around the G1/S transition. Cyclins of type A accumulate from late G1 phase and are destroyed before the metaphase of M. Cyclins of type B accumulate during S phase and G2 phase, and their destruction starts at the anaphase of M.

Cyclins D and E are regarded as the G1/S cyclins; cyclins B and A are also termed mitotic cyclins.

& G1/S cyclins: — Cyclins D1, D2, and D3 — Cyclin E Mitotic cyclins: — Cyclin A — Cyclin B

The main functions of cyclins in cell-cycle regulation are:

Activation of CDK: The role of the cyclins is to convert CDKs into an active state. This process confers specificity to CDK activation, because a specific cyclin preferentially binds and activates only a certain CDK (see Table 15.1). The concentration of the cyclins is an important factor in the control of CDK activity. Various mechanisms exist to control the level of cyclins available for CDK binding (see below). Contribution to substrate specificity of CDKs: The binding of protein substrates to CDK–cyclin complexes is not restricted to the CDK subunit. Rather, cyclins contain structural elements that mediate interactions with CKIs and with CDK substrates. Thus, the role of cyclins has to be extended to selection of binding substrates of CDKs. Cyclin functions independent of CDKs: Some cyclins perform functions independent of CDKs. This has been well established for cyclin D1, which has been shown to form physical associations with more than 30 different transcription factors or transcriptional coactivators. Proteins interacting directly with cyclin D1 include nuclear receptors, basic helix–loop–helix proteins and Smad proteins. Furthermore, cyclin D1 binds to histone acetyl transferases, histone deacetylases and chromatin-remodeling proteins, indicating a general role of cyclin D1 in modulating the activity of transcriptional regulators. 670 15 Cell-Cycle Control by External Signaling Pathways

& Functions of cyclins: — Activation of CDKs — Substrate binding and selection — Modulation of transcriptional regulators.

15.2.2.1 Regulation of Cyclin Concentration In the cell cycle, the different cyclins show characteristic concentration changes in which temporally deﬁned maxima in cyclin concentration are observed (Fig- ure 15.4). The amount of a distinct cyclin available for CDK activation in the nucleus is strictly controlled by the following mechanisms (Figure 15.5):

Figure 15.5 Processes influencing cyclin SCF complex and destruction by the ubiquitin- concentration. The upregulation of cyclin proteasome pathway. This phosphorylation concentration occurs mainly at the level of can be induced by activated cyclin---CDK gene expression. Growth factors transduce complexes. Cyclin destruction (e.g., cyclin B) signals via the MAPK pathway or other can be also mediated by the anaphase- pathways to transcription factors (c-Jun, c- promoting complex (APC). Another Myc) that activate for example, cyclin D mechanism for control of cyclin concentration transcription. Phosphorylation of cyclins (e.g., uses changes in the subcellular distribution. cyclin E) can provide a signal for binding of the 15.2 Key Elements of the Cell-Cycle Apparatus 671

Changes in cyclin expression (Section 15.4) in response to external signals. Targeted degradation in the ubiquitin pathway (Section 15.3.1): Most cyclins are the target of Ub-mediated proteolysis, and this degradation is a major mechanism for reducing cyclin concentrations at distinct cell cycle stages. Phosphorylation: Cyclins harbor multiple phosphorylation sites, each with distinct functions. Importantly, signal-directed phosphorylation of cyclins is a tool for targeting cyclins for proteolysis. Furthermore, cyclin phosphorylation inﬂuences the subcellular distribution of cyclins. Subcellular distribution: Cyclins show a complex pattern of subcellular distribu-

tion. Cyclin D1 for instance is localized to the nucleus during G1 phase and is distributed to the cytoplasm upon the onset of S phase.

& Cyclins are regulated via: — Gene expression — Phosphorylation — Ub-mediated proteolysis — Subcellular distribution.

15.2.3 Inhibitors of CDKs: The CKIs

Negative control of CDK activity in the cell cycle is performed by speciﬁc inhibitor proteins known as cyclin-dependent kinase inhibitors (CKIs). These are a heterogeneous family of proteins that may associate with a CDK or with a cyclin- CDK complex in a reversible manner, inhibiting CDK activity.

& CDK inhibitors (CKIs): — Negative regulators of CDKs — Prefer distinct CDKs — Two families: CIP1, Kip family Ink4 family

The CDKs are divided into two groups based on sequence homology:

CIP/KIP family p21CIP1 (also known as CIP1, Waf1) p27KIP1 (KIP1) p57KIP2

INK4 family p15INK4b p16INK4a p18INK4c p19INK4d 672 15 Cell-Cycle Control by External Signaling Pathways

15.2.3.1 Regulation and Function of CKIs

The CKIs are important control elements that regulate G1/S transition and are also involved in the control of G0/G1 transition when cells move from a quiescent state to a dividing state, and vice versa. The primary targets of CKIs are the CDKs, either free or complexed with cyclins, and the main function of CKIs is that of a negative regulator of CDK activity. As such, the CKIs are important entry points for signals that slow down or stop the cell cycle and induce

transition into the quiescent state, G0. The critical determinant for the regulation of CDK activity is the concentration of the CKI relative to that of the activating CDK subunits, the cyclins. Increasing the CKI concentration above the level of cyclin concentration will lead to a decrease in CDK activity and will slow down or stop progression through the cell cycle. CKI levels available in the nucleus are subject to internal controls and external controls in the form of antimitogenic signals. These controls comprise transcriptional, translational, proteolytic, and localizational mechanisms.

15.2.3.2 The CIP/KIP Family The members of the CIP/KIP family interact with all cell-cycle CDK–cyclin complexes (Figure 15.6). The mechanisms of interference with CDK activity appear to be diverse and to depend on the identity of the CDK–cyclin complexes. Whereas, inhibition by p27KIP1and p21CIP1 has been clearly shown for CDK2–cyclin A complexes in vivo, a more complex picture has emerged for the inﬂuence of both inhibitors on the activity of CDK4–cyclin D complexes. Low concentrations of p27KIP1 and p21CIP1 appear to stabilize CDK4–cyclin D complexes and are required for activation, whereas an inhibitory effect has been shown for higher CIP/KIP concentrations.

p27KIP1 This inhibitor plays a central role in the decision of a cell to either commit KIP1 to the cell cycle or to withdraw into the resting state, G0. Levels of p27 are high in the quiescent state but, upon entry of the cells into G1, these levels decrease sharply; conversely, the levels of p27KIP1 increase when cells leave the cell cycle and enter into a differentiated state. KIP1 Furthermore, the p27 inhibitor plays a central role in G1 progression and G1/ S transition by controlling the activity of CDK4 and CDK2 complexes (Sec- KIP1 tion 15.4.1). In G1 cells, an accumulation of p27 is induced by many antimitogenic signals including cell–cell contacts, TGFb-signaling and cAMP. KIP1 Further progression into S and G2 phase requires p27 downregulation by Ub- mediated proteasomal degradation. Overall, the activity and levels of p27KIP1 are subjected to multiple controls in a highly complicated fashion (for details, see Ref. [1]). The major controls are:

Ubiquitination by SCF (Skp1, cullin, F-box protein)-Skp2 E3 ligases (Section 2.8.3.4) and subsequent proteasomal degradation. Phosphorylation of p27KIP1 at T187 by cyclin E–CDK2 complexes (Section 15.4.2), as a prerequisite for ubiquitination. 15.2 Key Elements of the Cell-Cycle Apparatus 673

Figure 15.6 Regulation and attack points of complexes. Only the major routes of CKI cyclin-dependent protein kinase inhibitors regulation are indicated. p53, tumor (CKIs) in mammals. The Ink inhibitors are suppressor protein p53; TGFb, transforming activated by TGFb, and specifically inhibit the growth factor b; p21, p21CIP1; p27, p27KIP1; cyclinD---CDK4/6 complexes. The inhibitors p16, p16ink4A; CDK, cyclin-dependent protein p21, p27 and p57 are activated by p53 and by kinase; Cyc, cyclin. TGFb, and can inhibit all types of cyclin---CDK

Phosphorylation at Ser10, for example by ERK proteins (Chapter 12), to induce nuclear accumulation. Tyr-phosphorylation by Src family kinases to convert p27KIP1 from an inhibitor to an activator.

& CKIs are regulated via: — Gene expression — Phosphorylation — Ub-mediated proteolysis — Subcellular distribution. p21CIP1 The expression of mammalian p21CIP1 is tightly regulated by signals that control cell division. Furthermore, an outstanding property of p21CIP1 is its 674 15 Cell-Cycle Control by External Signaling Pathways

regulation at the transcriptional level by the tumor suppressor protein p53 (Section 16.7) during the DNA damage response. Increased levels of mRNA for p21CIP1 are observed on the treatment of cells with DNA-damaging agents, and this

transcriptional regulation is part of the DNA damage checkpoint during G1/S – phase (Section 15.6). In addition to its ability to associate with G1 cyclin CDK complexes, p21CIP1 also associates with the replication-accessory protein PCNA (proliferating cell nuclear antigen). PCNA is required for nuclear DNA synthesis, and functions in clamping DNA polymerase d to the DNA, thereby increasing the processivity of DNA synthesis. It is believed that this is another mechanism by which p21CIP1 can inhibit DNA synthesis and S-phase progression.

& p21CIP1: — Participates in DNA damage response — Induced by p53 — Inactivates G1-CDKs.

15.2.3.3 INK4 Proteins The inhibitors of the INK4 family bind to and inhibit CDK4 and CDK6 complexes (Figure 15.6). Generally, the INK 4 proteins compete with D-type cyclins for binding to the CDK. The p16INK4a binds to monomeric CDK6 at a site opposite to the cyclin-binding site and allosterically blocks binding of cyclin D. Furthermore, the ATP-binding site of CDK is deformed by the bound inhibitor. Regulation of INK4 abundance is cell type-speciﬁc and shows complex patterns.

& Ink4 inhibitors target mainly CDK4/6

Notably, the level of p15INK4b is subject to induction at the mRNA level by the antimitogenic cytokine TGF-b (Section 14.1). Properties as a tumor suppressor are attributed to the inhibitor p16INK4a, since the gene for p16INK4a is mutated in many tumor cell lines. Furthermore, silencing of the genes of INK proteins by aberrant CpG methylation is a frequent event in tumor cells.

15.2.4 Structural Basis of CDK Regulation

The CDKs may exist in either inactive and active states, with transition between the two states being controlled by cyclin and/or CKI binding and by phosphorylation/ dephosphorylation events (Figure 15.7).

15.2.4.1 CDK---Cyclin Complexes The structural basis for the activation of CDKs by cyclin binding and phosphorylation will be discussed using the example of the CDK2–cyclin A complex (for a review, see Ref. [2]). 15.2 Key Elements of the Cell-Cycle Apparatus 675

Figure 15.7 Principles of regulation of cyclin- take place by phosphorylation of Thr14 and dependent protein kinases. The figure shows Tyr15 (b) or by binding of a CKI (c). Other the principles of CDK regulation, using the inactive forms of CDKs are the CDK---cyclin CDK1 (here simply referred to as CDK) as an complex, in which Thr160 of the CDK is not example. The active form of CDK (a) is phosphorylated (d). In addition, the cyclin-free associated with the corresponding cyclin; forms of CDK are inactive (e). CDK, cyclin- Thr160 of CDK (or equivalent positions in dependent protein kinase; CKI, inhibitor of other CDKs) is phosphorylated, and Thr14 and CDK; CAK, CDK1-activating kinase. Tyr15 are unphosphorylated. Inactivation may

The catalytic center of CDK2 adopts the typical protein kinase fold (see Section 9.2.1 and Figure 15.8), composed of an N-terminal lobe and a mostly a-helical C-terminal lobe. Furthermore, the kinase domain of CDK2 harbors the critical structural elements required for full activation of most protein kinases, namely the activation loop (or T-loop) and the C-helix (or PSTAIRE helix). & Structures of CDK–cyclin complexes: — Typical kinase fold — Critical elements: Activation segment (T-loop) C-helix (PSTAIRE helix). 676 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.8 Structural basis of CDK regulation the PSTAIRE helix so as to place the glutamate by cyclins. In the absence of cyclin, the aC helix within the active site. The activation loop of CDK (also called the PSTAIRE helix) is adopts a near-active conformation upon cyclin rotated so as to move a crucial catalytic binding, and its subsequent phosphorylation glutamate out of the active site. This is further stabilizes the active form. Huse 2002 correlated with an inhibitory conformation of [2], Figure 2. Reproduced with permission of the activation loop. Cyclin binding reorients Elsevier.

Full activation of CDK2 requires phosphorylation of Thr160 within the activation loop and a correct orientation of the C-helix. In the absence of cyclin, CDK2 exists in an inactive conformation for two reasons: (i) the C-helix is misaligned; and (ii) the substrate binding site is blocked by the activation loop (Figure 15.8). The binding of cyclin A leads to a reorganization of the active site that includes a correct orientation of the critical glutamate, allowing for the productive binding of ATP, and a partial removal of the activation segment from the catalytic cleft. The cyclin–CDK complex possesses a basal protein kinase activity that is increased about 300-fold by phosphorylation at Thr160 (or at an equivalent position).

15.2.4.2 Phosphorylation of CDKs A major control of the CDKs is exerted by phosphorylation and dephosphorylation events. The CDKs of the cell cycle possess several phosphorylation sites, and these may have either an activating or inactivating effect.

& CDK phosphorylation: — Thr160(172)-P: activation — Thr14, Tyr15-P: inhibition

There are two major classes of phosphorylations:

Activation loop phosphorylation: Full activation of CDK2–cyclin A requires phosphorylation at Thr160 in the activation loop (or the equivalent positions Thr161 of CDK1and Thr172 of CDK4/6). The protein kinase responsible for this phosphorylation belongs to the family of CDKs, and is known as CAK (CDC2- 15.2 Key Elements of the Cell-Cycle Apparatus 677

activating kinase) composed of the catalytic subunit CDK7, cyclin H and the MAT subunit (see also Section 4.2.5). The CAK-catalyzed phosphorylation at Thr160 requires binding of the cyclin to CDK and leads to an almost 300-fold increase in protein kinase activity. Inhibitory phosphorylation at Thr14 and Tyr15: Phosphorylation of a tyrosine residue within the ATP-binding domain (e.g., Tyr15 of CDK1 and CDK2 and Tyr17 of CDK4) is inhibitory on the CDK activity. These tyrosine residues, together with adjacent threonine residues (e.g., Thr14 of CDK1 and CDK2), are phosphorylated by the Wee1 family of Ser/Thr-speciﬁc kinases following cyclin assembly, providing a ﬁne-tuning mechanism for the correct timing of CDK activation.

& Activation segment phosphorylation: — Catalyzed by CAK — Increases activity by orders of magnitude — Reorients catalytic center and substrate-binding site — Dephosphorylation by CDC25 phosphatase.

& Phosphorylation at Thr14, Tyr15: — Inhibits CDK — Catalyzed by Wee1 kinase — Removed by CDC25A, CDC25B, CDC25C phosphatases.

Phosphorylation at Thr14 and Tyr15 by Wee1 leads to an inactivation of the CDK, this control being of particular importance during G1/S and G2/M transitions (Section 15.5). The inactivating phosphorylation at Thr14 and Tyr15 can be reversed in a regulated manner by the protein dual-specificity phosphatases CDC25A, CDC25B, and CDC25C. The CDC25 phosphatases are protein phosphatases with twofold specificity that can cleave phosphate residues from phosphoserine and phosphotyrosine residues of CDKs or other proteins (see also Sections 9.7 and 10.4. Control of the CDC25 enzymes is achieved mainly by phosphorylation at specific Ser/Thr residues. Depending on the identity of the Ser/Thr residues, phosphorylation can have either an activating or inactivating effect on phosphatase activity. Some phosphorylation sites mediate an inhibition of the phosphatase by the binding of 14-3-3 proteins (Section 15.6). The phosphorylations are catalyzed by cyclin–CDK complexes or by other protein kinases that form a part of distinct signaling pathways.

15.2.4.3 Structural Basis of Inhibition by p27KIP1 Structural studies of a ternary complex composed of cyclin A, CDK2 and p27KIP1 have shown that the inhibitor interacts with both cyclin A and CDK2. The inhibition of kinase activity is explained by an alignment of the structural elements 678 15 Cell-Cycle Control by External Signaling Pathways

of p27KIP1 in the ATP-binding site of CDK2 that breaks up the glycine-rich phosphate-binding loop. In addition, the ATP-binding site is completely ﬁlled by residues of the inhibitor, so that ATP binding is no longer possible.

& p27Kip1: — Binds to CDK–cyclin complex — Distorts active site of CDK — Prevents substrate binding.

& Ink4 inhibitors: — Bind to CDK — Fix inactive CDK conformation and compete with cyclins

15.2.5 Multiple Regulation of CDKs

The CDKs serve as the central tool for controlling the cell cycle as they receive a multitude of signals and transmit signals to downstream substrates. The latter substrates trigger the large number of different activities that constitute the distinct functional, regulatory and morphological properties of the various phases of the cycle. The activity of the CDKs is regulated by various positively and negatively acting signals that are registered in the cell cycle and can bring about a halt in the cell cycle at various points (see Figure 15.7).

& CDK activity is controlled by: — Cyclin availability — Dephosphorylation by CDC phosphatases — CKIs — CDK expression.

The following have a positive effect on activity of CDKs and promote progression in the cell cycle:

Increase in cyclin concentration: by activation of transcription or inhibition of proteolytic degradation. Phosphorylation of CDKs at Thr160 or equivalent positions by CAKs. Dephosphorylation of CDKs at Thr14/Tyr15 by Cdc25 phosphatases. Redistribution of CKIs between different CDK complexes. Import of cyclins into the nucleus. Decrease in the concentration of CKIs at the transcription level, or by proteolysis. 15.2 Key Elements of the Cell-Cycle Apparatus 679

The following have a negative effect and can lead to a break in the cell cycle:

Decrease in the concentration of cyclins: by reduced transcription or by activation of proteolysis. Export of cyclins from the nucleus to the cytosol. Inhibition of Cdc25 phosphatases: these enzymes are required for removal of the inhibitory Thr14/Tyr15 phosphorylations of the CDK. Increase in the concentration of CKIs.

These regulation mechanisms cannot be considered in isolation; rather, it must be assumed that the individual mechanisms cooperate, that they demonstrate mutual regulation, and that feedback mechanisms are built in. All control elements can be activated – at least in principle – by external signals, the result being a complex network of cell-cycle control with many entry and exit points. The following sections are thus incomplete and describe only those elements that have been well proven experimentally.

15.2.6 Substrates of CDKs

Overall, the substrate selectivity of CDKs is determined in a complicated manner by the CDK itself that prefers a distinct consensus, the nature of the cyclin associated, and the availability of the substrate. As with most protein kinases, it is difficult to formulate a consensus sequence for phosphorylation by CDKs, though the sequence (K/R)-S/T-P-X-K (X: any amino acid) has been identified as such a consensus. In addition to the interactions with the target Ser/Thr of the substrate, both the CDK and the cyclin contact regions outside of the acceptor Ser/Thr residues. The region of the cyclin responsible for these interactions is also involved in the binding of CDK inhibitors and of protein substrates such as the retinoblastoma protein (Rb). An overview of the cell cycle-specific activation of CDKs and some important substrates are shown in Figure 15.9. At present, whereas comparatively few data are available for the G1 and S phase substrates of the CDKs, many fi proteins have been described that undergo speci c phosphorylation in G2/M phase.

15.2.6.1 Substrates in G1/S Phase The most important CDK substrates in G1/S phase are the tumor suppressor protein Rb and the Rb-related proteins p130 and p107, which are phosphorylated by the cyclin D1–CDK4/6 complex and by the cyclin E–CDK2 complex (Section 15.5.3). The protein Rb and its relatives are critical in preparing cells for entry into S phase. Other targets of the cyclin E–CDK2 complex comprise the p27KIP1 inhibitor and MCM proteins, which are involved in the regulation of DNA replication. 680 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.9 Substrates and phase-specific phosphorylation. p107 and p130, Rb-related activation of CDKs in the cell cycle. An proteins; Rb, tumor suppressor Rb; CDC25, overview is shown of the phase-specific CDC25 phosphatase; TFIIIB, transcription activation of the most important CDK---cyclin factor TFIIIB; TFIIH, transcription factor TFIIH; complexes and of selected substrates. The APC, anaphase-promoting complex; p27, arrows indicate activation and p27KIP1.

& Major Substrates of CDK4/6–cyclinD: — Rb — p130 — p107.

15.2.6.2 Substrates in G2/M Phase In M phase, the new phosphorylation of many proteins is observed that starts, in particular, from the CDK1–cyclin B complex. The phosphorylation affects proteins that are critical for entry into and for progression through M phase, such as the Cdc25 phosphatase and Emi1 that is an inhibitor of Cdc20, a subunit of anaphase- promoting complex (APC) (Section 15.4.2) Furthermore, proteins involved in the reorganization of the cytoskeleton, the nuclear membrane, and the formation of 15.3 Regulation of the Cell Cycle by Proteolysis 681 the spindle apparatus become phosphorylated by CDK1–cyclin B. As a consequence of these phosphorylation events vesicular transport is inhibited, accompanied by a general inhibition of transcription.

& Substrates of CDK1–cyclin B: — CDC25C — Inhibitor Emi1 — Many proteins involved in: Transcription Spindle formation Cytoskeleton organization.

15.3 Regulation of the Cell Cycle by Proteolysis

Summary Progression through the cell cycle is governed by the opposing forces of Ub- dependent proteolysis (Section 2.8.3.4) of the major cell cycle regulators and by the oscillating activation of CDKs. The changes in CDK activity are largely determined by the association of cyclins and CKIs, and the levels of these regulators are controlled by the Ub-E3 ligases APC and SCF complex. By setting the activity of CDKs to zero during distinct time windows of the cycle, the proteolytic machinery ensures the unidirectionality and irreversibility of the cycle.

SCF complexes primarily regulate progression from G1 into S phase, for example, by marking cyclins for degradation. APC is the major regulator of progression through and entry into M phase by destroying the mitotic cyclins, among others. Overall, a multi-layered control system of high complexity exists between APC, SCF, and the CDKs.

The Ub-dependent proteolysis of cyclins and CKIs represents a major tool for the control of cell-cycle progression, as well as of other cell-cycle regulators. The use of speciﬁc proteolysis as a tool to control the cell cycle has various advantages:

Proteolysis allows the simultaneous and complete inactivation of all functions of a multifunctional cell-cycle protein such as the cyclins. Proteolysis enables subunit-selective reorganization of heterooligomeric protein complexes. An example is the targeted degradation of a CDK inhibitor. The total substrate pool of regulatory enzymes of the cell cycle may be inactivated by proteolysis. The regulatory system of a cell-cycle section can be reset to the ground state by proteolysis. 682 15 Cell-Cycle Control by External Signaling Pathways

& E3 ligase complexes in the cell cycle: — SCF — APC

Two types of E2/E3 complex are of particular importance for cell-cycle control [3] ! (Figure 15.10). One complex, the SCF, not only regulates primarily G1 S progression but also plays important roles in the other phases of the cell cycle. The other, APC or cyclosome, is required for the separation of the sister chromatids at

anaphase and for the exit of cells from M phase into G1. Importantly, the activities of both types of complex are interconnected, which provides a means for coupling the two activities during the course of the cell cycle.

15.3.1 Proteolysis Mediated by the SCF Complex

Progress of the cell cycle through G1 phase and entry into S phase is associated with the Ub-dependent proteolysis of important regulatory cell-cycle proteins, mediated by multiprotein complexes called SCF complexes. These function as E3 enzymes (see Section 2.8) and are composed of three invariant subunits, namely

Figure 15.10 Roles of two distinct ubiquitin active from late G2 to mid-G1 phase and ligases in regulation of the cell cycle. The catalyzes the ubiquitination of mitotic cyclins ordered progression of the cell cycle is and securins which are anaphase inhibitors. In regulated by two ubiquitin ligases: the contrast, the SCF complex mediates the anaphase-promoting complex/cyclosome ubiquitination of G1 cyclins and CKIs. (APC/C) and the SCF complex. The APC is 15.3 Regulation of the Cell Cycle by Proteolysis 683

Skp1, CUL1, and the RING finger Protein RBX1, as well as a variable component, an F-box protein. The F-box proteins function as substrate-specific adapter subunits that confer substrate specificity by recruiting a particular target to the core ubiquitination machinery. The human genome encodes 69 F-box proteins which are divided into three classes, namely the Fbw, WD-rich, and Fbl F-box proteins. The CUL1 component functions as a scaffold that recruits the charged E2 enzyme and Skp1. In order to be active, the CUL1 subunit must be modified by neddylation (Section 2.8.6).

& SCF subunits: — Skp1 — Cullin — Rbx1 — F-box protein Major SCF substrates: — p21Cip1 — p27Kip1 — CycA — CycE — Cdc6 — CDC25 phosphatase.

As illustrated in Figure 2.16 of Chapter 2, the F-box proteins Skp2, Fbw7 and b-TrCp control the abundance of central cell-cycle regulators and other proteins involved in the promotion of proliferation. The regulators that are targeted for degradation include proteins, the degradation of which may be a requirement for an ordered progression through G1 and S phase. Examples include the inhibitors p21CIP1, p27KIP1, p57KIP2, cyclin A, cyclin E, cyclin D, and CDC25 phosphatase. The SCF complexes containing the WD-rich and Fbl F-box proteins are constitutively active and act on the speciﬁc substrates only after the substrate protein has been phosphorylated. Examples include the inhibitor p27KIP1 that must be phosphorylated by cyclin E/CDK2 on Thr187 for binding to the Skp2–SCF complex.

& The selection of SCF substrates often requires phosphorylation of the substrate at PEST sequences

The phosphorylation of p27KIP1 has a twofold function during Ub-mediated degradation. In order to be broken down, p27KIP1 must be transported out of the nucleus, and this requires phosphorylation of Thr187; such phosphorylation is also required for recognition by the Ub-conjugating system. Export from the nucleus and proteasome-mediated degradation are both controlled by phosphorylation in 684 15 Cell-Cycle Control by External Signaling Pathways

this case. Thus, the regulator for this degradation pathway is phosphorylation of the substrate by a regulatory protein kinase, and the Ub-ligase system may be constitutively active.

15.3.2 Proteolysis Mediated by the APC/Cyclosome

The APC (known also as the cyclosome) is another type of E3 ubiquitin ligase that mediates the proteolysis of important regulators of cell-cycle progression, with a major effect during mitosis. The activity of the APC is tightly regulated to control

cell-cycle progression, being high from late mitosis until late in the G1 phase, but low in S, G1, and early mitosis in mammalian cells. One hallmark of APC is its complicated structure, being composed of at least 11 different subunits [4].

& APC: — 11 subunits — Substrate selection mediated by CDC20 or Cdh1 subunits.

At least part of the activity and substrate speciﬁcity of the APC are dictated by phosphorylation and by the regulated association of coactivator proteins, of which two main types have been identiﬁed: the Cdc20 protein and the Hct1/Cdh1 protein. Accordingly, two main types of APC are found in the cell with distinct substrate preference and different functions in the cell cycle, namely the mitotic form APC– Cdc20 and the nonmitotic form APC–Cdh1.

& Mitotic APC: — APC–Cdc20 Nonmitotic APC: — APC–Cdh1

15.4

G1 Progression and S Phase Entry

Summary In order to enter S phase and start a new round of DNA replication, several

prerequisites must be fulfilled during G1: cells must be large enough to endure a new round of division; apoptotic signals (see Chapter 17) must be suppressed; and the components needed for DNA replication must be available. The signals

that control these functions are manifold. At the start of G1, the levels of D-type cyclins are low and mitogenic signals are required that activate transcription of the genes for D-type cyclins; this allows an inhibitory threshold imposed by CKIs

to be overcome. Ultimately, these signals initiate an activation of the G1 CDKs, namely CDK4/6 and CDK2. The CDK4/6–cyclin D and the CDK2–cyclin E 15.4 G1 Progression and S Phase Entry 685

complexes perform different functions in G1 progression. Whereas, the CDK4/6–cyclin D complexes prepare the cells for S phase entry, the final decision for entry into S phase is dependent on the activity of cyclin E– CDK2 complexes. A negative regulation of cell-cycle progression is mediated by cytostatic signals that limit cell proliferation in order to maintain tissue homeostasis. These

signals use CKIs to inactivate the G1 CDKs and stop G1 progression. In addition to the manifold signals that regulate G1 during normal cell proliferation, both G1 and S phase are subjected to quality control via DNA damage and DNA

replication checkpoints that intervene with G1 or S phase progression in the case of DNA damage and incomplete or erroneous DNA replication. A major target of active CDK4/6–cyclin D and CDK2–cyclin E complexes is the tumor suppressor protein Rb, which is found in both active and inactive states. Active Rb has the E2F transcription factors associated, which prevents the transcription of E2F-dependent genes. A progressive phosphorylation of Rb by CDK4/6–cyclin D and CDK2–cyclin E triggers the release of E2Fs and allows for the transcription of genes required for S phase entry and S phase progression.

15.4.1 CDK4/6 and the D-Type Cyclins

The CDK4/6–cyclin D and the CDK2–cyclin E complexes perform different – functions in G1 progression. Whereas, the CDK4/6 cyclin D complexes prepare the cells for S phase entry, the ﬁnal decision for entry into S phase is dependent on the activity of cyclin E–CDK2 complexes.

& CDK4/6–cyclin D: — Activated by Thr172 phosphorylation — Rb as substrate — Regulated by cyclin D expression.

Of the three cyclins of type D (D1, D2, D3), two (D2 and D3) do not occur inallcelltypes,whilecyclinD1hasacentralfunctionintheregulationofG1 phase in all cell types. For the full activation of CDK4, activating phosphorylation at Thr172 of CDK4 is also necessary, in addition to binding of cyclin D1 (Figure 13.12). This step is catalyzed by CAK. Furthermore, removal of the inhibitory phosphorylations at Tyr14 and Thr15 by CDC25 phosphatases is required for CDK4/6 activation.

15.4.1.1 Regulation of CDK4---cyclin D1 The following discussion concentrates on cyclin D1 that has a key role in binding and activating CDK4. In brief, the steps relevant for CDK4 regulation are (Figure 15.11): 686 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.11 Regulation of cyclin D. In the example of cyclin D1, the figure illustrates the activating and inactivating influences on D-type cyclins. For an explanation, see the text. The red and yellow phosphorylation symbols indicate inactivating and activating effects, respectively.

When cells leave M phase, the level of cyclin D1 is low and CDK4 is kept in an inactive state due to association with CIP/Kip or INK4 inhibitors. Progression

into G1 phase requires mitogenic signals that increase the amount of cyclin D1. Mitogenic signals induce at least one D-type cyclin at concentrations, allowing an inhibitory threshold imposed by INK4 and CIP/Kip proteins to be overcome. A constitutive activity of cyclin D1–CDK4 complexes is then observed during the

whole of G1 and during S phase. The D-type cyclins complexed to CDK4/6 have the task of integrating external signals into the cell cycle. Mitogenic signals, such as growth factors, activate transcription of the gene for cyclin D1 and thus increase the amount of cyclin D–CDK4/6 complexes. The activating signals can become active from the start of G1 onwards. The increase in cyclin D–CDK4/6 complexes is postulated also to sequester the p27KIP1 inhibitor bound to cyclin E–CDK2 complexes away from CDK2. This is thought to establish initial levels of active cyclin E–CDK2, which is necessary for entry into S phase. The only known essential substrate of CDK4 is the retinoblastoma protein Rb. The major task of cyclin D–CDK4/6 is to phosphorylate and inactivate Rb (Section 15.4.3). An initial inactivation of Rb by phosphorylation allows for the release of activating E2F proteins and the subsequent transcription of initial amounts of cyclin E.

& G1entry and G1progression are mainly regulated by: — CDK4/6–cyclin D — Cyclin D expression — CDK2–cyclin E — p27Kip1 — Ink4 CKIs — APC–Cdh1 activity. 15.4 G1 Progression and S Phase Entry 687

15.4.2 Function of CDK2---Cyclin E in S Phase Entry

Cyclin E binds and activates CDK2, and this has now been recognized as being a key regulator of G1/S transition, functioning in a non-overlapping fashion with cyclin D complexes. Until late G1, the level of cyclin E remains low and its expression is dependent on E2F transcription factors. In mitotically resting cells, and in cells that have just emerged from M phase, E2F factors are bound to Rb, or to its family members p107 and p130, and this binding turns E2Fs into repressors or inactive transactivators.

& Major substrates of CDK2–cyclin E: — Rb — p27Kip1.

The inactivation of Rb is performed in a sequential manner via phosphorylation by CDK4/6–cyclin D and CDK2–cyclin E. Following phosphorylation by CDK4/6– cyclin D, E2F dissociates from Rb, allowing E2F-dependent transcription and cyclin E expression. Levels of CDK2–cyclin E are then increased and show a maximal value at the start of S phase. Subsequently, the cyclin E–CDK2 activity falls off sharply within S phase.

& Control of cyclin E levels: — Transcription induced by E2F — Proteolysis induced by phosphorylation and Ub-ligation (SCF).

The activity of cyclin E–CDK2 complexes is mainly directed toward two substrates, the Rb protein and the inhibitor p27KIP1 (Figure 15.12). The CDK2– cyclin E complex phosphorylates Rb and promotes a further release of E2F from the repressed state, allowing the transcription of key proteins for G1/S transition. The gene for cyclin E is also induced by transcription factor E2F, which explains the – increase in cyclin E at the G1/S transition. Another important substrate of cyclin E CDK2 complexes is the inhibitor p27KIP1, the phosphorylation of which on Thr187 induces its ubiquitination and targets it for degradation in the proteasome.

15.4.3 Function of the Retinoblastoma protein (Rb) in the Cell Cycle

The Rb protein is a nuclear phosphoprotein of 105 kDa (reviewed in Ref. [5]) and belongs to a class of proteins called the pocket proteins. Currently, two relatives of Rb are known, namely p107 and p130, and these share many of the Rb protein’s biological properties. As demonstrated in knockout experiments, the three pocket proteins appear to perform genetically redundant or overlapping functions, particularly with regard to cell-cycle control. 688 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.12 Multiple regulation of cyclin E. controlled by the action of CDC25A The concentration of active cyclin E---CDK2 phosphatase, which cleaves off inhibitory complexes is determined by expression of the phosphate residues from CDK2. Important cyclin E gene, by binding of the inhibitors p15, substrates of active cyclin E---CDK2 complexes p16 and p27, and by its phosphorylation are the Rb protein and the inhibitor p27. status. The inhibitor p27 can be removed from Phosphorylation of the latter induces its p27---cyclin E---CDK2 complexes by ubiquitinylation and proteasome degradation. sequestering it to cyclin D---CDK4/6 complexes. Only the major regulatory influences on cyclin The amount of active cyclin E---CDK2 is also E are shown.

& Rb: — Tumor suppressor — Pocket protein — Related proteins: p107, p130 — Regulated by multiple phosphorylations.

The Rb protein and its relatives are central regulating units of the cell cycle. These proteins function as suppressors of cell growth and proliferation, and are involved in the transition into and maintenance of the differentiated state of cells [6]. The loss or deregulation of these functions is associated with a deregulation of cell division, and favors tumor formation. Hence, the Rb protein has the characteristics of a tumor suppressor protein that is mutated in various tumors (Section 16.6). 15.4 G1 Progression and S Phase Entry 689

Figure 15.13 Domain structure and and protein phosphatase 1 (PP1) are indicated. phosphorylation sites of the retinoblastoma The N terminus of Rb interacts directly with the protein, Rb. N, N-terminal region; R, regulatory pocket domain, thereby potentially generating region. A and B are small pocket regions that a closed configuration that can be regulated by mediate the binding of E2F proteins and of additional interactions. Phosphorylation of the viral oncoproteins. Binding sites for E2Fs C-terminal phosphorylation sites interrupts extend into the C-terminal region. The binding interactions mediated by the pocket region and sites for E2F1, CDK2---cyclin E, CDK2---cyclin A leads to the release of E2Fs.

15.4.3.1 Domain Structure of Rb The domain structure of Rb is shown in Figure 15.13. The Rb protein can be divided into at least three functional regions: an N-terminal region; a central pocket composed of two subdomains; and a C-terminal region. The N-terminal region appears to be required for oligomerization, as it interacts with the pocket region so as to induce a closed, inactive, state of Rb. The pocket region, which is composed of A and B subdomains, contains binding sites for E2F transcription factors, for the viral oncoproteins TAg, E1A, and E7 (Section 16.6.1), and for a large number of other cellular proteins. A nonspeciﬁc DNA-binding site, binding sites for E2F1, cyclin–CDK complexes and protein phosphatase 1 (PP1) are also found on the C- terminal region. Numerous Ser/Thr phosphorylation sites have been identiﬁed on Rb, and the different phosphorylation events appear to regulate distinct Rb functions.

& Major Rb functions: — Cell-cycle regulation — Control of differentiation — Regulation of E2F 690 15 Cell-Cycle Control by External Signaling Pathways

— Interaction with: Viral oncoproteins Tag, E1A, E7 MDM2 ligase Many other proteins.

The relatives of Rb, the 107 and p130 proteins, harbor additional insertions that mediate cyclin binding and inhibition of CDK4/6. This property is not found on the Rb protein.

15.4.3.2 Control of Rb by Phosphorylation Rb proteins control cell-cycle advancement through their interaction with the E2F family of transcription factors to negatively control the transcription of essential cell-cycle genes. This concept is basic to current thinking on Rb family function. The members of the E2F family coordinate the expression of various genes important for cell-cycle progression, and their control by Rb members and CDK

activity is a critical element of G1 progression and S phase entry. The link to the periodic activation of CDKs in G1 is provided by the phosphorylation status of Rb, which can be considered as a “switch” that transforms Rb from the active, unphosphorylated state to the phosphorylated, inactive state.

& Rb activity is controlled by multiple phosphorylations catalyzed by: — CDK4/6–cyclin D — CDK2–cyclin E — CDK2–cyclin A.

At the start of G1 phase, Rb exists in the active, underphosphorylated form and binds E2F members, leading to a repression of E2F-dependent genes. The inactivation of Rb and relief of repression is achieved when Rb is sequentially phosphorylated by the cyclinD–CDK4/6, cyclin E–CDK2, and also the cyclin A– CDK2 complexes. Rb contains more than 10 potential Ser/Thr phosphorylation sites, and these are phosphorylated by the various CDKs in a speciﬁc manner. Importantly, mitogenic signals induce – via activation of the cyclin–CDK complexes – the phosphorylation of Rb and thereby control the passage through G1 and the entry into S phase.

& Hypophosphorylated Rb: — Repression of E2F-dependent genes Hyperphosphorylated Rb: — Induction of E2F-dependent genes.

The repression of E2F-dependent genes is relieved on Rb phosphorylation, and the encoded proteins can be produced allowing for entry into and progression through S phase. 15.4 G1 Progression and S Phase Entry 691

15.4.4 The E2F Transcription Factors and their Control by Rb

The E2F proteins are heterodimeric transcription factors composed of E2F subunits and DP subunits [7]. Binding sites for E2F members are found in the promoters of many genes whose functions are needed for cell proliferation and whose products drive cell-cycle progression. Mammalian cells contain at least seven E2F family members (E2F1–7) and two DP family members. E2F1–5 associate with Rb family members, whereas E2F6 and E2F7 appear to act independently of Rb proteins.

& E2F: — Heterodimeric transcription factor with E2F subunit and DP subunit — Regulates genes required for S phase progression.

For simplicity, the E2F family is often divided into activator E2Fs (E2F1–3) and repressor E2Fs (E2F4 and 5). Repressor E2Fs occupy promoters in G0/G1 phase, and typically these proteins are complexed with Rb-family members. The relatives of Rb, p107 and p130 bind to E2F members different from that bound by Rb, and these associations occur at distinct stages of the cell cycle (Figure 15.14).

Figure 15.14 Activating and repressing p130) associate with different subsets of E2F E2Fs. Eight E2F transcription factor genes proteins. The binding of Rb to the C-terminal (E2f1---E2f8) have been identified in mouse activation domain of E2F1, E2F2 and E2F3a cells, each with the characteristic hallmark of a inhibits transcriptional activation. Rb proteins conserved DNA-binding domain. E2F1---E2F5 contain distinct binding sites for E2F proteins can either activate or repress transcription. and for transcriptional repressor complexes. E2F1, E2F2 and E2F3a seem to function Therefore, in association with E2F4, E2F5 or primarily as activators, whereas E2F4, E2F5 E2F3b, the Rb proteins can also recruit and E2F3b bind to E2F-regulated promoters chromatin-modifying enzymes to E2F- when targets are repressed. Like E2F6, E2F7 regulated promoters, thereby enabling and E2F8, they are thought to recruit repressor silencing of gene expression. Activating E2Fs complexes to DNA. E2F1---E2F5 contain are shown in green, and repressing E2Fs in C-terminal domains that mediate interactions red. DPs, DP1; PcG, polycomb group with the pocket domains Rb-family proteins. repressive complex. The three Rb-family members (Rb, p107 and 692 15 Cell-Cycle Control by External Signaling Pathways

Whereas, Rb is associated with E2F both in quiescent and actively dividing cells, p130

binds to E2F predominantly in cells that have entered the G0 state. & Two classes of E2F: — Activator E2Fs — Repressor E2Fs.

According to the prevailing model of E2F function, a rise in CDK activity during

G1 and the subsequent phosphorylation of Rb-family members, leads to a release of E2F-containing repressor complexes from E2F-regulated promoters, the binding of activator E2Fs, and the expression of the E2F target genes. In this way, Rb-family members provide an important connection between CDK activation and the expression of genes that are needed for cell proliferation. Examples of genes controlled by E2Fs include: Thymidine kinase Dihydrofolate reductase DNA polymerase a Cyclin A Cyclin E Transcription factor c-myc E2F1 Rb Proapoptotic protein Apaf1 (Chapter 17) Tumor suppressor ARF (Sections 16.6 and 16.7).

15.4.4.1 Control of E2Fs by Rb

In late M and in early G1 phase, Rb exists in an underphosphorylated or even unphosphorylated state. In this state, Rb is associated with repressive E2F–DP members and is bound to promoters of E2F-responsive genes, such that these genes are repressed. The repression of E2F activity by Rb appears to be mediated via two mechanisms (Figure 15.15):

Rb binds directly to the activation domain of the activator E2Fs and, in doing so, blocks the activity of this domain. Rb uses a protein interaction domain that is distinct from its E2F-binding site to associate with transcriptional repressor complexes. By forming these complexes, the Rb-family proteins serve as molecular adapters allowing chromatin-modifying enzymes to be recruited to E2F-regulated promoters and actively repress transcription.

& Rb–E2F complexes recruit chromatin-modifying proteins such as: — HDACs — Lys methylases 15.4 G1 Progression and S Phase Entry 693

Figure 15.15 Function of Rb and G1 cyclins target genes, which include the gene for cyclin upon G0/G1 transition and during G1 E, is now partially relieved and cyclin E---CDK2 progression. During G0, and in the absence of complexes accumulate, completing the mitogenic stimuli, Rb is in a phosphorylation of Rb. Hyperphosphorylated hypophosphorylated state and actively Rb dissociates from E2F, allowing a full represses genes by recruitment of histone transcription of the E2F target genes and deacetylases (HDACs). Mitogenic stimuli progression into S phase. The activated cyclin induce the accumulation of active E---CDK2 complex also phosphorylates the cyclinD---CDK4/6 complexes which promote inhibitor p27KIP1, inducing its ubiquitination cell growth and mediate a partial and proteasome degradation. TK, thymidine phosphorylation of Rb. Repression of E2F kinase; DHFR, dihydrofolate reductase.

Examples of chromatin-modifying proteins associated with Rb–E2F complexes include histone deacetylases, protein lysine methylase complexes such as SUV39/ HP1, and chromatin-remodeling proteins of the SWI/SNF type (Section 4.5). The nature of chromatin-modifying proteins associated with Rb family members depends on the identity of the Rb–E2Fcomplexes formed, and a transiently or permanently repressed state may be induced. The relief of repression is a dynamic and complex process that is thought to include the reversal or overriding of the repressive state of the chromatin by exchanging the repressive E2Fs for the activating forms, or the additional binding of transcription promoting complexes such as histone acetylases (HATs). The initial signal for transition of Rb from the unphosphorylated (or hypophosphorylated) active state to the phosphorylated, inactive state is provided by Rb phosphorylation under the inﬂuence of mitogenic signals, catalyzed by CDK2/4–cyclin D complexes. In this way, the repression of E2F-dependent genes is progressively relieved. As the 694 15 Cell-Cycle Control by External Signaling Pathways

gene of cyclin E is an E2F-regulated gene, the levels of cyclin E will begin to rise such that, ﬁnally, the cyclin E–CDK2 complex becomes mainly responsible for converting Rb into the hyperphosphorylated form and providing full relief of repression. The prior phosphorylation of Rb by cyclinD–CDK4/6 is essential for this step because cyclin E–CDK2 acts only on Rb proteins that are already partially phosphorylated.

15.4.5

Summary of G1 Progression

The essential steps driving G1 progression may be summarized as follows:

When cells enter G1, levels of D-type cyclins are low and G1 CDKs are inhibited by binding to CKIs. Progression through G1 requires mitogenic signals that induce cyclin D expression to levels that overcome inhibition of CDKs by CKIs. ﬁ – The rst kinase complexes to become active in G1 are cyclin D CDK4/6, and these begin to phosphorylate the Rb-family proteins. As cells approach the end of

G1, a partial phosphorylation of Rb allows some transcription by activator E2Fs that begin to stimulate the production of cyclin E; this, in turn, creates more cyclin E–CDK2 kinase activity. The full activation of cyclin E–CDK2 is impeded by interaction with and inhibition by p27KIP1. This is ultimately overcome by cyclin E–cdk2 phosphorylating p27KIP1 and results in p27KIP1 being targeted for degradation and allowing for a full activation of the kinase. Phosphorylation and inactivation of Rb is completed by cyclin E–CDK2 complexes, leading to the release of activating E2F members and the transcription of E2F target genes. A feed-forward loop ensures that cells advance to S phase irreversibly. This control relies on the cyclin E–CDK2 phosphorylation of pRB to release activator E2Fs that transcribe more cyclin E and generate more kinase activity towards pRB; this leads to more free E2Fs and, ultimately, even more cyclin E. Once cells have committed to S phase progression and initiated DNA synthesis, cyclin E–CDK2 will phosphorylate other cyclin E subunits; this will target them for degradation, bringing the surge in cyclin E–CDK2 activity to an end.

Overall, the proteins involved in G1 progression form part of a complicated network, and it has been possible to present only the key aspects of G1 regulation in the preceding sections. Each protein component of G1 control is subject to multiple regulations that originate from external signals, as well as from internal signals that ensure a correct completion of the cell cycle. Unfortunately, a detailed discussion of these aspects is not possible in this chapter.

15.4.6

Mitogenic Signals Regulating G1 Entry and Progression

Nontransformed cells require mitogenic signals in order to be able to transit from

the quiescent state into G1 and further into S phase. In these cells, the cyclin D1 15.4 G1 Progression and S Phase Entry 695 gene senses the mitogenic potential of the microenvironment during cell-cycle entry from quiescence, because its induction requires a coordinated signaling from the extracellular matrix and soluble growth factors. Conversely, the repression of cyclin D1 gene expression is a hallmark of cell differentiation. Actively dividing cells also require appropriate mitogenic signals. Following exit from M phase, both mitogenic and growth-promoting signals must become active in order to allow the cell to progress in G1 and enter S phase. The most signiﬁcant target of mitogenic signals is cyclin D1, which is key to the activation of CDK4/6 and G1 progression. The importance of cyclin D1 for cell division activity is supported by the observation that it has been identiﬁed as a major oncogenic driver in human cancer [8]. The regulation of cyclin D1 occurs at multiple levels, including the transcription of its gene, mRNA stability, translation, and nucleocytoplasmic translocation [9]. Most important are the signals that target cyclin D1 transcription. It is known that a variety of signaling pathways converge on the cyclin D1 promoter to regulate its cell cycle and context-dependent transcription [10]. Furthermore, cyclin D1 translation and stability are important attack points of mitogenic signals.

15.4.6.1 Transcriptional Control of Cyclin D1 The cyclin D1 promoter harbors consensus binding sites for a large number of different transcription factors that mediate the activation or repression of cyclin D1 transcription. Accordingly, a wide variety of signaling paths converge on the cyclin D1 promoters, and these paths are regulated by signals from either extracellular or intracellular sources in a cell-cycle- or context-dependent manner. Cyclin D1 transcription is regulated by the following major signaling pathways (see Figure 15.16):

Extracellular signals originating from cell–cell interactions, via integrins (see Section 13.4) Cytokines and their receptors, via the Jak/Stat pathway and via NFkB Notch, via CSL Wnt signaling, via b-catenin and TCF (Section 16.8) Growth factor signaling, Ras/MAPK/ERK pathway, via for example, transcription factors C-Jun, C-Fos and ATF PI3 kinase/Akt signaling, via NFkB Rac signaling, via NFkB Estrogen receptor signaling.

In addition to activating transcription factors, a specificrepressionofthegene for cyclin D1 has also been described. Negative regulators of D1 transcription include the transcription factors c-Myc and Oct-1, for example. Quite how the positive and negative regulations are coordinated is not well understood, but cell type-specific and locus-specific chromatin structures, and the availability of the transcription factors, will surely determine the final outcome of these regulations. 696 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.16 Transcriptional regulation of activating signals are shown as boxes in the cyclin D1. Transcription of the gene for cyclin upstream control region. The promoter region D1 is controlled by a composite promoter that also harbors binding sites that mediate harbors multiple binding sites for transcription transcription repression (these are not shown factors as determined by CHIP analysis and in the figure). For details, see Ref. [10]. For luciferase assays. The cyclin D1 gene is subject integrins, see Section 13.4; for cytokine to transcriptional control via the receptors, see Sections 13.1 and 14.3; for transmembrane receptors indicated. Only part Notch, see Section 14.2; for Wnt, see Section of the downstream signaling components are 16.8; for RTKs, see Chapter 10. included. The binding sites that convey

15.4.6.2 Control of Cyclin D1 Translation and Stability In addition to the activation of transcription, external mitogenic signals have been shown to enhance translation and reduce proteolysis of cyclin D, thus potentiating the induction of cyclin D1. This control occurs primarily via the Akt pathway. An increase in cyclin D1 translation is mediated by the PI3K–Akt–mTOR–S6K1 cascade (Section 9.4). The pathway that directs a reduced proteolysis of cyclin D1 involves PI3K–Akt–GSK3b (glycogen synthase kinase-3b); the latter is crucial to cyclin D stability because this enzyme phosphorylates cyclin D1 at Thr286, leading to cytoplasmic export and Ub-mediated degradation. The protein kinase Akt catalyzes the phosphorylation of GSK3b, decreasing its catalytic activity towards 15.4 G1 Progression and S Phase Entry 697 cyclin D1 and resulting in an increased nuclear accumulation and a reduced cytoplasmic degradation.

15.4.7

Negative Regulation of the G1/S Transition

In addition to mitogenic signals, antimitogenic signals are also processed during

G1 phase. These lead to an increase in the level of CDK inhibitors, or they may inﬂuence the phosphorylation state of the CDKs via the phosphatase Cdc25.

Antimitogenic signals can lead to a halt during G1 phase and bring the cell into a resting state. An antimitogenic signal originates, for example, from TGFb, from cAMP, from certain cell–cell contacts, and from DNA damage.

& G1/S progression is inhibited by CKIs in response to: — TGFb signals — cAMP signals — Cell–cell contacts — DNA damage checkpoints.

Negative regulation of the cell cycle in G1 phase is performed by inhibitors of the INK4 family, which preferentially bind and inactivate monomeric CDK4/6 complexes, preventing cyclin D activation. An accumulation of p16INK4a, for example, sequesters CDK4/6 complexes, preventing progress in G1 phase. The inhibitors p21CIP1and p57KIP2 are directed mainly against heterodimeric CDK2 complexes, and can thereby inactivate the cyclin E/cyclin A–CDK2 complexes, preventing crossing of the restriction point and entry into S phase. The balance between activated CDK4/6–cyclin complexes and the various inhibitors controls progress through G1 phase, with the concentration of inhibitors being regulated in a complex pattern by external cues. Examples of such external inﬂuences on CKI proteins include the induction of p21CIP1 by p53, the stimulation of the degradation of p27KIP1 by growth factors, and the induction of p15INK4b by TGFb.

15.4.8 Integration of Mitogenic Signals for Control of Cell Proliferation, Cell Growth, and Survival

ﬂ Overall, G1 is a period during which many signals intervene to in uence cell division and the deployment of a cell’s developmental program. Diverse metabolic, stress and environmental cues are integrated and interpreted during this period. On the basis of these inputs, the cell decides whether to enter S phase or to pause. Moreover, in multicellular organisms the behavior of a cell must obey dictums from its neighbors. To this end, during G1 the cell must make further decisions regarding whether to self-renew, to differentiate, or to die. 698 15 Cell-Cycle Control by External Signaling Pathways

Proliferation, cell growth and survival are distinct but intertwined functions needed for the passage of cells through the cell cycle. First, cells must receive mitogenic signals that allow cell division in the frame of the development of the organism. Second, the cells must be large enough for division, and growth- promoting signals are required to this end. Finally, cell death signals must be allowed to become active under conditions where the cell number is to be controlled and where accidents such as DNA damage call for cell death. & Signals required for G1/S progression: — Mitogenic signals (e.g., growth factors) — Anti-apoptotic signals — Growth-promoting signals.

As illustrated in Figure 15.17, the Ras and the PI3 kinase pathways are versatile pathways that achieve at least partial integration of these functions.

Figure 15.17 Control of G1/S transition by Ras ensure mitochondrial integrity; (c) By and Akt kinase pathways. Ras and Akt promoting cell growth via the translation pathways control cell proliferation via three initiation factor eIF4E. For the apoptotic major approaches. (a) By regulating the G1/S proteins Bad, Bim, Bcl2, BclXL, IAPs, see transition; (b) By suppressing apoptosis and Chapter 15. For eIF4E and 4EBP, see Section promoting cell survival. Here, the Ras and Akt 5.2. For IKK and NFkB, see Section 2.8.5.3. signals finally mediate caspase inhibition and Gsk3-b, glycogen synthase kinase 3 type b. 15.5 Transit Through S Phase and M Phase 699

Mitogenic signaling pathways are activated by external signals, such as growth factors, control inhibitor and cyclin concentrations, which in turn empower CDKs to drive the G1/S transition, and the Ras and PI3K/Akt pathways stand out in this respect [11]. As outlined in Chapters 6, 8, and 9, a large number of external signals can feed into these proliferation-promoting pathways (see Figure 15.17). Most of the mitogenic activities of Ras and PI3K/Akt pathways are directed towards expression of the D-type cyclins, allowing for the formation of active CDK4/6 complexes and sequestration of the inhibitor p27Kip1. One critical factor for cell-cycle progression is that of cell growth, as the cells must achieve a certain size before being able to pass through G1 and enter S phase. The PI3K/Akt kinase pathway is a major pathway that directs signals to the level of translation and nutrient uptake. Coordination of the mitogenic signals with cell survival is another important aspect of cell-cycle progression. Both, the Ras and PI3K/Akt pathways transmit anti-apoptotic signals by reducing the levels of apoptosis-promoting proteins such as Bcl2 and increasing the levels of apoptosis-inhibiting proteins.

15.5 Transit Through S Phase and M Phase

15.5.1 DNA Replication During S Phase

The replication of DNA in S phase is subject to strict control in the cell cycle, resulting in the following observations:

DNA replication is restricted to S phase. DNA is only replicated once in a cycle. The time sequence of DNA replication during S phase and mitosis is strictly maintained. If DNA damage is present, DNA replication can be halted (DNA damage checkpoint).

The control of DNA replication occurs at two levels in particular:

Availability of the replication components: At the start of and during S phase, the dNTPs and all proteins required for replication must be available in sufficient quantities. An important control function is performed here by the transcription factor E2F, which induces the different enzymes needed for replication (see above). Control at the initiation level: The replication of a DNA sequence starts at specific sequence sections of the DNA, known as replication origins. Control of origin activity occurs via specific protein complexes that are bound to the origins at certain times of the cell cycle. For replication initiation, two states of these 700 15 Cell-Cycle Control by External Signaling Pathways

protein complexes are important, known as the pre-replication complex (pre-RC) and the post-replication complex. For details on the control of S phase, see Ref. [12].

& Initiation of DNA replication is controlled by formation of: — Pre-replication complexes — Post-replication complexes.

15.5.2

G2/M Transition and Progression Through M Phase

Summary Entry into and progression through M phase is strictly regulated at two

checkpoints, the G2/M transition and the transition from metaphase to anaphase within M phase. Exit from G2 and entry into M depends on the activation of CDK1, while progression through M requires the targeted proteolysis of M phase regulators by the activity of a large E3 ligase complex, the anaphase-promoting complex (APC).

15.5.2.1 CDK1 Control at G2/M Entry into M phase is determined primarily by the activity of the cyclin B–CDK1 kinase complex, which is also known as the mitosis-promoting factor (MPF).

& G2/M transition depends critically on CDK1–cyclin B activation

The crucial regulatory elements of CDK1 activity are the concentration of cyclin B and phosphorylation/dephosphorylation events. Cyclin B concentrations are increased on entry into S phase to a threshold at which sufﬁcient cyclin B–CDK1 is available for triggering mitosis. However, for entry into mitosis the phosphorylation status of CDK1 is the most vital element. The critical phosphorylation sites on CDK1 are Thr161 within the activation loop, and Thr14 and Tyr15 within the ATP-binding site. – At the end of G2 phase, the cyclin B CDK1 complex exists in a threefold phosphorylated form, which represents an inactive state (Section 15.2.4.2; Figure 15.18). Whilst phosphorylation on Thr161 is catalyzed by CAK, Thr14 and Tyr15 are phosphorylated by the Wee1 kinase. In order to become active, the CDK1–cyclin B complex must be dephosphorylated at Tyr15 and Thr14, a reaction performed by Cdc25C phosphatase.

& CDK1–cyclin B: — Activated by CDC25 in positive feedback — Inhibited by Wee1 kinase.

Regulation of the phosphorylation status of CDK1 occurs mainly via the activity of Cdc25C and the Wee1 kinase, both of which enzymes are controlled by 15.5 Transit Through S Phase and M Phase 701

Figure 15.18 Control of M phase entry by which are activating and some are inhibiting. CDC25 phosphatase and Wee1 kinase. The Wee1 kinase is inactivated by phosphorylation, phosphorylation status of the CDK1---cyclin B subsequent ubiquitination, and proteasomal complex is determined by the opposing degradation. The activity of CDC25 activities of Cdc25 phosphatase and Wee1 phosphatase is upregulated by a positive kinase. CDK1---cyclin B is inactive when feedback loop involving CDK1---cyclinB. residues Thr14 and Tyr15 become The initial activation of CDK1---cyclinB may phosphorylated by Wee1 kinase, but is active be achieved by CDK2---cyclin A and Polo-like when these residues become kinase 1 (Plk1). The phosphorylation status dephosphorylated by CDC25 phosphatase. of Cdc25 is also controlled by the action of Transition into M phase requires inactivation phosphatases. Furthermore, DNA-damage of Wee1 kinase and activation of Cdc25 by signals mediate the inhibitory phosphorylation multiple phosphorylation events, some of of Cdc25. phosphorylation/dephosphorylation events triggered by upstream signals. An activated CDK1–cyclin B complex has been shown to phosphorylate and activate Cdc25C, providing a positive feedback loop between CDK1 and Cdc25C phosphatase. An initial activation of Cdc25C is achieved by upstream protein kinases that may be sensors of external signals. One such kinase appears to be the polo-like kinase Plk1, which can phosphorylate and thus activate the Cdc25C phosphatase. 702 15 Cell-Cycle Control by External Signaling Pathways

The protein kinase Wee1, which is responsible for the inhibitory phosphorylation of CDK1, is also subject to multiple regulations. Wee1 is inactivated during mitotic entry by proteolysis, translational regulation, transcriptional regulation, and by phosphorylation. A multisite phosphorylation by kinases to be identiﬁed cooperatively inactivates Wee1 and promotes Wee1 proteolysis.

15.5.2.2 Progression Through M Phase The activities promoting progression through M phase are mainly provided by the CDK1–cyclin B, CDK1–cyclin A complexes and the APC. As outlined in Section 15.3.2, the APC is an E3 ligase that targets speciﬁc substrates to proteasomal degradation. There are two forms of APC depending on the nature of the subunit necessary for substrate recognition: APC-Cdc20 and APC-Cdh1, where the Cdc20 and Cdh1 proteins are responsible for substrate binding and selection of APC, respectively. During the early stages of mitosis it is APC-Cdc20 that is relevant whereas, in late mitosis and early

G1, APC-Cdh1 has been shown to play an essential role in cell-cycle progression & Mitotic APC: — APC-Cdc20 Nonmitotic APC: — APC-Cdh1

For details of the role of APC-Cdh1 and the complicated network linking APC- cdc20 and APC-Cdh activity with cell-cycle progression, see Ref. [13]. & Major APC-Cdh1 substrates: — Cyclin A — Cyclin B — ORC1 — Cdc6 — Geminin.

15.6 DNA Damage and DNA Replication Checkpoints

Summary DNA damage and DNA replication checkpoints serve to stop cell-cycle progression in case of DNA damage or the stalling of DNA replication.

Activation of these checkpoints can result in arrest in either G1, S or G2 in order to prevent the replication of a damaged template. Furthermore, the progress of S phase can be slowed if the replication apparatus meets damaged DNA sites. The DNA damage-related checkpoints share a common logic and common components in all phases of the cell cycle. The presence of a DNA lesion or a DNA replication problem is detected by a sensor kinase in cooperation with a multitude of accessory proteins. These kinases transmit the signal with the help 15.6 DNA Damage and DNA Replication Checkpoints 703

of proteins named mediators or transducers to critical cell-cycle regulators that participate in cell-cycle control, DNA repair, transcription regulation, replication, and apoptosis. The major sensor kinases are the ATM and ATR kinases. The major mediators are the checkpoint kinases Chk1 and Chk2, which phosphorylate and activate downstream effectors such as the CDC25 enzymes, p53, and different repair enzymes.

The genomes of all organisms constantly suffer attack by agents that either damage DNA or lead to stop in DNA replication. In order to cope with these problems, cells can activate a complex network of interacting pathways that lead either to damage repair and the resumption of a normal cellular life cycle, or to programmed cell death (apoptosis). When the genetic material is damaged or when DNA replication is stalled, a delay in the progression of the cell cycle is initiated and the DNA damage response is activated. This response coordinates the activation of cell-cycle checkpoints, the appropriate DNA repair pathways, and numerous other responses. Details of cell-cycle checkpoints linked to DNA damage and aberrant DNA replication will be presented in the following subsections. & DNA damage checkpoints operate by stopping the cell cycle in: — G1 — S — G2/M — M.

The DNA damage and replication checkpoints induce a series of different physiological responses such as (see Figure 15.19; see also Figure 15.21):

Halting the cell cycle at G1,S,orG2 phases Slowing of DNA replication Increased transcription of repair genes Induction of programmed cell death (apoptosis).

The signaling pathways that lead from the appearance of DNA damage to a halt in the cell cycle involve an entire network of damage-response proteins that cooperate to protect cells against the potentially deleterious consequences of DNA damage. This complicated network of cell cycle checkpoints is known to be of utmost importance in the prevention of cancer, but only a brief outline will be provided at this point [14].

15.6.1 Components and Organization of DNA Damage Checkpoints

One major task of DNA damage response is the activation of cell-cycle checkpoints, that will temporarily halt cell-cycle progression while the damage is assessed and 704 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.19 The major components of the effectors as, for example, the CDC25 DNA-damage response. DNA damages such phosphatases. When activated, the CDC25 as double-strand breaks or larger unpaired enzymes inhibit CDK enzymes which leads to a regions are recognized by sensor protein stop in the cell cycle. Besides influencing cell- complexes that have the ATM or ATR proteins cycle progression, the sensor kinases activate as core sensor kinases. The damage-induced other transducers and effectors that are activation of ATM/ATR triggers the activation components of the DNA-damage response of transducer proteins such as the Chk1 and network, such as p53 and repair enzymes. Chk2 kinases that pass the signal on to 15.6 DNA Damage and DNA Replication Checkpoints 705 processed in response to several DNA-damaging agents. Cell-cycle checkpoints operate in all phases of the cell cycle, and based on the timing of these checkpoints and the components involved, it is possible to discern between G1 checkpoints, replication checkpoints, intra-S-checkpoints, and G2 checkpoints. A further checkpoint – the spindle assembly checkpoint – operates in M phase but will not be discussed at this point [15]. The DNA damage-related checkpoints share a common logic and common components in all phases of the cell cycle. The common logic includes the detection of DNA damage or a DNA replication problem via a sensor kinase, and transmission of the signal by proteins termed mediators or transducers to effectors that participate in cell-cycle control, DNA repair, transcription regulation, replication, and apoptosis (Figure 15.19). The major sensor kinases are the ATM and ATR kinases, while the major mediators include the checkpoint kinases Chk1 and Chk2. Prominent effectors include the CDC25 enzymes, p53, and different repair enzymes. Major effectors of cell-cycle control in DNA damage response are the CDC25 phosphatases that remove the inhibitory phosphorylations on Thr14 and Tyr15 of the CDKs, and thus are required for full activation of the CDKs, primarily of CDK2 and CDK1 (Figures 15.20 and 15.21). Damage-induced signaling leads to an

Figure 15.20 Regulation of CDK activity by Wee1 kinase and is removed by CDC25 ATR, Chk1, and Wee1. Phosphorylation of phosphatase enzymes. DNA damage leads to CDKs at Tyr15 inhibits CDK activity. The the inhibition of CDC25, preserving the inhibitory phosphorylation is introduced by inactive state of the CDK. 706 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.21 DNA-damage response p53-dependent, ATM/Chk2 targets p53 and pathways. DNA double-strand breaks (DSBs) Mdm2 which leads to p53 stabilization and are detected by the MRN complex and the induction of apoptosis (Section 16.7). associated cofactors, while single-stranded p53 and MDM2 are phosphorylated at Ser15 breaks (SSBs) are detected by the single- and Ser395, respectively. p53 is also stranded DNA-binding protein (RPA). This phosphorylated by ATR at Ser15. Furthermore, leads to an activation of the sensor kinases p53 is phosphorylated at Ser20 by ATM- ATM and ATR. Both kinases trigger a series of activated Chk2. Accumulation (by loss of downstream events that lead ultimately to the MDM2 inhibition) and stabilization or induction of apoptosis, induction of DNA activation of p53 (by P-Chk2 and p-ATM- repair, cell-cycle arrest, and senescence. mediated phosphorylation) exert p53- Apoptosis may be induced by ATM via two dependent G1/S arrest or apoptosis. PML, routes. In the p53-independent route, the promyelocytic leukemia protein, an organizer transducer kinase Chk2 activates transcription of nuclear bodies. MK2, MAP kinase-activated of apoptosis-inducing genes via the E2F1 protein kinase 2 transcription factor. In another route that is 15.6 DNA Damage and DNA Replication Checkpoints 707 inactivation of the CDC25 enzymes, allowing for the persistence of inhibitory phosphorylations introduced by the Wee1 kinases. In addition to targeting CDC25 enzymes, DNA damage-induced signaling also promotes the degradation of cyclins (e.g., cyclin D1) by activating components of the SCF complex [16]. An overview of the major signaling reactions induced by DNA damage is provided in Figure 15.21. Although, overall, these processes involve a large number of different proteins, details of only the major components are presented in the following subsections.

15.6.1.1 Recognition of DNA Damage or Sensing of Replication Stress The presence of DNA damage or stalled replication is sensed via the formation of specific protein complexes at the sites where DNA cannot be replicated correctly or where alterations in DNA structure and/or chromatin pose severe problems to DNA function. The cell cycle must be brought to a stop under these circumstances in order to allow repair of the damage. If repair is not possible, the cells may be directed to programmed cell death (apoptosis). The triggering of damage checkpoints has been best-characterized for DNA damaged by double-strand breaks and for damages that disturb DNA structure leading to the formation of single-stranded DNA (ssDNA). The protein complexes forming at the damaged sites differ in composition, depending on the type of damage. For instance, the presence of double-strand breaks is sensed by the Ku70 protein in cooperation with the Mre11-Rad50-Nbs1 (MRN) complex and the phosphorylated histone variant H2AX [17], whereas damages associated with the exposure of ssDNA are characterized by coating with the ssDNA-binding protein, RPA. The formation of these complexes initiates the activation of two major parallel pathways that respond to DNA damage, namely the ATM and ATR pathways. In this network, the sensor kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related) are located at the top of a checkpoint signaling cascade, which phosphorylate and activate a variety of downstream proteins to execute the DNA damage response. These two major sensor kinases, ATM and ATR, belong to the PI3 kinase-like family of Ser/Thr protein kinases (Section 9.4.1) and become phosphorylated and activated upon the detection of a DNA lesion (Figure 15.21). Proteins such as claspin and BRAC1 with Mediator function cooperate in this step. Both, ATM and ATR share the same consensus motif SQ/TQ for phosphorylation. More than 700 proteins containing this motif have been identified as potential substrates that are inducibly phosphorylated in response to DNA damage caused by ionizing radiation. The network of proteins influenced by ATM, either directly or indirectly, is illustrated in Figure 15.22. One reason for this multitude of substrates is that ATM phosphorylates many targets within each ATM-dependent pathway, and fine-tunes various processes by modulating a number of pathways within the same process [16]. 708 15 Cell-Cycle Control by External Signaling Pathways

Figure 15.22 ATM interactions. The yellow, MAPK pathways; green, Akt, NFkB, expanding protein kinase landscape of the mTOR pathways. The node sizes are DNA damage response [16]. Nodes represent proportional to the number of edges. protein kinases (squares), phosphatases Functional cross-talks (dependency, activation, (diamonds) and other proteins mentioned in inhibition, or physical interaction) were the text (circles). Colors denote pathways by depicted as edges between the proteins. which kinases were described: blue, DNA- Bensimon 2011 [16], Figure 1. Reproduced damage response; red, cell-cycle control; with permission of Elsevier.

& Major protein kinases involved in DNA damage checkpoints: — ATM — ATR — Chk1, Chk2.

15.6.1.2 Functions of ATM The ATM sensor kinase becomes activated primarily by the presence of double- strand breaks, leading to the formation of MRN complexes. ATM activation at these 15.6 DNA Damage and DNA Replication Checkpoints 709 sites appears to be complex and is poorly understood. The increased ATM activity at sites of DNA damage is responsible for the phosphorylation and activation of the downstream effector kinase, Chk2, the full activation of which also requires phosphorylation by members of the polo-like kinase family. ATM also phosphorylates the tumor suppressor protein p53, leading to its stabilization; the increased levels of p53 then stimulate transcription of the inhibitor p27 leading to an inhibition of CDKs and cell-cycle arrest.

15.6.1.3 Functions of ATR The ATR-Chk1 pathway is the principal direct effector of the DNA damage and replication checkpoints and, as such, is essential for the survival of many – though not all – cell types. In the ATR signaling pathway, RPA-ssDNA is the ligand that recruits ATR and other ATR signaling components to sites of DNA damage or replication stress. An obligatory accessory protein, ATRIP (ATR-interacting protein), is required for ATR binding to RPA-ssDNA; subsequently, a larger checkpoint complex forms that involves the cooperation of multiple proteins and leads to ATR activation. ATR signals the regulation of DNA replication, cell-cycle transitions and DNA repair through the phosphorylation of hundreds of substrates, including Chk1 and the MCM helicase complex. However, for most of these substrates the potential biological function has not yet been explored (for details, see Ref. [18]). For checkpoint activation by ATR, the most prominent substrates are the Ser/Thr kinases Chk1 and Chk2. ATR activates Chk1 via phosphorylation on two Ser residues that lie outside of the kinase domain, whilst for the activation of Chk2 the participation of ATM also appears to be required. Other substrates include Akt kinase, which is phosphorylated and activated by ATR/ATM in response to various DNA damages. As a consequence, the mTor/NFkB pathway (Section 2.8.5.3) is activated, leading to many different further reactions.

15.6.1.4 Chk1, Chk2, and Downregulation of CDC25 Phosphatase The activated effector kinases Chk1 and Chk2 phosphorylate specific substrates, the phosphorylation of which interferes with cell-cycle progression. However, which of these substrates ultimately becomes phosphorylated has been shown to depend on the phase of the cell cycle when the damage was first recognized. At all checkpoints, both transient and longlasting pauses in cell-cycle progression can be mediated by these mechanisms, so as to provide time for DNA repairs to be carried out, or for the cell to be led into programmed cell death (apoptosis). The ATM/Chk2 module is activated following DNA double-strand breaks, while the ATR/Chk1 pathway responds primarily to DNA single-strand breaks or bulky lesions. Both pathways converge on Cdc25, a positive regulator of cell-cycle progression, which is inhibited by either Chk1- or Chk2-mediated phosphorylation. In addition, a third effector kinase complex consisting of p38MAPK and MK2 (MAP kinase-activated protein kinase 2) has been identified. This pathway is activated downstream of ATM and ATR in response to DNA damage. MK2 has been shown to share substrate homology with both Chk1 and Chk2 [19]. 710 15 Cell-Cycle Control by External Signaling Pathways

At the G1/S and G2/M transitions, the CDC25 phosphatases remove the critical inhibitory phosphorylations on Cdk/Cyclin complexes, namely Thr14 and Tyr15, in the ATP-binding loop of CDK1 and CDK2. Chk1 and Chk2 then downregulate the CDC25 enzymes by inducing their Ub-mediated proteasomal degradation. For example, upon intra-S phase checkpoint activation, Cdc25A is phosphorylated on Ser76 by Chk1 and subsequently undergoes SCF-dependent ubiquitination and proteasomal degradation.

& ATM kinase: — Responds to DNA double-strand breaks — Substrates: Chk1, p53, BRCA1, NBS1.

15.6.2

The Mammalian G1DNA Damage Checkpoint

At this point, only a brief outline will be provided of the G1/S and G2 checkpoints. Arrest in G1 phase can be achieved in at least two ways: (i) by targeting the tumor suppressor protein p53 (Section 16.7); or (ii) by targeting the activator of the cyclin E(A)/CDK2 kinase, the CDC25A phosphatase (Figure 15.23). The dominant checkpoint response to DNA damage in mammalian cells – – traversing through G1 is the ATM(ATR)/CHK2(CHK1) p53/MDM2 p21 pathway, which is capable of inducing sustained, sometimes even permanent, G1 arrest. Both, ATM and ATR directly phosphorylate the p53 transcription factor within its amino-terminal transactivation domain on Ser15, Thr18 and Ser20. In addition, the ubiquitin ligase MDM2, that normally binds p53 and ensures a rapid p53 turnover, is targeted after DNA damage by ATM/ATR. These modiﬁcations of p53 and Mdm2 contribute to the stabilization and accumulation of the p53 protein, as well as to its increased activity as a transcription factor.

& G1DNA damage checkpoints operate via: — p2 Cip1 induction — p53 stabilization — Inactivation of CDC25A and subsequent inhibition of CDK2–cyclin E.

The key transcriptional target of p53 is the p21CIP1/WAF1 inhibitor of cyclin-

dependent kinases, which silences the G1/S-promoting cyclin E/Cdk2 kinase and thereby causes G1 arrest. This not only leads to an inability to initiate DNA synthesis but also preserves the Rb/E2F pathway in its active, growth-suppressing

mode, thereby causing a sustained G1 blockade. Thus, the G1 checkpoint response targets two critical tumor suppressor pathways governed by p53 and pRB.

The other G1 checkpoint response operates by a downregulation of the CDC25A phosphatase. Activation of CHK1 and CHK2 leads to phosphorylation of the CDC25A phosphatase, an enzyme required for activation of the CDK2–cyclin E (or

A) complexes and for progression from G1 into S phase. The Chk1 (or 2)-mediated 15.6 DNA Damage and DNA Replication Checkpoints 711

Figure 15.23 Activation of DNA damage cell- p38MAPK/MK2 effector kinase complex cycle network. Activation of the upstream downstream of TAO kinases in response to activating PI3 kinase-like kinases ATM and ATR DNA damage. The three effector kinases, triggers signaling through the cell-cycle Chk1, Chk2 and MK2, are directly responsible checkpoint signaling cascade. ATM activates for inhibitory phosphorylations on members of the checkpoint effector kinase Chk2 by direct the Cdc25 family of phosphatases. phosphorylation on Thr68. Activation of ATR Additionally, Chk1 phosphorylates and requires TopBP1 and ATR is recruited to the activates Wee1 kinase. Wee1 directly mediates damaged DNA via its regulatory subunit the inhibitory phosphorylation of Cdks, which ATRIP. An efficient ATR-mediated activation of are normally removed through the enzymatic the downstream kinase Chk1 requires the activity of Cdc25 phosphatases to allow presence of claspin. Both, ATM and ATR progression through the cell cycle. appear to be required to activate the phosphorylation induces the ubiquitination of CDC25A by SCF complexes and its proteasomal degradation. The impact of these events on the cell-cycle machinery is faster in the CDC25A- degradation cascade which, unlike the slower-operating p53 pathway, does not require the transcription and accumulation of newly synthesized proteins. Thus, the CHK1/CHK2–CDC25A checkpoint is implemented rapidly, independently of 712 15 Cell-Cycle Control by External Signaling Pathways

p53, and delays the G1/S transition only for a few hours unless the sustained p53- dependent mechanism prolongs the G1 arrest.

15.6.3

The G2/M Checkpoint

The G2/M checkpoint prevents cells from initiating mitosis when they experience DNA damage during G2, or when they progress into G2 with some unrepaired ﬂ damage in icted during previous S or G1 phases. The critical target of the G2 checkpoint is the activity of the cyclin B/CDK1 kinase, the activation of which after various stresses is inhibited by ATM/ATR, CHK1/CHK2-mediated subcellular sequestration, degradation, and/or inhibition of the phosphatases CDC25B/C that

normally activate CDK1 at the G2/M boundary. In addition, other upstream regulators of CDC25B/C and/or cyclin B/CDK1, such as the Polo-like kinases PLK3 and PLK1 seem to be targeted by DNA damage- induced mechanisms.

Questions 15.1. What are the major external controls of the cell cycle? Describe at least one pathway that promotes cell-cycle progression. 15.2. Which major pathways can relay proliferation-promoting signals into the cell cycle? 15.3. Name at least one pathway that transmits anti-proliferative signals? Name the major signaling components of this pathway. 15.4. Describe the major structural characteristics of CDKs. What are the major regulatory phosphorylation sites, and which protein kinases are responsible for these phosphorylations? 15.5. Describe the concentration changes of the mammalian cyclins during the cell cycle. Which processes shape these concentration changes? 15.6. Which are the major classes of CKIs? Name the major processes that control CKI concentration and activity. 15.7. Which signals have a negative effect on CDK activity? 15.8. Describe the major structural features of the SCF complex. Name at least two substrates of SCF and explain the consequences of the Ub-mediated degradation of these proteins. 15.9. Describe the order of events – with the major components – allowing

transition from G1 into S. 15.10. How are the activating E2Fs regulated? Name the major target genes of these E2Fs. References 713

! 15.11. Describe the structure of Rb and its function in G1 S progression. 15.12. What are the major components of the DNA damage checkpoints, and how do these proteins convey signals to the cell cycle?

15.13. Describe the reactions operating in the G1 DNA damage checkpoint.

References

1 Lu, Z. and Hunter, T. (2010) Ubiquitylation 10 Klein, E.A. and Assoian, R.K. (2008) and proteasomal degradation of the p21 Transcriptional regulation of the cyclin D1 (Cip1), p27(Kip1) and p57(Kip2) CDK gene at a glance. J. Cell Sci., 121 (Pt 23), inhibitors. Cell Cycle, 9 (12), 2342–2352. 3853–3857. PubMed PMID: 19020303. PubMed PMID: 20519948. Pubmed Central 11 Massague, J. (2004) G1 cell-cycle control and PMCID: PMC3319752. Epub 2010/06/04. cancer. Nature, 432 (7015), 298–306. eng. PubMed PMID: 15549091. Epub 2004/11/ 2 Huse, M. and Kuriyan, J. (2002) The 19. eng. conformational plasticity of protein kinases. 12 Li, C. and Jin, J. (2010) DNA replication Cell, 109 (3), 275–282. PubMed PMID: licensing control and rereplication 12015977. Epub 2002/05/23. eng. prevention. Protein Cell, 1 (3), 227–236. 3 Mocciaro, A. and Rape, M. (2012) Emerging PubMed PMID: 21203969. Epub 2011/01/ regulatory mechanisms in ubiquitin- 05. eng. dependent cell cycle control. J. Cell Sci., 125 13 Qiao, X., Zhang, L., Gamper, A.M., Fujita, (Pt 2), 255–263. PubMed PMID: 22357967. T., and Wan, Y. (2010) APC/C-Cdh1: from Pubmed Central PMCID: 3283867. cell cycle to cellular differentiation and 4 Skaar, J.R. and Pagano, M. (2009) Control of genomic integrity. Cell Cycle, 9 (19), 3904– cell growth by the SCF and APC/C ubiquitin 3912. PubMed PMID: 20935501. Pubmed ligases. Curr. Opin. Cell Biol., 21 (6), 816– Central PMCID: PMC3047751. Epub 2010/ 824. PubMed PMID: 19775879. Pubmed 10/12. eng. Central PMCID: 2805079. 14 Giglia-Mari, G., Zotter, A., and Vermeulen, 5 Henley, S.A. and Dick, F.A. (2012) The W. (2011) DNA damage response. Cold retinoblastoma family of proteins and their Spring Harb. Perspect. Biol., 3 (1), a000745. regulatory functions in the mammalian cell PubMed PMID: 20980439. Pubmed Central division cycle. Cell Div., 7 (1), 10. PMCID: PMC3003462. Epub 2010/10/29. 6 Poznic, M. (2009) Retinoblastoma protein: a eng. central processing unit. J. Biosci., 34 (2), 15 Smith, J., Tho, L.M., Xu, N., and Gillespie, 305–312. D.A. (2010) The ATM-Chk2 and ATR-Chk1 7 van denHeuvel, S. and Dyson, N.J. (2008) pathways in DNA damage signaling and Conserved functions of the pRB and E2F cancer. Adv. Cancer Res., 108,73–112. families. Nat. Rev. Mol. Cell. Biol., 9 (9), 713– PubMed PMID: 21034966. Epub 2010/11/ 724. PubMed PMID: 18719710. 03. eng. 8 Kim, J.K. and Diehl, J.A. (2009) Nuclear 16 Bensimon, A., Aebersold, R., and Shiloh, Y. cyclin D1: an oncogenic driver in human (2011) Beyond ATM: the protein kinase cancer. J. Cell Physiol., 220 (2), 292–296. landscape of the DNA damage response. PubMed PMID: 19415697. Pubmed Central FEBS Lett., 585 (11), 1625–1639. PubMed PMCID: 2874239. PMID: 21570395. Epub 2011/05/17. eng. 9 Witzel, I.I., Koh, L.F., and Perkins, N.D. 17 Langerak, P. and Russell, P. (2011) (2010) Regulation of cyclin D1 gene Regulatory networks integrating cell cycle expression. Biochem. Soc. Trans., control with DNA damage checkpoints and 38 (Pt 1), 217–222. PubMed PMID: double-strand break repair. Philos. Trans. 20074063. R. Soc. Lond. B Biol. Sci., 366 (1584), 714 15 Cell-Cycle Control by External Signaling Pathways

3562–3571. PubMed PMID: 22084383. 19 Reinhardt, H.C. and Yaffe, M.B. (2009) Pubmed Central PMCID: PMC3203453. Kinases that control the cell cycle in Epub 2011/11/16. eng. response to DNA damage: Chk1, Chk2, and 18 Nam, E.A. and Cortez, D. (2011) ATR MK2. Curr. Opin. Cell Biol., 21 (2), 245–255. signalling: more than meeting at the fork. PubMed PMID: 19230643. Pubmed Central Biochem. J., 436 (3), 527–536. PubMed PMCID: 2699687. PMID: 21615334. 715

16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

16.1 Basic Characteristics of Tumor Cells

It is now generally accepted that cancer is a genetic disease affecting primarily the genes that control cell proliferation. The unrestrained proliferation of cancer cells following from these mutations ﬁnally allows cancer cells to invade beyond normal tissue boundaries and metastasize to distant organs, with the accumulation of a large number of mutations distributed over the whole genome. All cancers are thought to arise by an evolutionary process that has the characteristics of a Darwinian evolution, leading from a single clone to the full-grown cancer in a multistep process. This evolution occurs within the microcosm provided by the tissues of a multicellular organism. Analogous to Darwinian evolution occurring in the origins of species, cancer development is based on two constituent processes: mutation and selection. The continuous acquisition of heritable genetic variation in individual cells by more-or-less random mutation leads to heterogeneous cell populations which adapt to different microenvironments through selection of the most suitable variants. The selection may eliminate cells that have acquired deleterious mutations, or it may allow the survival of cells carrying mutations that confer a proliferative advantage and survive more effectively than their neighbors. Eventually, a single cell acquires a set of mutations that allows it to proliferate autonomously, to invade tissues, and to metastasize.

16.2 Mutations in Cancer Cells

Summary Cancers arise by an evolutionary process that is driven by mutation and selection, analogous to Darwinian evolution. The mutations found in cancer cells can be classified as inherited or somatic mutations. How many and which type of somatic mutations accumulate depends on endogenous and exogenous exposures, and on the repair capabilities of the cell. Typically, cancer cells acquire

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 716 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

mutations at a higher rate as compared to normal cells, which leads to the accumulation of a large number of genetic changes in the cancer cell. The more- or-less random mutations are inherited to the daughter cells, which results in a large heterogeneity within the cancer cell population. The genomic mutations comprise small- and large-scale mutations, and often become visible as genomic instability and gross changes in chromosome numbers and structures. In addition to the genomic changes, epigenetic changes may drive cancer evolution. Heritable changes in DNA methylation, chromatin structure and miRNA expression have been shown to shape the phenotype of cancer cells. Genes that are key to the development of cancer cells are also called “cancer genes.” At least 400 genes have been classified as cancer genes, and these can be further categorized as oncogenes and tumor suppressor genes.

The spectrum of mutations found in cancer cells contains inherited mutations and somatic mutations acquired during development in utero and the replenish- ment of body tissues in postnatal life. Additional mutations accumulate during the evolution from the cancer cell clone to the fully grown tumor. The rate of acquisition and the type of somatic mutations that accrue depend to a large part on exogenous and endogenous exposures that cause DNA damage. To what extent a DNA damage becomes manifest as a somatic mutation depends on the efficiency of the DNA repair systems. It is generally assumed that cancer cells have impaired DNA repair capabilities, and consequently exhibit an increased mutation rate. Whether a mature cancer clone emerges in an individual person is also influenced by the set of genomic sequence variants present in the fertilized egg from which the individual develops, and that are therefore found in all somatic cells. These “germline” mutations may lead to an inherited predisposal for tumor formation. The inherited mutations can influence cancer susceptibility in a number of ways, including directly altering the growth of the cancer clone, altering the mutation rate in somatic cells, or modulating the metabolism of carcinogens.

16.2.1 Genetic Changes in Cancer Cells

Compared to the normal progenitor cell, tumor cells harbor a large number of mutations. Cytogenetic studies, analyses of copy number changes, the sequencing of selected protein-coding genes and whole genome sequencing have revealed a broad spectrum of mutations in cancer cells that differ markedly between individual cancers. It is now known that the genomes of most adult cancers usually harbor between 1000 and 10 000 somatic substitutions or mutations. Moreover, some cancer types, such as leukemias, have very few substitutions whereas other cancers, such as lung cancers, occasionally carry more than 100 000 mutations.

& Tumor cells harbor a large number of different genetic changes 16.2 Mutations in Cancer Cells 717

The multiple mutations observed in tumors include a broad spectrum of reorganizations and changes in genetic information observable on both small and large scales.

16.2.1.1 Small-Scale Changes These mutations include:

simple base substitutions; insertion or deletion of bases; and inversion, duplication or deletion of DNA sequences.

16.2.1.2 Large-Scale Changes and Genetic Instability Especially in the later phases of tumor formation, an increasing genetic instability is observed. There are various forms of genomic instability; most cancers have a form that is termed chromosomal instability (CIN), which refers to the high rate by which chromosome structures and numbers change over time in cancer cells compared to normal cells. Other forms of genomic instability include microsatellite instability (MSI), and forms of genomic instability that are characterized by increased frequencies of base-pair mutations. The latter types of genomic instability have been linked to mutations in DNA repair genes. CIN is visible particularly as a change in chromosome structure and number, such as:

the loss or duplication of whole chromosomes; multiplication of the chromosome set; chromosome translocations: deletion, addition or exchange of individual chromosomes; and ampliﬁcation of DNA sequences: changes in copy number of genes or larger chromosome sections.

Changes in the chromosome structure are often observed in tumors of the blood- forming system, the leukemias and lymphomas, and are almost always found in the later phases of aggressive solid tumors. There is one example where an apparently catastrophic event leads to the shattering of a chromosome and rejoining of the chromosome fragments [1]. These extensive reorganizations have far-reaching consequences for growth behavior and functional performance. However, the mechanisms that trigger genomic instability are only poorly characterized.

16.2.2 Epigenetic Changes in Tumor Cells

As outlined in Section 4.5, epigenetic mechanisms are essential for tissue-speciﬁc gene-expression patterns. Which part of the genome is available for transcription at 718 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

a given developmental stage is determined by the cooperation of diverse epigenetic mechanisms that create heritable changes in the gene expression patterns of cells. Most of these heritable changes are established during differentiation, and are stably maintained through multiple cycles of cell division, enabling cells to have distinct identities while containing the same genetic information. Currently, three major systems are known that are involved in epigenetic control:

DNA methylation (see Section 4.5.8) Histone and chromatin modiﬁcation (see Section 4.5) RNA-associated silencing: micro RNAs (see Section 5.3).

Malfunction of the epigenetic controls can result in the dysregulation of gene expression profiles, leading to the development and progression of disease states, including cancer. It is now well established that the initiation and progression of cancer involves epigenetic abnormalities along with genetic alterations [2]. Research investigations into the epigenetics of cancer cells have provided much data relating to cancer cell-specific patterns of global DNA methylation, global histone and chromatin modifications, and protein-binding to gene-regulatory regions. These studies have revealed numerous epigenetic alterations in cancer cells that can be considered as alternatives to mutations and chromosomal alterations in disrupting the gene functions that are essential for cell proliferation and homeostasis.

& Epigenetic changes in tumor cells: — Altered DNA methylation — Altered histone and chromatin modification — Altered miRNA expression.

The epigenetic changes in cancer cells include global DNA hypomethylation, the hypermethylation and hypomethylation of specific genes, loss of imprinting, alterations in the modification and positioning of histones, and changes in the expression of small regulatory RNAs. Each of these can lead to aberrant malfunctions of genes critical for tumor initiation and progression [3]. Accordingly, epigenetic changes have been identified as putative cancer biomarkers for early detection, disease monitoring, prognosis, and risk assessment. One critical question in cancer epigenetics is whether these changes are the cause of cell transformation, or rather the consequence of it. This question cannot be answered yet and the interplay between mutagenic changes in key cancer genes and changes in epigenetics is not really understood.

16.2.2.1 Altered DNA Methylation Methylation at CpG sequences is a major tool for controlling gene expression operating via changes in chromatin structure (see Section 4.5.9). To date, all tumors examined – whether malignant or benign – have shown two major altered patterns 16.2 Mutations in Cancer Cells 719 of DNA methylation, namely a global DNA hypomethylation and hypermethylation of CpG islands at promoter regions. Global hypomethylation – that is, a loss of methylcytosines – has been clearly correlated with different stages of cancer progression and metastasis in various tumor types. The potential cellular consequences of global hypomethylation are diverse, ranging from chromosome instability and genetic mutation to the reactivation of various cancer-related genes. In the same cancer cells that harbor genome-wide DNA hypomethylation, hundreds of genes simultaneously exhibit DNA hypermethylation of the promoter CpG islands, which is associated with stable states of transcriptional silencing. Examples of tumor suppressor genes inactivated by CpG hypermethylation include the genes for the Rb protein, for p14ARF, for APC, and for the BRCA protein (see later sections of this chapter). In many tumors, a loss of function of tumor suppressor genes can only be explained by hypermethylation of the promoters, as no changes in DNA sequence could be found in these genes. Other genes whose function is disrupted by aberrant DNA methylation in tumors include repair enzymes and cell-cycle regulators. Changes in DNA methylation can also lead to the activation of oncogenes. In line with the altered methylation patterns in cancer cells is the identiﬁcation of the gene encoding DNA methyltransferase 1 (DNMT1) as an oncogene that is frequently mutated in cancer cells. Some examples of the hypermethylation of genes linked to tumor formation are listed in Table 16.1.

Table 16.1 Genes that are frequently mutated and/or hypermethylated in cancer (only subsets of genes that are known to be frequently silenced in one or more cancer types are shown).

Gene Function p14, p15 CDK inhibitors

PTEN PtdInsP3 phosphatase MLH1 DNA mismatch repair APC Wnt signaling CDH-1 Cell adhesion, loss during tumor metastasis NF1 GAP involved in Ras signaling TIMP3 Tissue inhibitor of metalloproteinase 3 BRCA1 DNA repair STK11 Serine/threonine kinase 11, mutated in hereditary cancers MGMT DNA repair

Abbreviations and links: p14, p15, CDK inhibitors of Ink family (see Section 13.2.6); PTEN see Section 6.6.3; MLH1, mutL homolog 1, see Section 14.5; APC, adenomatosis polyposis coli, see Section 14.9; CDH1, E-cadherin cell adhesion protein, see Section 14.9; NF1, neuroﬁbromin 1, GTPase-activating protein, see Section 9.5; TIMP3, tissue inhibitor of metalloproteinase 3; BRCA1, breast cancer 1, early onset, see Section 14.5; STK11, serine/threonine kinase 11; MGMT, O6- methylguanine-DNA methyltransferase. 720 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

16.2.2.2 Altered Histone Modifications in Cancer A large number of histone posttranslational modifications (PTMs) have been identified that either activate or inactivate gene transcription (Section 4.5). The modification pattern of the core histones dictates to a large extent the transcriptional activity of genes, and these modifications have been shown to survive mitosis and have been implicated in “chromatin memory.” Global changes in the methylation and acetylation patterns of chromatin are general features of cancer cells, and many genes involved in histone and chromatin modification have now been identified as genes frequently mutated in cancer cells [4]. Examples include the genes for the histone H3 K36 methylase SETD2, the H3 lysine demethylases KDM6A and KDM5C, and components of protein complexes that restructure chromatin. These observations highlight a potentially important link between somatic mutation and epigenetic abnormalities that are present in many cancers.

16.2.2.3 Micro RNAs and Cancer Micro RNAs (miRNAs) are known to repress thousands of target genes and coordinate normal processes, including cellular proliferation, differentiation, apoptosis and stress response (Section 5.3). The aberrant expression or alteration of miRNAs also contributes to a range of human pathologies, including cancer. There is now abundant evidence that the altered expression and activity of miRNAs contributes to the initiation and progression of cancer [5]. The strong association of miRNAs with cancer is supported by the following observations:

The genes for miRNAs are often located in small chromosomal alterations (ampliﬁcations, deletions) in tumors. miRNA genes have been found in chromosomal breakpoints associated with cancer. miRNA transcription can be silenced by the aberrant methylation of CpG islands at promoters and by the loss of histone acetylation.

Overall, the global changes in miRNA expression that are seen in tumors are thought to arise through multiple mechanisms (for details, see Ref. [5]). miRNAs typically function through the repression of target genes. This repressive function can convey either oncogenic or tumor-suppressing properties on miRNAs: tumor-suppressor miRNAs can negatively regulate protein-coding oncogenes, whereas oncogenic miRNAs often repress known tumor suppressors. Alterations in miRNA expression can therefore lead to an accumulation of oncogenes or the downregulation of tumor suppressor genes (Figure 16.1). As shown in Table 16.1, the altered pattern of miRNA expression observed in many tumors mirrors the deregulation for critical protein-coding oncogenes or tumor suppressor genes. Overall, miRNAs form part of the network of oncogenes and tumor suppressor genes in cancer cells, and have both direct and indirect links to the many components of this network 16.2 Mutations in Cancer Cells 721

Figure 16.1 Cellular targets and functions of metabolism (GLS, L-glutaminase). miRNAs Myc-regulated miRNAs. The oncogenic that repress oncogenic proteins therefore transcription factor Myc has been shown to function as tumor suppressor miRNAs. Myc repress miRNAs (in blue) that negatively also can activate miRNAs (in red) that regulate the function of proteins involved in negatively regulate proteins with tumor- cell survival and proliferation (Bcl-2, suppressing function such as PTEN (Sec- Section 17.3; CDK6, Section 15.2.1; CCND2, tion 8.6). When deregulated, these miRNAs cyclin D2, Section 15.2.2, KRas, Section 11.5), can have oncogenic functions. Note that Myc epigenetic modifications (EZH2, member of can repress let-7, either directly or indirectly, the Polycomb group repressive complex, PcG; through LIN28 activation. Conversely, let-7 can Section 3.5.5; DNMT3, DNA methyl also repress MYC, which closes a regulatory transferase 3, Section 4.5.9) and cellular circle. Reproduced from Ref. [6].

An example of the intricate link between miRNAs and cancer genes is provided by the transcription factor Myc, a prominent oncogene that is activated in many tumors (see Section 16.4.2.5). In addition to regulating a large number of protein- coding genes, Myc regulates the transcription of many miRNA genes which allows for the control and orchestration of signaling pathways involved in cell survival, proliferation, metabolism, and epigenetic modiﬁcations (see Figure 16.1).

16.2.3 Cancer Genes: Drivers and Passengers

The large number of somatic mutations found in cancer cells raises the question about the contribution of particular mutations for tumor initiation and progression. Each somatic mutation in a cancer cell genome, whatever its structural nature, may be classiﬁed according to its consequences for cancer development. In a tumor progenitor cell, a subset of mutations termed driver mutations will eventually fall in a set of genes, called cancer genes, that are key to the control of cell proliferation, differentiation, death, and communication with the tissue microenvironment [4]. 722 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

The “driver mutations” are positively selected during cancer evolution, and they confer a growth advantage on the tumor progenitor, allowing it to expand much more than normal cells from the same tissue, and finally to develop all the properties of a full-grown tumor. The remainder of mutations are “passengers” that do not confer growth advantage, but simply happened to be present in an ancestor of the cancer cell when it acquired one of its drivers. Thus far, the large majority of base substitutions in most cancer genomes appear to be passengers which, by definition, do not confer any growth advantage. The number of driver mutations – and hence the number of abnormal cancer genes – in an individual cancer is not well established. Varied approaches have identified approximately 400 somatically mutated cancer genes that contribute to neoplastic change in one or more types of cancer. Although this number corresponds to about 2% of the protein-coding genes in the human genome, it may have been underestimated and there may be many more drivers than can be unambiguously identified by using current methods.

16.2.3.1 Oncogenes Versus Tumor Suppressor Genes A common classiﬁcation of cancer genes is based on whether they function in a dominant or recessive manner at the level of the cancer cell. Dominant cancer genes require only one of the two parental alleles present in a normal cell to be mutated, whereas recessive cancer genes require the mutation of both parental alleles. Dominant cancer genes are usually constitutively activated by mutation and are called oncogenes, and these now comprise more than 80% of the currently known cancer genes. Recessive cancer genes are also termed tumor suppressor genes, and the mutation of these genes typically results in an inactivation of the encoded protein – that is, a loss of function. Examples of prominent oncogenes and tumor suppressor genes are presented separately in Sections 16.4 and 16.5.

16.2.4 Carcinogenesis as an Evolutionary Process

It is now generally accepted that cancer development is the outcome of a process of Darwinian evolution occurring among cell populations within the microenvironments provided by the tissues of a multicellular organism. Analogous to Darwinian evolution occurring in the origins of species, cancer development involves two basic processes:

More-or-less random mutation results in the continuous acquisition of heritable genetic variation in individual cells, creating a pool of genetically divergent cells. The resultant phenotypic diversity is subject to selection imposed by the microenvironment or chemotherapy. The selection may weed out cells that have acquired deleterious mutations, or it may foster cells carrying alterations that confer the capability to proliferate and survive more effectively than their 16.2 Mutations in Cancer Cells 723

neighbors. Occasionally, a single cell acquires a set of advantageous mutations that allows it to proliferate autonomously, to invade tissues, and to metastasize.

The evolution of a pre-cancerous cell to a fully grown, aggressive tumor includes the accumulation of up to 100 000 genetic and epigenetic alterations within the cells that constitute the tumor. As a result, the tumor exhibits common physiologic changes that can be described by the hallmarks summarized in Section 16.3. Due to the large number of genetic alterations, each particular tumor will show a characteristic pattern of mutations and will be composed of genetically divergent cells.

& Characteristics of tumor cells: — High mutation rate — Genetic instability — Epigenetic alterations — Dysfunction of cell-cycle checkpoints and/or repair enzymes — Growth advantage — Independence of mitogenic signals — Loss of contact inhibition — Survival in foreign tissues.

The processes that initiate, contribute to and propagate the malignant phenotype can be summarized as follows (Figure 16.2).

Initiation of the cancer process: In an initial step, pre-cancerous cells are converted into cancer-prone cells due to mutations caused by exposure to intrinsic or extrinsic DNA damage. These mutations will also affect the key components of DNA repair and DNA damage checkpoints, leading to an increased accumulation of further genetic changes. The cells accumulate mutations at a higher rate than do normal cells expressing a mutator phenotype (reviewed in Ref. [8]). As a consequence, the pool of cells that are prone to further tumor progression is enlarged. Furthermore, the malfunction of DNA repair and the checkpoints that couple DNA damage to cell cycle arrest and apoptosis allow for heritable chromosomal instability. Epigenetic changes in tumor progenitor cells have been also postulated as early events in tumorigenesis, as these can serve as surrogates for genetic changes and can lead to aberrant regulation of genes involved in cell proliferation and homeostasis. Given that some epigenetic alterations behave as heritable traits, cancer evolution is also shaped by heritable epigenetic differences. Growth advantage, selection and clonal expansion of tumor cells: According to a model of clonal succession, somatic evolution in cancer proceeds as a stepwise series of clonal expansions. This process is triggered by the acquisition of driver mutations that confer strong ﬁtness gain and cause clonal homogenization, as the more advanced clone out-competes its less ﬁt parental and sister clones. However, the scenario of linear clonal succession is now replaced by models of 724 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.2 Model of the transition of a the immune system and from chemotherapy normal cell into a heterogeneous population of will ultimately lead to the appearance of tumor tumor cells. The continuous accumulation of cells that have acquired the whole set of mutations leads to a population of tumor cells tumor hallmarks as described in Ref. [7]. The composed of multiple subclones with sequence of events shown is hypothetical and divergent tumorigenic potential. Selection may depend on the type of the tumor cell and pressure from the cellular environment, from its progenitor. 16.3 Common Physiologic Changes in Tumor Cells: The Hallmarks of Cancer 725

complex subclonal architectures where increased mutation rates and enhanced genetic and epigenetic plasticity creates a pool of tumor cells composed of a large number of subclones. These variants are able to coexist and provide a repertoire of cells that may respond in a plastic way to changes in selection pressures. At this stage, the tumor is only clonal in the sense that it was originally derived from a single progenitor cell. The pool of tumor cell variants must be considered as a highly flexible collection of different cells with the potential for future changes in the presence of selective pressures. This heterogeneous and dynamic population is the target of selection for cells that carry driver mutations conferring a competitive advantage of proliferation and growth in the surroundings of the progenitor cell. Formation of solid tumors and metastasis: Eventually, subclones will appear that have the ability to proliferate independently of signals from the neighboring cells and have lost contact inhibition. Thus, in a late stage of tumorigenesis, tumor cells can acquire the potential to survive in a foreign cellular environment and form organ-like structures. A solid tumor frequently measures 1 cm3 and encompasses between 108 and 109 cells, each containing tens of thousands of clonal, subclonal, and random mutations. The genetic heterogeneity within the tumor is accompanied by a functional heterogeneity of the individual cells that carry selected alterations, for example, altered metabolism and the ability to form blood vessels, along with the instability mutations. The coexistence of genetically diverged clonal subpopulations along with heterogeneous protein function is thought to foster tumor adaptation and therapeutic failure. This process is essentially Darwinian: genetically and epigenetically diversified heritable phenotypes are tested by selection, which causes the preferential outgrowth of clones with higher-than-average fitness. Evolution of the tumor in a later stage appears to proceed in a branched manner: the subclones within the tumor show different patterns of mutated oncogenes and tumor suppressor genes. In any cell of the tumor, none of the genes is invariably mutated and there is no set of mutated genes that are diagnostic of a specific tumor. Similar to evolutionary processes in natural populations, tumor evolution is characterized by complex dynamics that produces unique and unpredictable patterns of clonal architecture.

Although there is general agreement that epigenetic and genetic changes are key steps in tumor initiation, controversy persists concerning the timing and the nature of the initial tumor-causing events, and the sequential order of the above- mentioned events is still uncertain. Due to the clonal heterogeneity of cancer cell populations, the ability to metastasize may be already present in subclones at early stages of tumor progression.

16.3 Common Physiologic Changes in Tumor Cells: The Hallmarks of Cancer

There are more than 100 distinct types of cancer, and subtypes of cancers can be found within speciﬁc organs. The vast catalog of cancer cell genotypes has been 726 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.3 Acquired capabilities of cancers. Hanahan 2000 [9], figure 1. Reproduced with permission of Elsevier.

suggested to be a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth [9] (Figure 16.3):

Self-sufﬁciency in growth signals Insensitivity to growth-inhibitory (antigrowth) signals Evasion of programmed cell death (apoptosis) Limitless replicative potential Sustained angiogenesis Tissue invasion and metastasis.

This list has now been expanded by two further hallmarks [7]:

Reprogramming of energy metabolism Evasion of immune destruction.

Each of these physiologic changes represents the successful breaching of an anticancer defense mechanism hardwired into cells and tissues, and is the result of a long selection process. The linkage of the hallmarks of cancer to the dysregulation of signaling pathways and cell-cycle checkpoints will be discussed brieﬂy in the following subsections for the ﬁrst three of the above-mentioned hallmarks of cancer. This subset of hallmark capabilities belongs to the core hallmarks of cancer cells, and it involves well-established linkages to the signaling pathways presented 16.3 Common Physiologic Changes in Tumor Cells: The Hallmarks of Cancer 727 in this book. Much progress has been achieved over the past decade in the characterization of the other distinguishing hallmarks of cancer cells. However, these complicated topics are beyond the scope of this book and the reader is referred to Ref. [7] for more detailed information.

16.3.1 Self-Sufficiency in Growth Signals

Normal cells require mitogenic growth signals before they can progress through G1 into S or move from a quiescent state into an active proliferative state. The pathways that transmit mitogenic signals are manifold and have been outlined in Chapters 10–14. Of outstanding importance for proliferation regulation are the pathways that lead from the cell surface to the cell cycle. There are many examples that cancer cells acquire a state that is no longer dependent on external mitogenic signals mediated by these pathways. Tumor cells can acquire the ability to induce and sustain proliferative signaling in a number of ways:

Autocrine stimulation: Cancer cells may produce growth factor ligands themselves, to which they respond by expressing the cognate receptor. This creates a positive feedback signaling loop called autocrine stimulation (Figure 16.4). Alternatively, cancer cells may stimulate the tumor-associated stroma cells to supply the cancer cells with growth factors.

Figure 16.4 Autocrine loops in tumor factor can then bind and activate a mitogenic formation. Due to an error in the control of signal chain. In this situation, the cell creates transcription, growth factors may be produced the mitogenic signal itself. There is evidence and secreted in the cell which would normally that the growth factors can become active only be formed in low concentrations, or not at intracellularly. The mechanism behind this is all. If the cell also possesses the receptors unknown. corresponding to the growth factor, the growth 728 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Aberrant activation of transmembrane receptors: Growth factor receptors with intrinsic or associated tyrosine kinase activity are overexpressed in many tumors, often eliciting ligand-independent signaling. The latter can also be achieved through structural alteration of receptors; for example, truncated versions of the EGF receptor lacking part of its cytoplasmic domain ﬁre constitutively. Aberrant activation of intracellular signaling networks: The constitutive activation of components of signaling pathways operating downstream of growth factor receptors may obviate the need to stimulate these pathways by ligand- mediated receptor activation. Alterations of the intracellular proliferation- promoting signaling paths may affect both the activating components as well as the components that have a dampening or braking effect on signal transmission. Here, a central role is ascribed to the Sos–Ras–MAPK pathways (Chapters 11 and 12), the integrin pathways (Section 12.4) and the PI3 kinase pathway (Section 9.4). Constitutive activation of these pathways is now recognized as a classical signature of many cancer types. Furthermore, the importance of negative feedback loops for proliferation control is increasingly recognized. Such negative feedback loops normally operate to dampen various types of signaling, and thereby ensure a homeostatic regulation of the ﬂux of signals transmitted within the intracellular circuits. Defects in these feedback mechanisms are capable of enhancing proliferative signaling. Examples include the tumorigenic mutation of Ras protein (Chapter 11) compromising its GTPAse activity, and the PTEN phosphatase that functions as brake in proliferative signaling through the PI3K/ Akt pathway (Section 9.4).

16.3.2 Insensitivity to Antigrowth Signals

Within a normal tissue, multiple antiproliferative signals operate to maintain cellular quiescence and tissue homeostasis; these signals include both soluble growth inhibitors and immobilized inhibitors embedded in the cell membrane. These growth-inhibitory signals, much like their positively acting counterparts, are received by transmembrane cell-surface receptors coupled to intracellular signaling networks. Cancer cells must circumvent the powerful programs that negatively regulate cell proliferation. Accordingly, many of these programs depend on the actions of cancer genes that are classiﬁed as tumor suppressor genes. Of the many known tumor suppressors, the Rb and p53 proteins play a central role in controlling cell proliferation or, alternatively, activating senescence and apoptotic programs. As outlined in Section 15.4.3, Rb, when in the underphosphorylated state, blocks proliferation by shutting down the E2F-dependent expression of genes essential for

progression from G1 into S phase. Many of the antimitogenic signals that keep Rb in the active, hypophosphorylated state are mediated by the TGFb-pathway (see Section 14.1) that has been found to be disrupted in a variety of ways in different types of human tumors. The TGFb-pathway leads – among others – to an increased 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 729 expression of the gene for the inhibitor p15INK4B and the subsequent inhibition of the G1 CDKs (CDk4/6, CDK2) that are responsible for inactivation of Rb (Section 14.4.7). Whereas, Rb transduces growth-inhibitory signals that originate largely outside of the cell, p53 receives inputs from stress and abnormality sensors that function within the cell’s intracellular operating systems (Section 16.6). These inputs can activate p53 to halt further cell-cycle progression until these conditions have been normalized. Alternatively, in the face of alarm signals indicating overwhelming or irreparable damage, p53 can trigger apoptosis.

16.3.3 Evasion of Programmed Cell Death (Apoptosis)

The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation but also by the rate of programmed cell death (apoptosis) (Chapter 17). It is now well established that apoptosis is a major barrier to cancer that must be circumvented, and an acquired resistance towards apoptosis is a hallmark of most and perhaps all types of cancer. Tumor cells evolve a variety of strategies to limit or circumvent apoptosis. Most common is the loss of p53 tumor suppressor function, which eliminates this critical damage sensor from the apoptosis-inducing circuitry. Alternatively, tumors may achieve similar ends by increasing expression of antiapoptotic regulators (Bcl-2, Bcl-xL) or of survival signals (IGF1/2), by downregulating proapoptotic factors (Bax, Bim, Puma), or by short-circuiting the extrinsic ligand-induced death pathway (Chapter 17).

16.4 Signaling Proteins Mutated in Cancer: Oncogenes

Summary Cancer genes classified as oncogenes generally have a dominant character. The mutation of a proto-oncogene to an oncogene is phenotypically visible when only one of the two copies of the gene in a diploid chromosome set is affected by the mutation. The dominant mutation is accompanied by a “gain of function”;it typically amplifies or increases the yield of a function in growth regulation. There are various mechanisms by which a gene can acquire oncogenic properties during cancer evolution. Typically, function and/or regulation of the affected protein (or miRNA) is compromised by oncogenic mutations. Furthermore, epigenetic changes also contribute to the activation of oncogenes. The structural changes that lead to the activation of oncogenes may affect the activity, regulatory properties, stability, subcellular location and protein interactions of the encoded protein. Other mechanisms of oncogene activation include the formation of oncogenic fusion proteins and gene amplification. Nearly all crucial 730 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

regulators of the pathways controlling cell proliferation, DNA repair and apoptosis can be activated to oncogenes. Examples include RTKs, non-RTks, Ras proteins, cyclins, repair proteins, and transcription factors.

& Dysregulation of cell proliferation: — Increased mitogenic signaling: activation of oncogenes — Decreased anti-mitogenic signaling: inactivation of tumor suppressor genes.

& Oncogenes: — Arise by activating mutations from proto-oncogenes — Enhance mitogenic signaling — Have a dominant character.

Oncogenes have been identified historically as genes involved in retroviral tumorigenesis, and can result in a transforming or immortalizing phenotype on experimental transformation in cellular model systems. These genes arise by activating a mutation of their precursors, the proto-oncogenes, and typically lead to a gain of function in signaling pathways linked to tumorigenesis. Many retroviruses carry mutated versions of cellular proto-oncogenes; in this case the viral oncogenes are prefixed with a v (e.g., v-src, v-sis), whilst the corresponding proto- oncogenes are prefixed with a c (e.g., c-src, c-sis).

16.4.1 Mechanisms of Oncogene Activation

& Oncogenes are activated by: — Epigenetic changes — Concentration increase — Structural mutation — Formation of hybrid proteins.

Two pathways of oncogenic activation can be roughly differentiated (Figure 16.5). On the one hand, the structure of the coded protein may be affected; on the other hand, activation may lead to an increase in the concentration of the protein.

16.4.1.1 Oncogenic Activation by Structural Changes The spectrum of structural mutations that can confer oncogenic properties on a protein is very diverse and includes simple amino acid substitutions and larger 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 731

Figure 16.5 Mechanism of activation of proto- and unprogrammed function of the signal oncogenes to oncogenes. Proto-oncogenes protein coded by the proto-oncogene. In the may be converted into oncogenes via the case of structural change, the proliferation- concentration increase pathway or the promoting activity of the oncoprotein results structural change pathway. In the case of the from changed activity, altered regulation or concentration increase, there is an excessive formation of a hybrid protein. structural alterations. The function of the affected protein may be altered for example, by changing its:

activity (see Ras protein; Section 11.5); subcellular localization (see Abl tyrosine kinase; Section 10.3.3); interactions with upstream and downstream effectors; and regulatory properties (see Raf kinase; Section 11.6).

The latter point is illustrated by the transforming v-raf gene, where the N- terminal sequence section of Raf kinase is missing, on which both the autoinhibitory function and the phosphorylation sites of Raf kinase are localized.

16.4.1.2 Oncogenic Fusion Proteins In many tumors, a reciprocal exchange of DNA sections on different chromosomes is observed. During this translocation of chromosomes, gene fusions may occur, leading to the formation of chimeric proteins within which there are often structural portions that originate from signal proteins. The function of the signal 732 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

protein portion is removed from normal regulation in the chimeric protein, and can have a tumor-promoting effect. The chimeric proteins arising from chromosome translocation frequently represent a characteristic of a particular tumor type. Often, tyrosine kinases and transcription factors are affected by the gene fusions. One prominent example is the Philadelphia tranlsocation in Burkitt’s lymphoma, where a fusion protein including Abl tyrosine kinase is formed with aberrant kinase activity (Section 10.3.3).

& Oncogenic fusion proteins: — Arise from chromosome translocations — Often comprise a fusion of a transcription factor with a protein kinase.

16.4.1.3 Activation by Protein Concentration Increase A change in the gene expression or stability of a proto-oncogene product may lead to an increase in the cellular concentration of the protein. Because of the increased concentration, a mitogenic signal mediated by a proto-oncogene product may be amplified. The mechanisms leading to increased concentrations of oncogenes are varied, and include all processes that have been discussed in Chapters 2, 4, and 5 as being relevant for protein expression and stability. Most important for increased oncogene concentrations appear to be gene amplification, promoter mutation, epigenetic changes, and alterations in Ub-mediated degradation. The importance of the latter point is shown by the observation that many components of the Ub- modification machinery have been identified as cancer genes.

& Factors contributing to concentration increase of oncogenes: — Promoter mutation — Epigenetic changes — mRNA stabilization — Gene amplification — Reduced Ub-dependent proteolysis.

The activation of proto-oncogenes by unprogrammed expression is often associated with chromosome translocations in leukemias and lymphomas (see Myc protein; Section 16.4.2.5). Another tumor-promoting mechanism based on the deregulation of transcription of growth factors is the formation of autocrine loops. During the course of tumor formation, an unprogrammed expression of growth factors may occur in cells in which there would normally be little or no expression of these proteins. If these cells express the appropriate growth factor receptors, the growth factors may bind to these and create a stimulus of division. As a consequence, the cell is no longer dependent on the supply of an external growth factor and then produces its own growth factor and division stimulus (Figure 16.4). 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 733

16.4.2 Examples of the Functions of Oncogenes

Sequencing of the protein-coding exons of more than 2000 individual cancers worldwide has provided a wealth of information on the distribution of oncogenic mutations among genes assumed to be critical for cancer development. From these data, a first insight has now been gained into the frequency of mutated oncogenes in different cancer types, and the contribution of particular oncogenes for cancer initiation and propagation. A key question in correlating oncogenic mutations with different cancer types relates to a possible oncogene signature in cancer – that is, whether there is a preponderance of mutations of certain cancer genes in a particular cancer type. Surprisingly, it proved difficult to establish a consistent pattern of oncogenic mutations in cancer cells. In fact, massive efforts of high-throughput sequencing of cancer genomes have shown that few genes were mutated, amplified or deleted at high frequencies in various cancer types. Specifically, in each cancer type, about four genes were altered in more than 20% of the tumors analyzed. This appears to be an emerging feature of the landscape of somatically mutated cancer genes, of which a relatively limited set are commonly mutated and a substantial number is mutated infrequently. Among the most frequently mutated genes in cancers are genes for the tumor suppressors p53 and ATM kinase, and for oncogenes encoding the Ras protein, the B-Raf protein kinase, regulatory subunit of PI3 kinase, p110, the non-receptor tyrosine kinases Abl and Src, and the transcription factor Myc. Furthermore, the systematic sequencing of cancer genomes has allowed direct identification of cancer genes through an elevated prevalence of base substitutions and small insertions or deletions. Among these, many dominant oncogenes have been identified that would not have been found by other approaches. Some are on biological pathways previously implicated in cancer development. Others – for example, IDH1, which encodes isocitrate dehydrogenase 1, a component of the Krebs cycle; or FOXL2, which encodes a tissue-specific transcription factor – would not have been expected to feature in oncogene lists. More than 300 dominant oncogenes have been identified to date, for which there is sufficient evidence to support a causal role in sporadic or familial cancer development when mutated. These genes have been altered by several types of genetic alterations, including point mutations, deletions, and rearrangements. Additional insight into the importance of these oncogenes for cancer development was gained from an integrated genome-wide screen of amplification and overexpression of cancer genes in human cancer [10]. This analysis has identified 77 cancer genes for which there is clear evidence for involvement in the development of cancer. Most of the cancer genes identified in this way are involved in pathways known to be important for the regulation of growth and proliferation. As outlined in Section 16.3, alterations of the proliferation-promoting signaling chains often fall into the hallmark signature of sustained proliferation and insensitivity to anti-oncogenic signaling. However, multiple changes in the other 734 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Table 16.2 Examples of oncogene products involved in signal transduction pathways.

Signal transduction pathway Oncogene product

Growth factors: TGFb Transmembrane receptors: Erb2B/Neu Adaptor proteins: Shc, Crk Nonreceptor tyrosine kinases: Src kinase Protein phosphatases: Cdc25A Small regulatory GTPases: Ras protein Cytoplasmic Ser/Thr protein kinases: Raf kinase Lipid phosphatases: PI3 kinase E3 ubiquitin ligases: Cbl Cyclins: Cyclin D1 Transcription factors: Myc, Jun, Fos

hallmark signatures must occur at the same time to allow the evolution of a full cancer phenotype. Selected examples of oncogenic mutations of key components of signaling pathways will be presented in the following subsections in greater detail, highlighting the principles that underlie oncogenic activation. Examples of signaling proteins for which oncogenic mutations have been demonstrated are summarized in Table 16.2.

16.4.2.1 Oncogenic Receptor Tyrosine Kinases: The ErbB2/neu Receptor More than the half of the known receptor tyrosine kinases have been repeatedly found in either mutated or overexpressed forms in human malignancies, including sporadic cancers. Most activating mutations lead to a ligand-independent, constitutive activation of the tyrosine kinase activity of the receptor. As outlined in Chapter 8, receptor tyrosine kinases normally exist in a repressed state and require ligand-induced autophosphorylation for activation. Oncogenic receptor tyrosine kinases often escape from this control and induce an inappropriate activation of downstream signaling components that leads to enhanced cell proliferation and increased cell survival. A well-studied example is provided by the EGF/ErbB2 family of receptor tyrosine kinases. This family comprises four members: EGF receptor, (EGFR, ErbB1), ErbB2 (Her2, Neu), ErbB3 and ErbB4 (reviewed in Ref. [11]). About a dozen ligands are known, including EGF, transforming growth factor a (TGFa), and the neuregulins. Oncogenic activation of members of the EGF/ErbB2 receptor family due to structural mutations, overexpression or gene ampliﬁcation is found in many tumors, and the ERB family receptors are now major targets of speciﬁc antitumor drugs.

& Oncogenic activation of EGF receptor family members: — ErbB2 frequently overexpressed in breast cancers — Leads to enhanced EGF receptor signaling and inappropriate MAPK and PI3 kinase signaling. 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 735

As an example, a significant fraction of lung cancer cases carry mutations in the EGFR. Dependent on the type of lung cancer, these mutations comprise missense mutations and deletions in the ligand-binding domain and the intracellular kinase and regulatory domains, along with amplification of the mutant genes. Interest- ingly, certain mutations within the kinase domain are strongly correlated with the resistance of tumor growth to the antitumor drug gefitinib, a small-protein kinase inhibitor [12]. ErbB2 is another member of this family that is found to be oncogenically activated in many cancers, especially in breast cancer. The gene for ERbB2 is amplified or overexpressed up to 100-fold in almost 30% of all breast cancer cases, and the extent of amplification and overexpression correlates with a poor prognosis. No external ligand is known for ErbB2, and heterodimerization with the three other receptors of the EGF/ErbB2 family is required for ErbB2 signaling. Overexpression of ErbB2 in cancer cells disturbs the complex network of receptor interactions and induces the formation of ErbB2-containing heterodimers. These have a potent oncogenic effect due to an inappropriate activation of mitogenic and survival pathways, such as the MAPK pathways and the PI3 kinase pathway. One approved therapy of breast cancer uses a humanized antibody named trastuzumab (Herceptin) that is directed against the extracellular domain of ErbB2. Trastuzumab downregulates ErbB2 activity by mechanisms that remain to be elucidated.

16.4.2.2 Oncogenic Nonreceptor Tyrosine Kinases Many of the nonreceptor tyrosine kinases were discovered because the mutated form of the protein is the product of a viral oncogene. The most prominent examples are the Src tyrosine kinase and the Abl tyrosine kinase (Section 10.3). The relationship of the Abl tyrosine kinase with the Philadelphia chromosome translocation in lymphocytes has been especially well investigated [13]. The Philadelphia translocation is a chromosome translocation affecting the c-abl gene of chromosome 9 and the bcr gene of chromosome 22. The translocation leads to the formation of a hybrid gene composed of the bcr gene, which codes for a Ser/Thr- speciﬁc protein kinase, and the c-abl gene. Consequently, the two alternative fusion proteins p210BCR ABL and p180BCR ABL are created, which are characteristic of various forms of chronic myelogenous leukemia (CML). Although the fusion protein Bcr/Abl is essential for the initiation, maintenance and progression of CML, progression of the disease to an aggressive phase requires additional genetic and/or epigenetic abnormalities.

& Philadelphia translocation: Fusion protein comprising Abl nonreceptor tyrosine kinase and BCR Ser/ Thr protein kinase

During the translocation, a part of the c-abl gene is fused to the ﬁrst exon of the bcr gene (Figure 16.6). The p180BCR ABL hybrid protein demonstrates an increased tyrosine kinase activity, and has an altered subcellular location in that it is 736 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.6 Formation of a hybrid Fusion genes are produced on chromosome oncoprotein, illustrated by translocation of the 22, coding for various fusion proteins. The Abl tyrosine kinase. The gene for the Ser- most important fusion proteins are the p180- specific protein kinase BCR is fused with a part and p120-BCR-Abl hybrid proteins, which have of the c-abl gene in the process of the increased Tyr kinase activity and an altered Philadelphia chromosome translocation. subcellular location.

predominantly found in the cytosol, whereas c-Abl normally exerts its function in the nucleus. The Bcr/Abl hybrid is found exclusively in the cytosol, where it activates pathways normally under the control of receptor tyrosine kinases. Among the pathways activated by cytoplasmic Bcr/Abl are the MAPK, Jak/Stat, and PI3 kinase pathways.

& Bcr/Abl hybrid: — Changed subcellular localization — Increased MAPK, PI3K and Jak/Stat signaling.

Most treatments of CML are directed against the kinase activity of BCR/Abl. The drug Gleevec (imatinib, STI571; see Figure 10.24) speciﬁcally inhibits the kinase activity of Bcr-Abl and is a generally well-tolerated, ﬁrst-line therapy for this malignancy. However, clinically acquired resistance mutations against imatinib are 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 737 a major challenge and there are strong efforts to develop second-generation inhibitors for the kinase activity of BCR-Abl [14].

16.4.2.3 Oncogenic Activation of Ras Signaling Pathways Oncogenic mutations of the signaling axis downstream of receptor tyrosine kinases are very common in cancers, and overstimulation of these pathways then creates signals that enhance cell proliferation and increase cell survival. Most prominent are mutations in the small regulatory GTPases of the Ras superfamily pathways. Oncogenic activation has been documented many times for the example of the Ras proteins (Section 11.5), and mutated Ras proteins (mostly Ki-Ras) have been detected in about 30% of solid tumors. Most Ras mutations either delete or impair the negative control exerted by the GTPase activity of the Ras–GAP complex. These oncogenic mutations render Ras insensitive to the negative control by GAPs. The lifetime of the active Ras–GTP state is increased, which allows a prolonged and persistent signaling to the downstream effectors, among which the Raf kinase, PI3 kinase and Ral GDS are the most important (see Chapter 11; Table 11.2). Another frequently activated oncogene in the Ras signaling pathway is B-Raf kinase, that can be constitutively activated by a number of missense mutations, among which the V66E mutation, located within the ATP binding site, is most prominent (Section 11.6.3). Other oncogenic mutations of B-Raf include the relief of autoinhibition by deletions or substitutions at the N terminus.

16.4.2.4 Oncogenic Activation of Cyclins Oncogenic activation of cyclins is mostly observed for the D-type cyclins, which play a central role in the transition from G0 to G1 and for G1 progression [15]. Increased levels of D-type cyclins and of CDK4/6 activity are frequently found in tumors because of gene translocation, overexpression, and ampliﬁcation of the cyclin D genes. Furthermore, hypomethylation of the gene for cyclin D1 has been reported for tumors. Inappropriately elevated levels of D-type cyclins will increase the number of cells that leave G0 phase and enter into G1 phase. Furthermore, high levels of D-type cyclins make cells more independent of nutrient supply and will therefore add another growth advantage to cancer cells. Several mechanisms are known that enhance cyclin D activity. The expression of cyclin D1 is, for example, stimulated by the Ras/MAPK pathway and is also regulated via the APC pathway (Section 16.8). Furthermore, cyclin D levels are controlled by ubiquitin-dependent proteolysis, by subcellular localization, and by phosphorylation (Section 15.2.2).

16.4.2.5 Oncogenic Transcription Factors: Myc A large number of proto-oncogenes code for transcription factors required for progression of the cell cycle and/or for the differentiation of the cell. Often, transcription factors are translocated in chromosomal rearrangements, and hybrid transcription factors are then formed with an altered expression level and regulation. Furthermore, translocation can bring transcription factors into the 738 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

vicinity of strong promoters, leading to overexpression. Well-studied examples of oncogenic mutated transcription factors involve, thefos, junand myc genes, and the

genes for the3 receptor T and the vitamin A acid receptor. Of these, c-myc has now been identi ﬁed as one of the most frequently mutated oncogenes in human cancer [16]. Oncogenic activation of c-myc is achieved in various ways, including gene translocation (as in Burkitt’ s lymphoma), gene ampliﬁcation, missense mutations, stable overexpression due to promoter mutations, and epigenetic alterations.

The Myc Transcription Factor Myc is a multivalent transcription factor that controls thousands of genes (http:www.myc-cancer-gene.org) and thereby inﬂ u- ences nearly all of the functions of a cell.

& Transcription factor Myc: — Frequently mutated in cancers — Controls transcription between cell cycles — Forms heterodimers with Mad, Max and Miz1 proteins in a complex network.

The Myc protein belongs to the family of basic helix-loop-helix proteins, and forms heterodimers via its leucine zipper motif with another helix-loop-helix motif transcription factor, the Max protein. The formation of Myc–Max heterodimers is a prerequisite for binding to the cognate DNA element, termed E box, in the promoters of the Myc target genes and activation of these genes. The Max protein can heterodimerize with other helix-loop-helix proteins such as the Mad protein or the Mxi-1 protein. The various heterodimers have different effects on transcription activity of the corresponding genes. The c-Myc–Max dimer activates transcription, while the Max–Mad or Max–Mxi-1 dimers repress rather than activate transcription. In a normal cell, there is a balanced equilibrium between the different dimers. When bound to the canonical E-box 50-CACGTG-30 on target promoters, the Myc–Max heterodimers interact in a dynamic fashion with multiple components of the RNA polymerase holoenzyme, with transcriptional cofactors (including coactivators and corepressors), and with various chromatin-remodeling and -modifying complexes. These interactions also include components of the elongating RNA polymerase complexes [17].

Cooperation with Other Transcription Factors The E-box is also the target site for other transcription factors, such as the sterol response-element binding protein (SREBP) and the hypoxia-induced transcription factor (HIF). Myc binding to the E-box alone is insufﬁcient for driving the expression of its target genes, and cooperation with other transcription factors appears to be required for stimulating expression of the targets. This illustrates the complexity of Myc function and regulation. 16.4 Signaling Proteins Mutated in Cancer: Oncogenes 739

Regulation of Myc Transcription Transcription of the Myc gene is strictly controlled and is subject to both positive and negative regulation by many pathways that convey extracellular growth signals. Myc expression is activated by RTK signaling pathways, Notch signaling, Wnt signaling, TCR signaling, and its expression is downregulated by for example, the TGFb pathway (Figure 16.7). Aberrant activation of these pathways will lead to inappropriate levels of Myc and aberrant transcription of Myc targets. Furthermore, Myc is subject to posttranslational modifications (PTMs) at multiple sites by phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These modifications affect Myc functions in a dynamic manner and mediate control via a diversity of upstream signaling events. For example, the activating phosphorylation at T58 is catalyzed by glycogen synthase kinase 3 (GSK3), a component of the PI3K/Akt pathway (Section 9.4), such that S62 of Myc becomes phosphorylated upon stimulation of the Ras/MAPK pathway. Special importance has been now attributed to transcriptional programs operating downstream of Myc. Myc promotes both pro- and anti-tumorigenic responses that are normally well balanced. Mutations in Myc may shift the balance to favor either the anti-oncogenic or the oncogenic state by activating distinct subprograms. Such a subprogram has been shown to involve the sumoylation- activating enzymes (SAEs) 1 and 2 [19]. SAE2 is required for a transcriptional subprogram of Myc enriched in proteins controlling spindle integrity, while the inactivation of SAE2 switches the subprogram of Myc from activated to repressed. The subprogram appears to cooperate with oncogenic Myc to maintain the expression of Myc target genes involved in mitotic fidelity. This example underscores the complexity of Myc functions which include transcriptional subprograms that may be segregated and may deliver either pro-oncogenic or anti-oncogenic signals.

Transcriptional Targets of Myc Myc regulates the transcription of huge number of genes and thereby controls all major functions of the cell [17,20]. Downstream Myc targets include genes involved in the control of cell growth and proliferation, DNA damage response, ribosomal biogenesis, mitochondrial biogenesis, and energy metabolism. Moreover, Myc targets include genes for rRNA, tRNA, and diverse set of miRNAs (Figure 16.1). The involvement of Myc in the regulation of energy metabolism and protein biosynthesis indicates that Myc functions as a master regulator that coordinates energy metabolism with biomass accumulation in preparation for DNA replication and cell division. Among Myc-regulated genes important for cell cycle control are the genes for cyclin D1, cyclin B1, CDK4, BRCA1, ARF, and others. Clearly, Myc is an important control element for G1 progression, and appears to couple DNA replication to processes preserving the integrity of the genome. Furthermore, Myc has been shown to function also as a repressor of transcription of genes, such as the gene for the kinase inhibitors p15INK4b and p21. This repression is performed in cooperation with another DNA-binding protein named Miz1. 740 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.7 Myc regulates cell growth and WNT pathway is depicted with APC negatively proliferation. The MYC proto-oncogene is regulating b-catenin, which upon nuclear depicted downstream of receptor signal translocation participates in the transactivation transduction pathways, which elicit a positive of MYC, such that loss of APC results in or negative regulation of the MYC gene. MYC constitutive oncogenic MYC expression. For produces the transcription factor Myc, which Wnt/b-catenin, see Section 16.8; for RTK, dimerizes with Max and binds target DNA see Chapter 10; for TGFb receptor, sequences or E boxes (with the sequence 50- see Section 14.1; for T-cell receptor, see CANNTG-30) to regulate transcription of genes Section 13.3. Adapted from Ref. [18]. involved in cell growth and proliferation. The 16.5 Tumor Suppressor Genes: General Functions 741

16.5 Tumor Suppressor Genes: General Functions

Summary Tumor suppressor genes help to prevent tumor formation by performing a dampening, braking function in signaling pathways that regulate cell proliferation and cell homeostasis. The elimination or downregulation of the tumor suppressor function by mutations leads to a preponderance of growth- promoting signals and thereby favors tumor formation. Mutations of tumor suppressor genes are often recessive. On the mutation of one allele, the remaining intact allele on the other chromosome continues to perform the growth-suppressing function. Only when both alleles are inactivated does the tumor-suppressing function cease to work. Suppression of tumor formation can be achieved by processes at various levels, including repair checkpoints, cell- cycle regulation, apoptosis, and processes that are required in the more secondary hallmarks of cancer, for example, blood vessel formation and metastasis. Well-characterized tumor suppressors include the retinoblastoma protein Rb, the tumor suppressor ARF (Section 16.6), and p53 (Section 16.7).

& General functions of tumor suppressor genes: — Negative regulation of cell proliferation — Often participate in cell-cycle checkpoints and in DNA repair.

One classiﬁcation of tumor suppressor genes involved in cell-cycle control and DNA repair is based on the function of the encoded proteins (Figure 16.8):

Anti-oncogenes encode proteins that antagonize the growth-promoting activities of oncogenes. Examples include the p16Ink4 inhibitor that negatively regulates the activities of the oncogenes cyclin D1 and CDK4/6, and PTEN phosphatase that antagonizes the action of PI3kinase. DNA damage checkpoint genes also function as tumor suppressors. Prominent examples are the ATM kinase and p53 that induce cell death or senescence in response to DNA damage or replication stress. Caretaker genes perform their tumor-suppressing function as part of DNA repair or mitotic checkpoint systems. These proteins help to maintain genomic stability. Well-known examples are proteins involved in nucleotide excision repair such as XPA, mismatch repair such as MUTH-L, and double-strand break repair such as BRCA1. Inherited defects in these tumor suppressor genes will favor the acquisition of a “mutator phenotype” [21], as it requires the mutational inactivation of the other allele only for a complete loss of function. There are many ways by which the tumor-suppressing functions of tumor suppressor proteins can be either inactivated or weakened. The major routes involve reduced 742 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.8 Three classes of tumor such as MLH1 (mutL homolog 1, involved in suppressor genes. Tumor suppressor genes mismatch repair), BRCA1 (breast cancer classified as anti-oncogenes antagonize the susceptibility 1), MYH (also known as growth-promoting activities of oncogenes. MUTYH, A/C specific DNA glycosylase) and For example, the tumor suppressor genes XPA (xeroderma pigmentosum group A) encoding pRb and p16Ink4 antagonize the encode proteins that help to maintain genomic function of CDK4 and cyclin D1, which are stability and thereby help to suppress tumor oncogenic. DNA damage checkpoint genes, formation. Some tumor suppressors have coding for example ATM or the tumor more than one function and could fit into more suppressor p53, induce cell death or than one of the classes described here; thus, senescence in response to DNA damage or the distinction proposed here is based on DNA replication stress. Caretaker genes (DNA primary function. DSB, double-strand break. repair genes and mitotic checkpoint genes,

expression, structural and functional changes, and an increased instability of the tumor suppressors.

A number of tumor suppressor genes are known with no direct relationship to the regulation of the cell cycle, repair, or apoptosis. Some of the tumor suppressor genes listed in Table 16.3 are involved in the organization of the cytoskeleton or in cell–cell interactions, and appear to be relevant in later stages of tumor formation where tumor cells invade foreign tissues and form organ-like structures.

Table 16.3 Characteristics of some tumor suppressor proteins.

Gene, protein Function

p53 DNA repair, apoptosis Rb Cell cycle control NF1, neuroﬁbromin GAP in Ras signaling BRCA1, BRCA2 DNA repair, for example, of double-strand breaks Wt-1 Transcription factor with Zn-binding motif PTEN Phosphatidylinositol phosphate phosphatase, blockade of PI3 kinase signaling CDH1, E-Cadherin Cell adhesion APC Binds to b-catenin; Wnt-signaling MSH2 Repair of DNA mismatches 16.6 Tumor Suppressors: Rb and ARF Proteins 743

& Tumor suppressor proteins can be inactivated by — Reduced expression due to: Altered transcriptional regulation Changes in promoter structure Epigenetic silencing. — Structural changes leading to Altered transcriptional regulation Changes in phosphorylation and other PTMs Altered subcellular distribution Altered interactions with upstream and downstream effectors Altered enzymatic activity. — Increased instability due to changes in Ub-mediated proteolysis.

The details of selected tumor suppressors that have been found as mutated in many tumors, and to which key functions in tumor suppression are ascribed, are presented in the following sections.

16.6 Tumor Suppressors: Rb and ARF Proteins

16.6.1 Rb in Cancer

The retinoblastoma gene was the ﬁrst tumor suppressor gene to be identiﬁed and characterized in humans. The product of the retinoblastoma gene, the retinoblastoma protein Rb, along with its cousins p107 and p130, regulates G1/S transition, facilitates differentiation, and restrains apoptosis, all of which processes are relevant to tumor formation [22].

& Tumor suppressor function of Rb: — Regulates G1/S transition via interaction with E2F — Rb–E2F interaction is controlled via Rb phosphorylation.

The best-characterized function of Rb in cell-cycle regulation is the transmission of mitogenic and antimitogenic signals down to the transcription level (Sec- tion 15.4.3). This regulatory function can be described roughly by a two-state model:

In the active state, Rb is hypophosphorylated and suppresses the transcription of E2F-dependent genes necessary for entry into S phase. In the inactive state, Rb is hyperphosphorylated, and the transcription of E2F- dependent genes is possible which allows progression into S phase. By restraining the cell-division activity in its hypophosphorylated state, Rb acts as a tumor suppressor. 744 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

The loss of Rb functions weakens these controls, dissociating the cell-cycle machinery from extracellular signals, dampening the ability of proliferating cells to exit the division cycle, and compromising the execution of Rb-dependent differentiation programs in certain tissues. The number of proteins that direct signals to and convey signals from Rb/E2F is very large, and functional inactivation of the tumor-suppressing function of the Rb network is thus possible in many ways [23]. The major methods of Rb inactivation include (Figure 16.9):

Unprogrammed activation of the G1CDKs and cyclins: Via the overexpression of cyclin D1 Loss of inhibitory signals: CKIs inhibit the phosphorylation of Rb and maintain

Rb in the active state to mediate a halt in the G1 phase. Of the various CKIs, mutations in the inhibitor p27KIP1 and, in particular, the p16INK4a protein (Section 15.2.3) are associated with tumor formation. Furthermore, alteration of the anti-growth signaling function of the TGFb-pathway (Section 14.1) also leads to low levels of CKIs and, consequently, to the inactivation of Rb. Binding of viral oncoproteins: DNA viruses that can trigger tumors are found in the classes of the polyoma viruses (e.g., SV40), the adenoma viruses, and the human papilloma viruses (HPV). These viruses encode proteins with the properties of oncoproteins. The oncoproteins are the T antigen (TAg) of the SV40 virus, the E1A protein of the adenoma virus, and the E7 protein of HPV. The three proteins have in common the ability to bind to the hypophosphorylated form of Rb. This leads to the release of E2F transcription factors and unprogrammed transcription of the E2F target genes. Genetic inactivation of Rb: The genetic inactivation of Rb is observed in many tumors. The mutations involve generally extensive structural changes in the Rb gene. Epigenetic inactivation of Rb: As for many other tumor suppressor proteins, Rb levels can be downregulated by epigenetic silencing of the Rb gene via DNA hypermethylation and altered chromatin modiﬁcation.

& Tumor suppressor function of Rb may be inactivated via: — Enhanced CDK/CycD/CycE signaling — Loss or inactivation of CKIs — Binding of oncoproteins — Mutation of Rb gene — Epigenetic inactivation of Rb.

16.6.1.1 Rb and Apoptosis One function that is equally important for tumorigenesis is the ability of Rb to protect differentiating cells (which contain high levels of Rb) against apoptosis. The link from the Rb pathway to apoptosis appears to be provided by E2F1, which controls not only the genes required for S-phase progression but also those 16.6 Tumor Suppressors: Rb and ARF Proteins 745

Figure 16.9 Errors in regulation of the tumor suppressor protein Rb. The figure shows a simplified version of well-characterized mechanisms by which errors in regulations of the Rb function can occur. involved in the regulation of apoptosis. Among the latter are the genes for the proapoptotic protein Apaf1 (Section 17.5) and for the tumor suppressor ARF which forms part of the network that regulates p53 function (Section 16.6).

& Rb protects cells from apoptosis via its influence on E2F1 746 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

AlossofRbfunction– in the absence of other mutations – has been shown by knockout studies in mice to result in increased apoptosis and in growth arrest. If, at the same time, other key regulators of apoptosis (e.g., the p53 protein or ARF) are mutated and inactivated, then apoptosis is reduced and cells are driven to hyperproliferation. In accordance with this model, nearly all cancers contain mutations both in the Rb pathway and in the pathways that link proliferation with apoptosis. This example illustrates the intense networking that exists between the various growth- and cell death-controlling pathways, and also underlines the cooperativity of mutations in key regulators of cell survival and cell death.

16.6.2 The p16INK4a Gene Locus and ARF

Investigations into tumor cells indicate that proteins coded by the p16INK gene locus function as tumor suppressors and are of great importance for tumor development [24]. The gene locus for p16INK4a codes for two proteins, namely the p16INK4a inhibitor (Section 15.2.3) and the ARF protein (ARF ¼alternative reading frame: p19ARF in mice; p14ARF in humans). The ARF protein is not homologous to the p16INK4a protein, although both originate from the same gene locus; rather, it arises by alternative splicing and by the use of a different reading frame. Both proteins have a growth-inhibiting function, though with different points of attack.

& Tumor suppressor ARF (p14ARF): — Stabilizes p53 — Binds to Mdm2 — Inhibits downregulation of p53 by Mdm2.

While the p16INK4a protein inhibits the cyclin D–CDK4/6 complex and brings about a halt in the cell cycle via the Rb protein, the ARF protein activates the transcription factor p53 that triggers the expression of many apoptotic inducers and cell-cycle inhibitory genes. ARF stabilizes and stimulates p53 activity by neutralizing two Ub ligases, the Mdm2 protein and the ARF-BP1. Both proteins are E3 ligases speciﬁc for p53 and inhibit its tumor suppressor function. Following aberrant oncogene activation, ARF is induced and downregulates the proteasomal degradation of p53, thereby promoting p-53-dependent cell cycle arrest or apoptosis. The oncogenic overexpression of ARF is linked for example, to aberrant alterations of the Rb/E2F pathway, as ARF is one of the transcriptional targets of the E2F transcription factors. Furthermore, oncogenic activation of the Ras-MAPK pathway and of Myc has been linked to an increased transcription of ARF (Figure 16.10). 16.7 Tumor Suppressor Protein p53 747

Figure 16.10 Control of the p53 pathway by inhibiting the negative regulation imposed by the alternative reading frame (ARF). the ubiquitin ligases MDM2 and ARF-BP1, Hyperproliferative signals generated by thereby inducing cell cycle arrest or apoptosis. oncogene activation induce the accumulation The mechanism by which ARF controls MDM2 of ARF. In turn, ARF stabilizes p53 and activity towards p53 is multifaceted, depending stimulates its transcriptional activity by on the activated signals and on the tissue type.

16.7 Tumor Suppressor Protein p53

Summary The most frequently observed genetic changes in human tumors affect the gene for a nuclear phosphoprotein of 393 amino acids, which is known as the p53 protein, after its molecular weight. The tumor-suppressing function of p53 is based on its ability to induce cell cycle arrest and apoptosis when the cells are exposed to a wide variety of genotoxic and nongenotoxic stress signals that can originate from either external or internal sources. The p53 protein integrates these signals and activates a complex signaling network, primarily by inducing the transcription of a large number of target genes in a differential fashion. p53 activation by stress signals facilitates DNA repair and promotes apoptosis or senescence, which provides for an efficient mechanism to prevent the accumulation of abnormal cells, particularly those with heritable DNA damage. 748 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Therefore, p53 has been said to have the function of a “guardian of the genome.” Loss of p53 function by inactivation of the p53 network cancels a central control element that ensures the integrity of the genome. In unstressed cells, p53 is present in low amounts and in an inactive state, due to negative control by the Mdm2 protein. Exposure of various stresses leads to an increase of p53 concentration and activity and transcription of target genes. p53 is a transcription factor that activates the transcription of target genes by binding to the cognate DNA recognition element in the control region of these genes. Among the genes regulated by p53 are those implicated in DNA repair, senescence, antioxidant responses and cell-cycle progression, among others. Depending on the intensity and nature of the stress signals, the p53-mediated responses lead to increased DNA repair, cell cycle arrest, senescence, or apoptosis. Stress signals that mediate p53 activation include DNA damage, replication stress, and oncogenic stress. These signals are transmitted to p53 mostly by PTMs, including phosphorylation, acetylation, methylation and ubiquitination that affect the interaction of p53 with partner proteins within the p53 network. A major regulator within this network is the Mdm2 protein that promotes p53 inactivation. In addition to a guardian function, p53 performs regulatory tasks in normal cells by controlling the transcription of genes involved in metabolism, growth regulation, and development.

p53 can be classified as the most prominent tumor suppressor protein. Mutations of the p53 gene are observed in over 50% of all human tumors. Defects in the p53 gene in the germline lead to a hereditary tendency to develop various tumors, especially of the connective tissues, notably Li–Fraumeni syndrome, named after its discoverers. Families have been identified that inherit p53 mutations and develop cancers with 100% penetrance. p53 is now placed at the center of a large network that allows cells to deal with a variety of stresses, including those implicated in tumorigenesis, and can be considered as the “number one” tumor suppressor protein. First insights into the tumor-suppressing functions of p53 were obtained from its ability to induce cell cycle arrest and apoptosis upon exposure of the cells to genotoxic stress. Inactivation of the p53 network enables the cell to continue in the cell cycle with damaged DNA, yet without DNA repair taking place. Furthermore, failure of the apoptotic control function permits the survival of cells with damaged DNA. Both effects lead to an increased susceptibility of the genome to accumulate further mutations. The cells can also divide under conditions in which serious changes of the genome are present, such as DNA amplification and chromosome rearrangement. Indeed, p53 is inactivated in most cancers, underscoring the importance of p53 as a tumor suppressor. However, it is now well-established that p53 plays numerous roles under normal conditions and under low and transient stresses, in addition to the “guardian function.” 16.7 Tumor Suppressor Protein p53 749

16.7.1 Overview of p53 Function

According to a generally accepted model, the p53 protein acts as a checkpoint responding to a wide variety of genotoxic and non-genotoxic stress signals that can originate from either external or internal sources. The primary function of p53 is that of a nuclear transcription factor that binds in a sequence-speciﬁc fashion to cognate recognition elements on target genes, to either activate or suppress expression of the encoded protein or RNA. In addition, functions of p53 have been identiﬁed that are not linked to gene transcription. For example, p53 can promote apoptosis through transcription-independent mechanisms (these functions will not be discussed in the following subsections). The p53 protein is present in almost all tissues at low concentrations and in an inactive, repressed state. Several types of stress induce p53 accumulation and activation, and this serves to protect the cells against the potentially disastrous consequences of stresses. It also occurs as a normal mechanism of defense against neoplastic transformation of the cell. The stresses that activate p53 can be broadly divided into three classes (Figure 16.11):

Genotoxic stress: DNA damage by UV, X-rays, carcinogens, and cytotoxic drugs, for example. Oncogenic stress: Aberrant activation of growth factor-signaling cascades Non-genotoxic stress: Replication stress, translation stress, metabolic stress such as hypoxia, and the depletion of ribonucleotides.

In this system, p53 functions as a node for organizing whether the cell responds to the various types and levels of stress with the following biological outcomes:

Induction of apoptosis Growth arrest and cell cycle arrest Stimulation of DNA repair and promotion of genome stability Induction of senescence Altered metabolism Autophagy.

The biological responses induced by p53 activation target the major functions of the cell. Most importantly, p53 also performs vital regulatory tasks in the normal, unstressed situation of a cell by controlling the transcription of many genes involved in metabolism, growth regulation, and development. Which of these responses dominates depends on the cell type and the nature and intensity of the stress. The stress signals that direct p53 to activate a distinct transcriptional program are transmitted primarily via PTMs of p53 induced by the activation of various stress-signaling cascades. 750 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.11 Input signals of p53 activation epigenetic landscape of the target gene and downstream transcriptional and biological promoter, among others. The transcriptional outputs. The complex signaling network with output of p53 is responsible for determining p53 at its center is shown in a highly simplified which cellular process(es) occur in response to fashion. In response to various inputs (top of distinct genotoxic insults. PAI-1, plasminogen figure), the p53 protein becomes stabilized. activator inhibitor 1, a secreted mediator of On stabilization of p53, various transcriptional senescence; TIGAR, p53-induced glycolysis outputs can be realized which may be and apoptosis regulator. For p21KIP1, see determined by the strength of the p53 Chapter 15; for Puma, see Chapter 17. response element, the PTM status of p53, Adapted from Ref. [18]. specific p53 binding partners, and the 16.7 Tumor Suppressor Protein p53 751

& Tumor suppressor p53: — Prevents cell-cycle progression — Activates apoptosis. Inactivation of p53: — Allows cell-cycle progression in the presence of DNA damage — Prevents apoptosis.

Activation of the p53 network can occur at varying degrees, and different subnetworks can be affected, depending on the type of stress signal responsible for p53 activation and subsequent transcriptional stimulation. One review lists 129 direct transcriptional targets of p53 [25], and there are likely to be many more genes to be discovered as transcriptional targets which are speciﬁcally activated by p53. Furthermore, the number of genes of which the expression is altered indirectly upon the induction of p53 is likely to be in the thousands. In some respect, the complexity of p53 transcriptional function is comparable to that of the transcription factor Myc, which also acts as a master regulator of a huge number of genes.

16.7.1.1 Cousins of p53 Proteins related to p53 have been identiﬁed that are also part of the p53 regulatory network. Two proteins, named p63 and p73 [26] have been found to be “cousins” of p53; both are activated by similar stresses as p53 and elicit some, but not all, of the biological responses of activated p53.

16.7.2 Structure and Biochemical Properties of the p53 Protein

The p53 protein is a transcriptional activator that shares many properties with typical eukaryotic transcriptional regulators (Section 4.4). It binds site-speciﬁcally to its cognate DNA element and regulates the transcription of neighboring genes. In doing so, p53 interacts with and is regulated by a large number of proteins, including proteins of the chromatin-remodeling and transcription machinery. Furthermore, p53 is subject to multiple PTMs.

& Properties of p53: — Transcriptional activator — Regulated by PTMs.

As shown in Figure 16.12, distinct domains can be identiﬁed in the p53 protein, and deﬁned biochemical functions can be assigned to them:

N-terminal domain: This comprises a transactivation domain (TAD; residues 1–60), composed of two subdomains TAD1 and TAD2, and a proline-rich regulatory (PRR) domain (residues 63–97). 752 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.12 Domain structure of p53. NLS, nuclear localization signal; NES, nuclear export signal.

Sequence-speciﬁcDNA-binding domain (DBD): This incorporates residues 100–300, and includes the binding site for the corresponding DNA element. Adjacent to the DBD is a linker region (residues 301–323) and a tetramerization domain (residues 323–355) that is responsible for the reversible association of p53 into tetramers. C-terminal domain (CTD): The CTD (residues 356–393) is ascribed a negative regulatory function. It is a highly basic domain that can interact nonspeciﬁcally with either DNA or RNA. Furthermore, signals for nuclear localization, sites for PTMs and for the binding of interacting proteins are found in the CTD.

& Structural domains of p53: — N-terminal domain: transactivation — Core domain: DNA-binding, tetramerization — C-terminal domain: negative regulation.

16.7.3 Structure of p53

Central to the function of the p53 protein is its ability, as a transcription activator, to specifically bind to a cognate DNA recognition element in target genes and to activate – or sometimes repress – their transcription. Over 80% of cancer-derived p53 mutations are found within the protein’s DNA-binding domain, underscoring p53’s main role as a sequence-specific DNA-binding protein. The most frequent mutations either interfere with the DNA binding or lead to a structural destabilization of the central core of p53. p53 binds as a tetramer to its recognition element composed of two inverted ¼ pentamers RRRC(A/T)(T/A)GYYY(Xn)RRRC(A/T)(T/A)GYYY, where R purine, Y ¼pyrimidine, and n ¼ 0–13 (Figure 4.12). The determination of the structure of full-length p53 alone and in complex with DNA has been very difficult because only the DNA-binding domain and the tetramerization domain are natively folded, whereas the N-terminal domain and the CTD are intrinsically unstructured. As 16.7 Tumor Suppressor Protein p53 753 shown by a variety of biophysical measurements, the DNA-binding domain and the tetramerization domains of the p53 tetramer bind as dimers to the recognition element, forming a core from which the transactivation domains project outward with flexible orientations [27].

& Structure of p53: — Tetramer — Only core domain natively folded — N- and C-terminal domains are unstructured.

One important feature of the ability of p53 to recognize and bind DNA is the presence of two DNA-binding sites, one in the central DBD and one in the basic CTD. Modiﬁcation of the CTD negatively regulates the core domain’s ability to bind to short oligonucleotides in vitro, and acetylation of the CTD can enhance the transactivation of p53 targets.

16.7.4 PTMs of p53

The p53 function is embedded in a finely tuned regulatory network that employs various signaling pathways for the reception of activating signals and directs the p53 response to different downstream effector pathways. In this process, PTMs of p53 are the major tools for the regulation of p53 function, and an array of PTMs is found on p53 both during normal homeostasis and in stress-induced responses. Intriguingly, more than 36 different amino acids within p53 have been shown to be modified in various biochemical and cell culture studies, indicating an enormous complexity of p53 regulation. In normal, unstressed cells, p53 is present in a repressed state and only in low amounts, primarily due to negative control by the E3 ligase MDM2 (Section 16.7.5). Exposure to various stresses initiates derepression and a concentration increase of p53, allowing it to exert its transcriptional regulatory function. Transition from the repressed state to the active state, and vice versa, is accompanied by a series of PTMs that influence in a differential manner the stability, subcellular localization, DNA-binding activity, and binding to effector proteins of p53 [28]. To date, PTMs associated with p53 have included phosphorylation, acetylation, methylation, ubiquitination, neddylation, and sumoylation (Figure 16.13).

16.7.4.1 Phosphorylation At least 18 Ser/Thr residues on p53 have been reported to be modiﬁed by phosphorylation [28]. Many of these sites cluster in the N-terminal region, and phosphorylation of this region stabilizes p53 by interfering with the ability of MDM2 to ubiquitinate p53 and induce its proteasomal degradation. The N- terminal phosphorylation of p53 occurs rapidly in response to various stresses to stabilize and activate p53. As outlined in Chapter 15, stresses such as DNA damage 754 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.13 Multiplicity of p53 signals. Furthermore, additional posttranslational modifications in response to phosphorylation and acetylation sites, as well DNA damage and stress. More than 36 amino as major sites of methylation (Me), and acids of p53 are reported to be modified under neddylation (N8), are indicated. For DNA-PK, the influence of various DNA damages and ATM/ATR, see Chapter 15. For MAPKAP2, stress signals. The major sites of p53 ERK2, JNK, p38k, see Chapter 12. CK, casein phosphorylation (P), ubiquitination (Ub), and kinase; CBP/p300, histone acetyltransferase, acetylation (Ac) are shown with the see Section 4.5.2; TAF1, see Section 4.2.5. corresponding major modifying enzymes and Adapted from Ref. [28]. 16.7 Tumor Suppressor Protein p53 755 and replication stress activate the protein kinases ATM, ATR, DNA-PK, Chk1, and Chk2, and these kinases have been implicated in phosphorylation of distinct sites within the N terminus of p53 allowing for a stop in the cell cycle or triggering apoptosis. Other protein kinases responsible for phosphorylation of the N-terminal sites include members of the ERK/MAPK family such as p38 and JNK that become activated by stresses such as UV light and glucose deprivation. The multiplicity of phosphorylation sites in the N terminus and the variety of protein kinases involved are shown in Figure 16.13. Some protein kinases phosphorylate a single, distinct site, whereas others can phosphorylate several sites. Many questions on the contribution of individual sites to stabilization of p53 in vivo remain unanswered, however. For instance, the contribution of a particular phosphorylation site to p53 stabilization is uncertain, and it may well be that multiple phosphorylations are required for substantial p53 stabilization. Furthermore, it is not presently understood how the phosphorylations are orchestrated and how a particular protein kinase selects among the multiple sites.

16.7.4.2 Acetylation p53 harbors multiple Lys-acetylation sites, two of which are located in the DNA-binding domain while the others are concentrated on the extreme N-terminal tail. Generally, the acetylation of these sites correlates with p53 stabilization and activation. Furthermore, some of these sites have been implicated in target gene selection and transcription activation. The acetyl transferases responsible for this modification include the p300/CBP, PCAF and TIP60 histone acetylases (Section 4.5.2). These enzymes form part of larger protein complexes that are recruited to promoters upon the formation of transcription pre-initiation complexes. It is possible that DNA-bound p53 becomes acetylated by the histone acetylases as part of a complex set of acetylation reactions, including the acetylation of histones and other chromatin components. Most importantly, the same lysine residues that have been shown to be acetylated can be also modified by methylation and ubiquitination, which points to a complex set of combinatorial possibilities for p53 lysine modification with the mutual exclusion of modifications at a particular site.

16.7.4.3 Methylation Both arginine and lysine residues of p53 become methylated in response to various stimuli. The methylation of speciﬁclysineresidueshavebeenshown to have both repressive and activating effects on p53 function. For example, dimethylation of K370 and monomethylation of K372 activate p53, whereas methylation of K370, K373 and K382 represses p53. The methyltransferase SET8hasbeenimplicatedintherepressivemethylations.Becausethesame lysine residues methylated in the repressive response are also the target of activating acetylations, the two alternative modiﬁcations appear to be used to switch p53 from a repressive into an activating mode (and vice versa). Moreover, the same lysine residues are the target of ubiquitination which will induce proteasomal degradation of p53. 756 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

16.7.4.4 Ubiquitination p53 has been found to be both monoubiquitinated and polyubiquitinated [29]. The polyubiquitination of p53 occurs mainly on six key lysine residues located in the CTD (Figure 16.13), and this modification induces the proteasomal degradation of p53. At least six different E3 ligases have been implicated in polyubiquitination, of which Mdm2 plays a prominent role in controlling p53 levels (see Section 16.7.5). The Mdm2 protein is a RING-finger E3 enzyme that binds to the N-terminal domain of p53 and mediates its ubiquitination, which in turn efficiently and rapidly blocks p53 signaling. Signal-induced phosphorylation of p53 blocks this interaction. The ubiquitination of p53 also serves other purposes than proteosomal degradation. The monoubiquitination of p53 at multiple lysine residues promotes the nuclear export of p53, thereby blocking any transcriptional activity. Further- more, ubiquitination by the atypical E3 ligase E4F1 increases the fraction of p53 associated with chromatin, and this association coincides specifically with p53- mediated transcriptional activation of genes involved with cell cycle arrest. Overall, the ubiquitination of p53 is critical for maintaining appropriate protein levels at all times and under all conditions.

16.7.4.5 Neddylation, Sumoylation The PTM of p53 includes also its conjugation with the ubiquitin-related proteins Nedd and Sumo (Section 2.8.6). Neddylation inhibits the transcriptional activity of p53, but the functional consequences of sumoylation are unclear. It is important to bear in mind that the PTMs on p53 listed above are of a transient nature only. Enzymes that remove the modiﬁcations also form part of the p53 network, but the contribution of these enzymes to the dynamic nature of the modiﬁcations is not well established.

& PTM of p53: — 18 P-sites — Acetylation — Lys-methylation — Ubiquitination — Sumoylation — Neddylation — ADP-ribosylation.

16.7.5 Control of p53 by Mdm2

A major inﬂuence on p53 activity is ascribed to its control by the E3 ligase, Mdm2 [30]. These two proteins form part of a complicated network that integrates multiple stress inputs to provide correct reactions that help to cope with the stresses. Any imbalances within this network will favor tumor formation. 16.7 Tumor Suppressor Protein p53 757

16.7.5.1 Mdm2 is an Oncogene Mdm2 overexpression due to amplification is observed in about 10% of all human cancers, and overexpression via mechanisms distinct from amplification also occurs in many human malignancies. Most of the oncogenic activity of Mdm2 can be explained by its impact on p53 activity and level. However, other findings indicate that Mdm2 also has a role in tumorigenesis, independent of p53. Mdm2 downregulates p53 by two major mechanisms:

Control of p53 stability: In unstimulated cells, p53 levels are low and p53 is unstable. The main factor contributing to p53 downregulation in the absence of stresses comes from ubiquitination and proteosomal degradation of p53 [29]. Many studies have shown that Mdm2 is the predominant and critical E3 ubiquitin ligase for p53. Mdm2 ubiquitinates p53 at its C terminus, which promotes nuclear export and subsequent proteasomal degradation. Repression of p53: The Mdm2 N terminus binds p53 and represses its ability to promote transcription. The cousin of Mdm2, Mdmx, is also involved in the direct repression of promoter-bound p53.

Upon oncogenic activation of Mdm2, the levels and activity of p53 will be kept low, even under stress conditions, and the guardian functions of p53 will be lost. Importantly, Mdm2 also functions independently of p53 in pathways that inﬂuence genome stability and apoptosis and thus contribute to tumorigenesis [31].

16.7.5.2 Negative Feedback Between p53 and Mdm2 Together, p53 and Mdm2 function in a negative feedback loop, with p53 driving the transcription of Mdm2 during times of normal homeostasis and maintaining low levels of p53 protein (Figure 16.14). The promoter of the Mdm2 gene carries p53 binding sites as well as binding sites for other transcription factors as for example, NFkB. p53 transactivates the Mdm2 promoter and promotes Mdm2 expression; the elevated Mdm2 then downregulates p53 by binding to p53 and inhibiting its transcriptional activity on the Mdm2 promoter. Furthermore, Mdmd2 ubiquitinates p53, leading to its nuclear export and proteasomal degradation. In this way, a negative feedback loop is formed that keeps both Mdm2 and p53 levels low under unstressed conditions. It is inevitable that, in response to stress, this loop must be disrupted so as to allow p53 to accumulate and be activated sufﬁciently, in order to exert its growth-inhibitory activities.

& Regulation of Mdm2: — Binding of Mdm2 to ARF prevents ligase action — Phosphorylation of p53 prevents Mdm2 binding.

Upon DNA damage or other type of cellular stresses, the repression and destabilization of p53 by Mdm2 is relieved. In this respect, two major approaches 758 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Figure 16.14 The MDM2-p53 network. The tumor suppressor p19ARF, which is coded for level of p53 is strongly regulated by its by the p16Ink4 gene locus. p19ARF binds to interaction with MDM2. Binding of MDM2 to MDM2 and sequesters it from the feedback p53 targets p53 for proteolytic degradation, loop, thus helping to increase p53 thus keeping p53 concentrations low. DNA concentration. The pro-apoptotic and anti- damage induces phosphorylation of p53; the proliferative function of p53 may be impaired phosphorylated p53 is no longer bound by by mutation of p53, by overexpression of MDM2, proteolysis is decreased, and the rising MDM2, or by a loss of p19ARF function. The p53 levels initiate apoptosis and lead to growth p16Ink4 gene locus is of specific importance in arrest. A negative feedback loop exists between this network because it also codes for the p53 and MDM2 since p53 controls the inhibitor p16Ink4 which inhibits the expression of MDM2 at the level of cyclinD---CDK4 complexes. transcription. Another control is exerted by the

have been identiﬁed that contribute to increases in p53 activity and levels in response to various stresses:

Downregulation of Mdm2 by the tumor suppressor ARF: One of the products of the p16 gene locus, the tumor suppressor protein ARF (Section 16.6.2), binds Mdm2 and blocks Mdm2-mediated p53 ubiquitination and nuclear export. This leads to the concomitant stabilization and accumulation of p53. Phosphorylation of p53: Stress-induced phosphorylation of p53 inhibits the binding of Mdm2 to p53, leading to its stabilization (Section 16.7.4). 16.7 Tumor Suppressor Protein p53 759

& MdmX: Binds to p53 and inhibits its transcriptional function

& Other E3 ligases involved in p53 ubiquitination: — Pirh1 — COP1.

16.7.5.3 Other Controls of Mdm2 Function The function of Mdm2 is controlled at multiple levels, with one important level affecting the stability of Mdm2. As an E3 ligase, Mdm2 can catalyze self- ubiquitination, thus inducing its own proteasomal degradation, but this activity can be inhibited by a number of other proteins, including de-ubiquitinating enzymes such as HAUSP and the Mdm2 relative MdmX. The latter protein also binds, together with Mdm2, with p53 to form a trimeric complex at p53-regulated promoters, which in turn inhibits the transcription of p53 target genes. The relief of MdmX-mediated p53 inhibition is achieved by phosphorylation of MdmX, catalyzed by the damage-induced protein kinases ATM and Chk2. This phosphorylation promotes ubiquitination and the degradation of Mdmx by Mdm2. Mdm2 functions are also regulated by multiple PTMs. A number of Ser/Thr phosphorylation sites have been identiﬁed on Mdm2 that become phosphorylated in response to a variety of genotoxic stresses. Overall, the phosphorylation of Mdm2 correlates inversely with its activity towards p53, which is explained by the phosphorylation-induced export of Mdm2 from the nucleus. Several protein kinases have been implicated in these phosphorylations, including Akt kinase and ribosomal kinase (RSK), among others.

16.7.5.4 p53-Independent Functions of Mdm2 Mdm2 is a multifunctional protein that, in addition to its E3 ligase activity towards p53, also has many other substrates including the p53 relatives p63 and p73, the tumor suppressor Rb, and the transcription factor, FOXO, among others [31]. Furthermore, Mdm2 interacts with and regulates the activity of other regulatory proteins, including ribosomal proteins, dihydrofolate reductase, transcription factor E2F1 and CKI inhibitor p21Kip1. Several Mdm2-interacting proteins function in pathways that impact on genome stability, which indicates that Mdm2 deregulation has the potential to inﬂuence multiple aspects of genomic stability through proteins other than p53. & Mdm2: — Negatively regulates p53 — E3 ligase — Mediates Ub-modification of p53 — Under transcriptional control by p53 in negative feedback loop. 760 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

16.7.6 Genes Regulated by p53

The “classical” view of p53 focused on its tumor suppressing functions. However, this is currently being challenged, as roles for p53 in normal cellular homeostasis and cancer cell homeostasis become increasingly recognized. Furthermore, the range of biological functions of p53 has been expanded to include – in addition to its conventional transcription-activating functions – multiple other processes. For example, p53 has been shown to be involved in transcription repression, in the regulation of translation and homologous recombination, metabolic regulation, cellular development, and even aging. The ability to regulate such an enormous diversity of cellular processes can be traced back to the differential ways by which p53 responds to various stresses and normal cellular signals. Within a complex regulatory network, p53 can trigger a huge diversity of transcriptional and nontranscriptional responses, allowing for graded responses that will ensure the survival of the cell under mild stresses, but will induce cell death in case of severe stresses. As outlined above, the stabilization and activation of p53 occurs mainly at levels of p53 transcription and PTMs that both provide many possibilities to generate a graded response.

16.7.6.1 Selection of Target Genes One of the most intriguing questions in the ﬁeld of p53-mediated transcription is how the protein is able to discriminate between its many promoters in a stimulus-, locus-, and context-speciﬁc manner. The problem of target gene selection by transcriptional activators was discussed in detail in Section 4.3.8. For p53-mediated transcriptional activation, the following factors contribute to target gene selection and diversity of responses:

p53 recognition elements: The nature of the p53 recognition elements themselves, and the identity of further recognition elements within composite p53 promoters, can influence the binding of p53 and the subsequent recruitment of coactivators (for a general discussion of this point, see Section 4.3.8). The range of functional p53 binding sites includes many elements with one or more base-pairs that do not match the consensus [32]. Furthermore, noncanonical recognition elements have also been described. Importantly, the binding of p53 to a target promoter is not sufficient for transcription activation. A number of studies have shown that a significant portion of p53 is bound to target promoter, such as for p21Kip1, already in the absence of stress. p53 appears to be exist in a repressed DNA-bound state in these cases, possibly due to interactions with Mdm2 and MdmX. p53 PTMs and promoter selection: p53 is regulated spatially and temporally by many PTMs, that integrate the various cell-signaling pathways which converge on the p53 protein. These modifications function as signals for the recruitment of coactivators or corepressors, as chromatin-modifying enzymes, and for the establishment of specific chromatin structures around p53 target promoters, all of which contribute to target activation. As the availability of coactivators, 16.7 Tumor Suppressor Protein p53 761

corepressors and chromatin-modifying enzymes may depend on the cell type, a wide range of cell type-specific responses can be generated. Furthermore, the local chromatin architecture will influence target gene activation. The impact of individual modifications on promoter selection and activation is poorly understood, and only for some modifications could any correlation with the transcriptional outcome be established (Figure 16.15). For example, phosphorylation at Ser46 correlates with the expression of AIP1 and induction of the apoptotic program, whereas acetylation at K120 and K164 is associated with the activation of PUMA (Chapter 17). Finally, acetylation at K320 correlates with p21Kip1 expression and the induction of cell cycle arrest. p53 interaction partners: During the course of promoter binding and transcription activation, p53 interacts with a large number of different proteins or protein complexes in a dynamic manner. These interaction partners include components of the transcription machinery such as general transcription factors and the mediator complex, as well as noncanonical histones (Chapter 4). In addition, many cofactors have been described that selectively alter p53’s transcriptional program. These binding partners function either by altering the ability of p53 to recognize a specific subset of recognition elements, or by influencing the ability of p53 to recruit transcriptional coactivators at certain loci [18]. One prominent binding partner of p53 is the tumor suppressor BRCA1. By interacting with the

Figure 16.15 Selective impact of p53 factor), SET7/9, lysine acetyl transferases modifications on transactivation. Several (Section 2.6.2); PRMT, protein arginine examples are depicted of residues within p53 methyl transferase (Section 2.7); HIPK2, that can be modified by acetylation (Ac), Homeodomain-interacting protein kinase 2 phosphorylation (P), ubiquitination (Ub), or that is negatively regulated by Mdm2. For AIP, methylation. The preferential activation of the Puma, see Chapter 17. p21, CDK inhibitor, indicated target genes (and others, not see Chapter 15. TAD, transactivation domain; depicted) can result in specific cell fates, such PP, proline-rich region; L, Linker; Tet, as apoptosis or cell cycle arrest. TIP60, MOF, tetramerization domain [33]. CBP/p300, PCAF (p300/CBP associated 762 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

CTD of p53, BRCA1 can selectively direct p53 to activate the transcription of genes necessary for cell cycle arrest and DNA repair. Local chromatin structure: The architecture of the promoter, local histone deposition and the epigenetic landscape at target genes are known to be crucial determinants of target gene activation by transcription factors. This also applies to target gene selection by p53.

All of these factors are interlinked and function in concert to determine the nature of the transcriptional response.

16.7.7 Pathways Involved in the Activation of p53

& Types of stress that lead to p53 stabilization and activation: — Genotoxic stress — Oncogenic stress — Nongenotoxic stress.

A large number of stresses can activate p53 (see Figure 16.11), with stress signals being transmitted to p53 primarily by means of PTMs introduced by modifying enzymes that are part of the stress-activated signaling pathways. The PTMs regulate p53 both spatially and temporally, primarily by phosphorylation and acetylation at the N and C termini, and also within the DNA-binding domain. These modifications integrate the various cell-signaling pathways that converge on the p53 protein. Whilst the response triggered is specific for the nature and intensity of the stress, the contribution of individual modifications to the outcome of the response is mostly uncertain and the precise combinatorial impact of the numerous p53 modifications on transactivation has to date been only weakly characterized.

16.7.7.1 Activation of p53 by DNA Damage Genotoxic stress, as manifested by, for example, the formation of DNA adducts and DNA strand breaks, activates p53 mostly via DNA damage checkpoints. It is the main purpose of this control to prevent the replication of damaged DNA, which is a potentially mutagenic process. The protein kinases ATR, ATM, Chk1, Chk2 and DNA-PK are involved in these regulations (Section 15.6.2). By phosphorylating p53 on speciﬁc Ser residues, these kinases interrupt the negative feedback between p53 and Mdm which allows for p53 accumulation. The DNA damage-activated kinases enforce the activation of p53, furthermore, by phosphorylating the MDM2 proteins, which leads to an enhanced ubiquitin-dependent degradation of MDM2.

16.7.7.2 Activation of p53 by Oncogenic Stress The aberrant activation of proto-oncogenes creates a stress situation which is also termed oncogenic stress. Normal cells are protected against the detrimental 16.7 Tumor Suppressor Protein p53 763 consequences of oncogenic stress by linking oncogenic pathways to the pro-apoptotic action of the p53 pathway. The aberrant activation of proto-oncogenes to oncogenes results in an activation of the p53 pathway and allows for destruction of the cell by apoptosis. The main target of regulatory inputs during oncogenic stress is the MDM2 protein, which is downregulated under excessive survival signals, allowing for the accumulation and activation of p53 and subsequently the initiation of apoptosis. In this pathway, the ARF protein (Section 16.6.2) has a key function (see Figures 16.10 and 16.14) of binding to MDM2, thereby reducing p53 degradation. Various signals induce an increase in the ARF protein, including transcriptional activation via the Rb/E2F pathway, the Ras/MAPK pathway, and the Myc transcription factor. As noted in previous chapters, these pathways receive and transmit a multitude of survival signals. However, by indirectly stabilizing p53, ARF plays a key role in eliminating any cells that develop proliferative abnormalities, thereby protecting the organism from cancer development.

& DNA damage and p53 activation: — DNA damage activates sensor kinases ATR, ATM and Chk2 that phosphorylate and stabilize p53 — Oncogenic stress activates p53via ARF and Mdm2.

16.7.7.3 Activation of p53 by Nongenotoxic Stress A host of nongenotoxic stresses such as replication stress, translation stress, metabolic imbalances, and under- or oversupply with oxygen can also activate p53 responses. Some links between these stresses and p53 appear to be provided by a subspecies of the Jun-N-terminal kinase (JNK2; Section 12.4.2), which is activated by various stresses and enhances p53 stability by phosphorylation on Thr81. Overall, however, these linkages are only poorly characterized.

16.7.8 Classification of p53 Target Genes

At least 130 direct p53 target genes have been identiﬁed [25], while the number of indirectly affected genes probably reaches thousands, placing p53 as the number- one gene mediating stress signaling in mammals. Among the target genes are not only protein-coding genes; rather, p53 also controls the expression of micro RNAs and long noncoding RNAs. Depending on the nature and severity of stress, p53 responses can activate different programs that help to cope with the stress and minimize consequences for the cell and for the whole organism. Each program involves the activation of a distinct set of genes, often with overlapping members (see Figure 16.11). Only the main features of the p53-activated programs are presented in the following subsections (for further information, see Refs [18,34]). The nature of p53-mediated responses is thought to be dictated by the pattern of PTMs imprinted on p53 by the various stresses. Which, and how many, of 764 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

the different p53 targets and subprograms will be activated is thought to depend on the exact pattern of the PTMs and the cell type. However, the rules that govern the selection of subprograms in a cell type-speciﬁc manner are only poorly understood. Depending on the nature of the inducing stress signal, the following classes of target genes can be turned on by p53 to execute p53-mediated functions at different levels (Figure 16.16). It should be noted that the classiﬁcation of target genes given below is arbitrary, and that some targets are shared between the different classes.

16.7.8.1 p53 Targets Activated in Unstressed Cells and Under Low Intrinsic Stress One class of p53 target genes comprises genes that protect p53 from excessive activation. In addition, this class of genes provides protection against intrinsic stresses that are inevitably associated with the normal functions of the cell. It is necessary to protect cells from excessive p53 activation and to provide a low-level answer of p53 in the case of “soft insults” that do not require a strong p53 response. Of the genes activated by p53 under normal conditions, the mdm2 gene stands out as a crucial regulator of p53 (Section 16.7.5). In nontransformed cells, the p53 levels are low because of the negative feedback between p53 and Mdm2. Given that activated p53 is potentially dangerous to cell viability, the negative feedback is essential to keep p53 in check, but this control most likely does not involve the PTM of p53. In cells such as stem cells with on-going proliferation, activation of the p53 response must be dampened so as to allow normal growth and development, while retaining the capacity to induce any response to stress associated with oncogenesis. One mechanism by which the p53 response is suppressed in normal cells appears to employ the phosphorylation of MDM2 by Akt kinase. The latter enzyme is activated by growth factor receptor pathways and transmits survival signals (Section 9.4). The Akt-mediated phosphorylation of MDM2 has been shown to promote the nuclear localization of MDM2, increasing nuclear levels of the latter and promoting an inhibition of the p53 response. However, normal cells experience internal stresses, such as fluctuations in nutrient supply or benign breaks in DNA, that occur during normal DNA replication. How then is it possible for p53 to discern between harmless stresses and severe stresses that require the induction of apoptosis or cell cycle arrest? A study of normal, proliferating cells has identified pulses of activating acetylations and repressing methylations on p53 during the cell cycle, without causing any significant changes in p53 concentrations [35]. This indicates that transient p53 modifications, rather than concentration changes, are employed to cope with intrinsic stresses.

16.7.8.2 p53 Targets Activated by Moderate Stresses Another class of p53 target genes is induced by moderate stresses such as metabolic aberrations, nutrient undersupply, replication and translation stress, and moderate DNA damage. Under conditions of moderate DNA damage, when repair is possible, p53 engages a temporary program of cell cycle arrest and DNA repair. 16.7 Tumor Suppressor Protein p53 765

Figure 16.16 Examples of genes regulated by Repressed genes: APC, see Section 16.8; p53. For apoptotic targets, see Chapter 17. CDC25C, see Chapter 15; Survivin, baculoviral Senescence targets: p21, cell cycle inhibitor, IAP repeat; DNMT1, see Section 4.5.9.1. Cell Section 15.2.3. Metabolism targets: AMPK, cycle arrest targets: GADD45, growth arrest AMP-dependent protein kinase; Tigar, p53- and DNA damage-inducible gene 45; 14-3-3, induced glycolysis and apoptosis regulator. see Section 2.4.5, Table 2.1; DNA repair Antioxidant targets: ALDH, aldehyde targets: MSH2, mutS homolog 2, involved dehydrogenase; GPX, glutathione peroxidase. in mismatch repair; XPC, Xeroderma Autophagy targets: DRAM, damage-regulated pigmentosum complementation group C; autophagy modulator; Puma, see Chapter 17. FANCC, Fanconi anemia complementation Angiogenesis targets: Maspin, serpin group C. peptidase inhibitor; TSP1, thrombospondin1; 766 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

The protective programs activated by mild stresses allow the cells to pause and to repair any damage incurred, thereby limiting the propagation of oncogenic mutations and ensuring cell survival. Examples of genes activated by moderate stress include genes for the cell-cycle inhibitor p21 and for the repair proteins XPC and MSH2, among many others. An additional protective mechanism against mild stresses is the capacity of p53 to upregulate the expression of antioxidant genes (such as the gene for GPX1) which suppress the accumulation of reactive oxygen species, thereby maintaining genomic integrity. Further tumor-suppressing functions of p53-induced transcription have also been discovered. For example, p53 inhibits glycolysis and promotes oxidative phosphorylation to protect cells from metabolic reprogramming; this so-called “Warburg effect” is thought to be fundamental for malignant transformation (Section 16.7.9). p53 can also limit tumorigenesis through autophagy (“self- eating”), which can provoke cell death through the activation of genes such as AMP-activated protein kinase (AMPK) and damage-regulated autophagy modulator (DRAM). The latter gene encodes a lysosomal protein that induces autophagy.

16.7.8.3 p53 Targets Activated by Sustained or Severe Stress In response to sustained or severe stress signals, p53 engages a third class of target genes that are primarily involved in the triggering of two irreversible programs, namely apoptosis and senescence. These responses are triggered primarily by severe genotoxic oncogenic stresses by strand. The induction of apoptosis by p53 includes the activation of many genes involved in the intrinsic and extrinsic pathways of apoptosis, such as genes for death receptors, the gene for the pro-apoptotic protein Bax, and for the apoptotic regulators Noxa and Puma. Activation of these genes collaboratively promotes cell death. & Apoptotic genes regulated by p53: — Bax — Noxa — Death receptor: Fas — Puma.

In contrast, the gene for the antiapoptotic protein Bcl-2 (Section 17.3) is repressed by p53. These effects show that p53 promotes apoptosis by enforcing the proapoptotic signals and blocking the antiapoptotic signals. In another approach, p53 responds to potent stress by inducing cellular senescence through the transcriptional activation of target genes such as the inhibitor p21Kip1 (Section 15.2.3) and the tumor suppressor, PML. p53-induced senescence is an irreversible cell cycle arrest that helps to prevent malignant progression in the face of for example, aberrant oncogene activation. In many carcinomas and sarcomas, the senescence program is triggered instead of the apoptotic program. 16.7 Tumor Suppressor Protein p53 767

16.7.8.4 p53-Mediated Repression The p53 protein functions as a speciﬁc transcription activator, but it can also bring about a repression of distinct genes. The importance of gene repression by p53 is underscored by the observation that activating and repressing p53 response elements have characteristic nucleotide distribution patterns. Surprisingly, about 24% of the known p53-responsive elements have been classiﬁed as repressive [32]. Several mechanism have emerged for repression mediated by p53 [18]:

p53 binding to a repressive response elements leads to the recruitment of corepressor complexes. p53 can activate the transcription of a repressor protein that suppresses the downstream events necessary for cell-cycle progression. For example, p53 activates p21 expression which suppresses the expression of important cell cycle genes via the p21Kip1–CDK4–Rb–E2F axis (see Section 16.4). p53 binding to its recognition element may interfere with the function of other transcription factors that cooperate in a composite promoter.

Examples of genes repressed by p53 include the genes for the transcription activators c-Jun and c-Fos, the cytokine IL-6, the retinoblastoma protein Rb, the Bcl- 2 protein, the DNA-methyltransferase DNMT1, and for the phosphatase CDC25, among others. In addition, a number of miRNAs can be induced by p53 (Section 5.3.2; Figure 5.15), as well as repressive long noncoding RNAs.

16.7.9 Metabolic Regulation by p53

Aberrant metabolism plays a role in many diseases, particularly cancer, and p53 has now emerged as an important regulator of cellular metabolism during normal cellular homeostasis as well as cancer cell homeostasis [34]. Among the metabolic pathways regulated by p53 can be included glycolysis, oxidative phosphorylation, insulin sensitivity, nucleotide biosynthesis, fatty acid oxidation and anti-oxidant responses. Today, p53 is emerging as an important regulatory component in these pathways by activating or repressing key metabolic enzymes. In this way, p53 helps cells to deal with metabolic fluctuations, not only by balancing their proliferation and growth with nutrient availability but also by limiting the accumulation of further damage. Overall, p53 mediates an adaptive response during metabolic fluctuations and stresses. The control of glycolysis by p53 may be presented here as a brief example. An elevated rate of glycolysis under aerobic conditions drives anabolism and increases cell proliferation, and is a characteristic of stem cells and many cancers. p53 has the ability to limit flux through glycolysis, and favors use of the tricarboxylic acid (Krebs’) cycle for energy production, which maximizes energy production under conditions of nutrient deprivation. The loss of p53 function in tumors will direct the metabolism towards aerobic glycolysis – the Warburg effect – that underpins the growth of most cancer cells. 768 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

The mechanisms by which p53 inﬂuences the rate of glycolysis are diverse. For instance, p53 negatively controls glucose transporters, represses the promoter of the insulin receptor, and activates expression of the TP53-induced glycolysis and apoptosis regulator (TIGAR). The latter protein functions as a fructose-2,6- bisphosphatase that counteracts phosphofructokinase-1 and so lowers the rate of glycolysis. Selected p53-activated genes involved in metabolic regulation and homeostasis are shown in Figure 16.16.

& p53 is a transcriptional regulator that can function as an activator or repressor of transcription

& Important target genes of p53: — E3 ligase Mdm2 — p21Cip1 inhibitor — 14-3-3 sigma — Gadd45 — Many transcription factors.

& Apoptotic genes regulated by p53: — Bax — Noxa — Death receptor: Fas — Puma.

16.7.10 p53-Mediated Induction of miRNA

p53 activates the transcription of many miRNAs, and thereby controls pathways regulating its own stability and the production of tumor suppressor miRNAs. Furthermore, p53-regulated miRNA transcription activates different anti-oncogenic pathways (Section 4.3.2.5). At the same time, p53 can be negatively controlled by oncogenic miRNAs [5].

16.7.11 Summary of Tumor Suppression by p53

Tumor suppression by p53 involves the following responses (Figure 16.17): Irreversible responses:

Induction of apoptosis by inducing the transcription of pro-apoptotic proteins and the repression of anti-apoptotic proteins. 16.7 Tumor Suppressor Protein p53 769

Figure 16.17 Dual mechanisms of p53 and promote the repair of genotoxic damage. function in tumor suppression and aging. p53 Sustained stress or irreparable damage, on the can help to promote the repair and survival of other hand, induces the killer functions of p53 damaged cells, or it can promote the to activate cell death or senescence. Notably, permanent removal of damaged cells though the protector functions of p53 could contribute death or senescence. The ultimate result of to tumor development if not properly regulated p53 activation depends on many variables, (red, dashed arrow). Some of p53’s protector including the extent of the stress or damage. In functions may also help to enhance longevity, this model, basal p53 activity or that induced whereas the consequences of p53’s killer by low-stress elicits the protector responses functions can promote aging. After Ref. [36]. that support cell survival, control glycolysis,

Induction of senescence by inducing growth and cell cycle arrest for example, via p21Kip1 expression. Induction of apoptosis via direct interaction with apoptotic components.

Reversible responses that ensure cell survival:

Induction of DNA repair by stimulating expression of repair proteins. Upregulation of antioxidant responses. Adaptation of cell growth, by downregulation of protein biosynthesis. 770 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

Induction of autophagy. Adaptation of cell metabolism and homeostasis. The main mechanisms leading to abrogation of the tumor-suppressing functions of p53 include mutation and/or aberrant destabilization of p53: Tumorigenic mutations of p53: Most mutations of p53 observed in cancer cells are located in the DNA-binding domain. This interferes with the transcription- activating functions of p53, and may lead to inappropriate or incomplete activation of the p53-triggered programs. Other mutations of p53 may interfere with PTMs, and may impede the ability of p53 to receive activating signals and trigger the appropriate transcriptional response. Aberrant destabilization of p53: Inactivation of the Mdm2–p53 feedback by oncogenic mutations of Mdm2 will downregulate p53 levels and activity. As a consequence, the induction of for example, apoptosis or senescence, will be suppressed and cells can survive in the presence of DNA damage or under conditions of genomic instability. An important contribution to aberrant destabilization of p53 can also come from inactivation of the tumor-suppressing function of ARF that keeps Mdm2 activity in check.

16.8 Wnt/b-Catenin Signaling and the Tumor Suppressor APC

The extracellular signaling protein Wnt is at the top of signaling pathways that are key regulators of many developmental processes. The Wnt genes that code for secreted lipoproteins can initiate various pathways of which the canonical Wnt/ b-catenin pathway has been best-characterized [37]. This pathway is a critical regulator of stem cells, and is frequently altered in many human malignancies such as colorectal cancers, hepatocellular carcinomas, and gastric cancers. The Wnt/b-catenin pathway harbors two proteins with tumor-suppressing activity, the APC (adenomatous polyposis coli) protein and axin, as well as an oncogenic protein, namely b-catenin. & Tumor suppressor APC: — Regulates cell proliferation, cell adhesion, apoptosis, and cell differentiation — Involved in colon cancer formation — Cooperates with axin and b-catenin.

16.8.1 APC

The APC gene encodes a multifunctional protein involved in central biological processes, including cell adhesion and migration, proliferation, apoptosis, and differentiation [38]. Most functions of the APC protein are linked to the Wnt/b-catenin signal transduction pathway (Figure 16.18), which leads from 16.8 Wnt/b-Catenin Signaling and the Tumor Suppressor APC 771

Figure 16.18 Schematic overview of the Wnt Wnt signal reaches a cell, Wnt associates with signaling pathway. Central to wnt signaling is a the transmembrane receptor Frizzled (Fz), protein complex composed of b-catenin, APC, leading to activation of the cytoplasmic protein glycogen synthase kinase 3 (GSK3) and axin. Disheveled by an unknown mechanism. (a) In the absence of the extracellular signaling Disheveled inhibits b-catenin phosphorylation protein Wnt, b-catenin is phosphorylated by and degradation, and b-catenin now acts as a GSK3. Upon phosphorylation, b-catenin is transcriptional coactivator for the TCF family of primed for ubiquitinylation and subsequent transcription factors which activates target degradation by the proteasome. Reduced genes that regulate diverse cellular responses. levels of b-catenin permit repression of Wnt Levels of b-catenin can be also regulated by target genes by association of transcriptional p53 and by integrins. corepressors such as Groucho; (b) When a 772 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

transmembrane receptor-bound Wnt down to the level of transcription. In this pathway, signals are transduced from Wnt to the transcriptional regulator b-catenin, which controls the expression of numerous target genes, including the gene for cyclin D1 and the transcription factor Myc.

& Wnt/b-catenin signaling: — Delivers signals from the cell membrane to the level of transcription — b-catenin: transcriptional regulator under control of Wnt.

Low levels of b-catenin prevent transcription of the target genes, while high levels of b-catenin stimulate target expression. As the targets include critical regulators of cell growth and cell division, deregulation of the Wnt/b-catenin pathway has been implicated in various diseases, including cancer. Mutations in the APC tumor suppressor gene strongly predispose to the development of gastrointestinal tumors. Central to the tumorigenic events in APC mutant cells is the uncontrolled stabilization and transcriptional activation of b-catenin.

16.8.1.1 Downregulation of b-Catenin b-catenin may exist either in membrane-bound form in complex with the cell- adhesion molecule E-cadherin, or in a cytosolic form. In the absence of a Wnt signal, the expression of b-catenin targets is downregulated. Cytosolic levels of b-catenin are low because b-catenin is primed for Ub-dependent proteasomal degradation, which occurs in a cytosolic degradation complex composed of b-catenin, APC, glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1) and the scaffolding protein axin. A sequential phosphorylation of b-catenin by CK1 and by GSK3 at Ser/Thr residues marks b-catenin for ubiquitination by the E3 ligase b-TrCP and subsequent proteasomal degradation.

16.8.1.2 Activation of b-Catenin The stabilization of b-catenin requires disruption of the degradation complex, which is initiated by activation of the Wnt signaling pathway. Signaling through the Wnt pathway starts with binding of the extracellular Wnt ligand to two transmembrane receptors, the frizzled receptor, and the LRP coreceptor. Frizzled is a seven-helix transmembrane receptor, while LRP is a single-pass transmembrane receptor belonging to the class of low-density lipoprotein receptor-related proteins. The Wnt-induced signaling complex leads to the phosphorylation of LRP at ﬁve Ser/Thr phosphorylation motifs located in the intracellular domain of LRP. The phospho-sites created in this way then serve to recruit axin to the signaling complex, removing it from the degradation complex. As a consequence, b-catenin is uncoupled from the degradation complex, leading to the stabilization and accumulation of cytosolic b-catenin; the latter then travels to the nucleus where it activates its target genes in cooperation with the transcription factors TCF/LEF. Loss of APC function leads to an uncontrolled activation of the Wnt/b-catenin pathway and provides a proliferative advantage to the mutated cell. In addition, Questions 773 chromosomal instability is observed in cells with decreased APC function. The scaffold protein axin has been identiﬁed as a further tumor suppressor in this pathway, and b-catenin can be activated to an oncogene by mutation or aberrant expression. There is also a link to p53 function which downregulates b-catenin levels in response to DNA damage. Overall, an aberrant function of the Wnt/ b-catenin pathway is observed in almost all colorectal cancers, as well as in many other cancers.

Questions 16.1. Describe the spectrum of mutations found in cancer cells. Give examples of at least three such mutations, and describe the functions of the proteins affected. 16.2. Which epigenetic mechanisms may contribute to tumorigenesis? Describe the principal features of these mechanisms and explain how these processes may promote tumor formation. 16.3. What are the hallmarks of cancer? Give examples of the pathways affected for at least two of these hallmarks. 16.4. What is the deﬁnition of an oncogene? Which mechanism may contribute to the oncogenic activation of a gene? Give examples of at least two of these mechanisms. 16.5. EGF receptor members are involved in the formation of several types of tumor. Describe the function of these receptors and the signaling paths that are triggered by EGFRs. 16.6. Describe the properties of Abl kinase and its involvement in chronic myelogenous leukemia. 16.7. MYC is a frequently mutated oncogenic transcription factor. Describe the main structural properties and protein interactions of Myc. Name and describe at least two signaling pathways that control Myc function. 16.8. Describe the general characteristic of a tumor suppressor. Which classes of tumor suppressors do you know? Give an example of each of these classes. 16.9. Which mechanism may contribute to inactivation of the tumor suppressor Rb? 16.10. Describe the functions of the proteins encoded by the p16INK gene locus. 16.11. Describe the function of p53 in tumor prevention. Which domains are found on p53 and what is the function of these domains? 16.12. How does Mdm2 control p53 function? Which signaling pathways override this control, and how are these signals delivered into the Mdm2–p53 network? 774 16 Malfunction of Signaling Pathways and Tumorigenesis: Oncogenes and Tumor Suppressor Genes

16.13. Which classes of genes are under transcriptional control by p53? How does p53 activation trigger apoptosis, and how may p53 activation lead to a halt in the cell cycle? 16.14. Describe the logic and major reactions of Wnt/b-catenin signaling.

References

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17 Apoptosis

Eukaryotic cells can self-destruct in an orderly, highly controlled process known as apoptosis. The term apoptosis, which was ﬁrst coined following investigations of the nematode Caenorhabditis elegans, is of Greek origin and describes the “falling of leaves.” Activation of the apoptotic program involves the coordinated demolition of intracellular structures by members of the caspase family of proteases. This is accompanied by characteristic changes in cell morphology, such as condensation of the chromatin, the degradation of DNA, cell shrinkage, fragmentation of the cell nucleus, and disassembly into membrane-enclosed apoptotic vesicles. Apoptosis is based on a genetic program that forms an indispensable part of the development and function of an organism, and serves to eliminate any undesired or superﬂuous cells in a targeted manner. The functions of apoptosis target the major biological processes of the cell, including:

tissue homeostasis; the elimination of cells during differentiation and development; the elimination of cells during immune responses; and the elimination of damaged cells to avoid propagation of mutations and degeneration into tumor cells.

The major part of the apoptotic program, which exists in the cell in a latent, inactive form, requires only an apoptotic stimulus to activate the program and to induce apoptosis. Thus, apoptotic processes may be initiated within a short timescale, without the activation of transcription. Some forms of apoptosis are also known that are dependent on transcription. Due to the potential deleterious consequences of an inappropriate activation of apoptosis in normal cells, the apoptotic program is strictly controlled by the balanced input of pro-apoptotic and anti-apoptotic signals originating from internal and external sources. Many signals that feed into the apoptotic program are transmitted via cellular signaling pathways involved in the control of cell proliferation and homeostasis. Furthermore, all signaling pathways that form part of stress responses and DNA-damage checkpoints have links to the apoptotic program and can induce its activation.

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 778 17 Apoptosis

& Apoptosis: — Cellular program for the elimination of cells — Involves the proteolytic degradation of cellular structures by caspases — Controlled by internal and external signals — Linked to stress response and DNA-damage checkpoints.

A brief overview of the major aspects of the apoptotic machinery will be provided in the following subsections. As apoptosis has multiple linkages to nearly all physiological processes of the cell, only linkages between the major signaling pathways described in the preceding chapters and apoptosis will detailed at this point.

17.1 Overview of Apoptotic Pathways

At the center of the apoptotic program is a family of proteases named caspases. These enzymes are involved in the initiation and execution of the program, and can be activated by a large number of stimuli to induce cell death by degrading key cellular components. As a consequence of caspase activation, a number of key enzymes and structural proteins of the cell are degraded, leading to cell death. The stimuli that induce apoptosis are very diverse, and include DNA damage, stress conditions, and the malfunction of pathways regulating cell proliferation. There are two main pathways for activation of caspases: (i) an intrinsic pathway involving mitochondria; and (ii) an extrinsic pathway that involves death receptors.

& Major apoptotic pathways: — Death receptor-triggered pathway — Mitochondrial pathway.

The activation of apoptosis via mitochondria is the most frequently used apoptotic pathway. It is an intrinsic pathway where stress signals, DNA damage signals and defects in signaling pathways are processed. In contrast, the extrinsic pathway uses external signals transmitted via transmembrane receptors of the TNFb class (death receptor class; see Section 14.3) to trigger the apoptotic program. The external signaling proteins – the “death ligands”–bind to and activate the death receptors for triggering the apoptotic response. This pathway is mainly used in developmental processes and in the immune system. In addition, two other less well-characterized apoptotic pathways are emerging, namely endoplasmic reticulum stress-induced apoptosis and caspase-independent apoptosis; however, the details of these systems will not be presented here.

& Minor apoptotic pathways: — ER-stress-induced apoptosis — Caspase-independent apoptosis. 17.2 Caspases: Death by Proteolysis 779

Figure 17.1 The major pathways of apoptosis. multitude of intracellular signals including The extrinsic pathway uses extracellular death various stresses, DNA damage and ligands [Fas ligand, tumor necrosis factor inappropriate cell signaling lead to activation (TNF)] to activate transmembrane receptors of the proapoptotic protein Bax which induces (“death receptors” Fas-CD95, TNF receptor) release of cytochrome c from mitochondria, which pass the apoptotic signal to initiator formation of the apoptosome and activation of caspases (e.g., capsase-8) and to the effector the initiator caspase-9. Finally, the effector caspases (e.g., caspase-3; caspase-7). In the caspases are activated and cells are destroyed execution phase of apoptosis, various cellular by proteolysis. Apoptosis via this pathway can substrates are degraded, leading to cellular be controlled by various antiapoptotic collapse. The intrinsic pathway uses the proteins, including the Bcl-2 protein and mitochondrion as a central component for the inhibitors of apoptosis. activation of apoptosis. In this pathway, a

An overview of apoptosis is shown in Figure 17.1. The function and regulation of the components of apoptosis are discussed in more detail below.

17.2 Caspases: Death by Proteolysis

A family of specialized proteases, the caspases, is central to the apoptotic program [1] (the term caspase is a contraction of cysteine-dependent, aspartate-speciﬁc 780 17 Apoptosis

protease). These proteases employ a Cys residue as a nucleophile, and cleave the substrate after an Asp residue. The only known eukaryotic proteases with this speciﬁcity are the caspases themselves and the cytotoxic serine protease granzyme B that is present in T lymphocytes.

& Properties of caspases: — Proteases with Cys as nucleophile — Cleavage after Asp — Synthesized as inactive proenzymes — Require proteolysis for activation.

To date, 14 mammalian caspase sequences (named caspases 1–14) have been reported, and 11 of these are of human origin. With respect to function, the caspases are grouped into two biologically distinct subfamilies. The first subfamily mediates either the initiation (initiator caspases; caspase-2, -8, -9 and -10) or the execution (effector or executioner caspases; caspase-3, -6 and -7) of the apoptotic program. Members of the second subfamily (caspase-1, -4, -5, -11, -12, -13 and -14) are involved in the inflammatory processes by virtue of their ability to process proinflammatory cytokines.

& Caspase subfamilies are classified by function: — Apoptotic caspases — Inflammatory caspases.

Like many other proteases, caspases are synthesized as inactive proenzymes, but are rapidly activated by autoproteolytic cleavage or cleavage by other caspases at speciﬁc aspartic acid residues.

& Apoptotic caspases: — Initiator caspases: caspase-2, -8, -9, -10 — Effector caspases: caspase-3, -6, -7. Inflammatory caspases: — Caspase-1, -4, -5, -11, -14.

17.2.1 Initiator and Effector Caspases

The apoptotic caspases are generally divided into two categories, the initiator caspases, which include caspase-2, -8, -9, and -10, and the effector caspases, which include caspase-3, -6, and -7. An initiator caspase is characterized by an extended N- terminal prodomain (>90 amino acids), whereas an effector caspase contains 20– 30 residues in its prodomain sequence. 17.2 Caspases: Death by Proteolysis 781

17.2.1.1 Initiator Caspases Initiator caspases are the ﬁrst to be activated in response to a proapoptotic stimulus, and are responsible for activating the effector caspases by limited proteolysis. The activation of the initiator caspases occurs in multiprotein complexes by an autocatalytic process that does not necessarily require proteolysis of the procaspase form. One characteristic of the initiator caspases is the presence of homotypic CARD (caspase-recruitment domain) or DED (death effector domain) interaction domains at their N-terminal prodomain (Figure 17.2). These modules direct initiator procaspases to oligomeric activation assemblies in the cell.

& Initiator caspases: — Activated in multiprotein complexes — Contain CARD or DED domains in their prodomain.

17.2.1.2 Effector Caspases The apoptotic effector caspases are deﬁned by the absence of recognizable homotypic recruitment domains (Figure 17.2). Together, they are responsible for the majority of the limited proteolytic events that combine to create the characteristic cellular changes that direct the cell to death.

& Effector caspases: — Activated by initiator caspases — Contain small prodomains.

17.2.2 Mechanism of Caspases

The fundamental catalytic domain of all caspases is made up of one a-subunit (17–12 kDa) and one b-subunit (10–13 kDa), which form a heterodimer with an active site composed of residues from both subunits. Two heterodimers then may align to form a tetramer with two catalytic centers (Figure 17.2). Caspases belong to the Cys-family of proteases. A catalytic dyad comprised of a reactive cysteine and a histidine residue is used for substrate cleavage, and a covalent thioacyl intermediate is formed during catalysis.

& Enzymatic properties of caspases: — Catalytic dyad Cys/His — Formation of covalent Cys–substrate intermediate — High cleavage specificity.

The special feature of the caspases is their high cleavage speciﬁcity based on the recognition of a tetrapeptide located N-terminally to the canonical cleavage site Asp- X. By virtue of cleavage speciﬁcity, the caspases can be grouped into three families, 782 17 Apoptosis

Figure 17.2 Domain structure of human caspase that contains a DED (death effector initiator and caspases. (a) The structure of domain). Caspase-9 is an initiator caspase (like caspase-3 stands for the structure of the other caspase-2), that carries a CARD (caspase effector caspases, caspase-6 and caspase-7. recruitment domain). The four surface loops Caspase-8 (like caspase-10) is an initiator (L1---L4) that shape the active site are shown as 17.2 Caspases: Death by Proteolysis 783

which differ mainly in position P4 of the tetrapeptide recognized on the substrate protein.

17.2.3 Caspase Activation and Regulation

The unprogrammed activation of the caspases has serious consequences for the cell, and consequently the activation of caspases is strictly controlled. In the normal state of the cell, the caspases are maintained in an inactive state, but they can be rapidly and extensively activated by a small inducing signal. The control of caspase activity occurs at two levels. The first level involves a conversion of the caspase precursors, the proenzymes, to the active forms in response to inflammatory or apoptotic stimuli. The initiator and executioner caspases use different mechanisms for proenzyme activation (Figure 17.3). The initiator caspases are activated in large protein complexes, the death platforms, whereas the executioner caspases are activated by the initiator caspases. The second level of caspase control involves the specific inhibition by the binding of natural inhibitors.

17.2.3.1 Death Platforms The activation of initiator caspases typically occurs in large multiprotein assemblies, the molecular death platforms. Many of the interactions responsible for the assembly of the death platforms involve proteins that are members of the death domain superfamily that includes variants such as the death domain (DD), death effector domain (DED), and caspase recruitment domain (CARD) (for details, see Ref. [1]). The domains are found on several components of the apoptotic signaling pathways and mediate homotypic protein–protein interactions; that is, a given module will interact only with a member of the same family and not with members of the other families. As members of the same module are found on different proteins, these modules mediate the assembly of heterooligomeric protein complexes. J boxes. The position of the first activating determining residues R179, H237, C285 and cleavage is indicated by a red arrow, and R341 are brought into the characteristic cleavage of the prodomain regions is indicated structural arrangement for catalysis. C285 is by a black arrow. The position of the active the catalytic nucleophile and H237 represents site cysteine is shown as a red line at the the general base. The crystal structures reveal beginning of loop L2; (b) Processing and that the active enzyme is a dimer in which one subunit structure of caspases. Schematic (ab) unit that harbors the active site is related representation of the proteolytic activation of by a twofold axis to a second unit to form the ab caspases. Caspases are synthesized as single- active ( )2 dimer. By their cleavage specificity, chain precursors. Activation proceeds by caspases are grouped into three families. cleavage of the N-terminal peptide at Asp119 Caspases recognize a sequence of four amino and at the conserved sites Asp296 and Asp316 acids and cleave the substrate C-terminal to (all caspase-1 numbering convention), leading the aspartate residue, as indicated by the to the formation of a large a-subunit and a arrow. small b-subunit. The activity- and specificity- 784 17 Apoptosis

Figure 17.3 Maturation of initiator and higher oligomers of the initiators bound to the executioner caspases. Initiator caspases are death platforms. Executioner caspases are activated upon recruitment into the PIDD, processed by activated initiators to yield apoptosome or DISC death platforms, as tetramers composed of two small and two indicated. Activation occurs in dimers or large subunits.

DDs are able to assemble into oligomeric complexes, which vary in their stoichiometry depending on the particular DD and signaling pathway. This is essential for the second role carried out by CARD and DED modules, namely the recruitment of downstream effector proteins. The CARDs and DEDs interact homotypically with other CARD or DED domains that are present in many effector proteins, including caspases and kinases, bringing them into sufﬁcient proximity for activation. At present, three types of death platform are known [2]:

PIDDosome: This is a soluble death platform that triggers the activation of caspase-2. In this process, caspase-2 is recruited into and activated within a multiprotein assembly centered on the oligomerization of the DDs of two types of subunit, named RAIDD (receptor-interacting protein (RIP)-associated ICH-1/ CED-3-homologous protein with a death domain) and PIDD (p53-induced protein with death domain). DISC: The extrinsic apoptotic pathway is triggered by death-inducing signaling complexes (DISCs), which form when a death ligand binds to a death receptor such as the prototypic Fas receptors (see Section 17.5). Caspase-8 is activated in this process. 17.2 Caspases: Death by Proteolysis 785

Apoptosome: Formation of the apoptosome initiates the intrinsic apoptotic pathway (Section 17.4). The initiator caspase-9 is recruited and activated in the apoptosome, and this is mediated by interactions between CARD domains of the platform and CARD domains on caspase-9.

17.2.3.2 Activation of Initiator Caspases The initiator caspases receive proapoptotic signals and initiate the activation of a caspase cascade. They are activated during assembly into the death platforms, and their large prodomains are involved in this interaction.

& Initiator caspases are activated in multiprotein complexes: — Caspase 8 activated in DISC — Caspase 9 activated in apoptosome.

In the “off” state, initiator caspases are inert monomers that require homodimerization for activation (Figure 17.3). Physiologically, dimerization is facilitated by caspase recruitment to oligomeric activation platforms, the death platforms, that assemble subsequent to an apoptotic signal. Thus, activation of the initiator caspase appears to be possible without proteolytic cleavage, by dimerization within the death platform only [3].

& Interaction modules in apoptogenic multiprotein complexes: — DD (death domain) — DED (death effector domain) — CARD (caspase recruitment domain) — Pyrin domain.

17.2.3.3 Activation of Effector Caspases The activation of an effector caspase (such as caspase-3 or -7) is performed by an initiator caspase (such as caspase-9) through cleavage at speciﬁc internal Asp residues that separate the large (a-chain) and small (b-chain) subunits (Figure 17.2). As a consequence of the intrachain cleavage, the catalytic activity of an effector caspase is enhanced by several orders of magnitude.

& Effector caspase activation: — Cleavage at internal Asp sites — Catalyzed by initiator caspases.

17.2.3.4 Control by Inhibitor Proteins Caspases can be directly inhibited by the binding of inhibitory proteins. Currently, three families of proteins are known that are capable of ablating caspase activity in vivo and in vitro. Two of these families are of viral origin, whilst the third family – 786 17 Apoptosis

the inhibitor of apoptosis (IAP) family – comprises eight members in mammals [4]. The IAPs are conserved from ﬂies to humans, and regulate cellular apoptosis by direct caspase inhibition.

& IAPs (inhibitors of apoptosis): — Inhibit caspases — Eight distinct IAPs.

17.2.3.5 Caspase Substrates A large number of caspase substrates have been identiﬁed, some of which have a direct relationship to the survival of the cell. The caspase substrates can be grouped into different classes according to their function:

Procaspases: The triggering of caspase cascades involves the transactivation of a procaspase by already-activated caspases (Figure 17.2). Pro- and anti-apoptotic proteins: Examples of anti-apoptotic proteins degraded by caspases are the Bcl-2 and Bcl-xL proteins, which are cleaved by caspase-3 to generate C-terminal fragments that are pro-apoptotic. Caspase-8 cleaves the proapoptotic protein Bid (Section 17.4), generating a C-terminal fragment that induces release of cytochrome c from mitochondria. DNAase inhibitor ICAD (inhibitor of caspase-activated DNAase): The caspase- mediated degradation of ICAD relieves the inhibition of a DNAase responsible for DNA fragmentation. Structural proteins: gelsolin, lamin. Proteins important for cellular signaling, DNA repair and macromolecular synthesis: PKCd, Rb, Wee1 kinase, poly(ADP-ribose) polymerase (PARP).

& Caspase substrates: — Procaspases — Pro-apoptotic proteins (e.g., Bcl-2) — Antiapoptotic proteins (e.g., Bid) — DNAase inhibitor ICAD — Structural proteins — Signaling proteins — DNA repair proteins.

17.3 The Family of Bcl-2 Proteins: Gatekeepers of Apoptosis

Bcl-2 family members are the key regulators that control mitochondrion-mediated apoptosis [5]. Mammals possess an entire family of Bcl-2 proteins that includes both proapoptotic and antiapoptotic members. The ratio of antiapoptotic to proapoptotic molecules such as Bcl-2/BAX constitutes a rheostat that sets the threshold of 17.3 The Family of Bcl-2 Proteins: Gatekeepers of Apoptosis 787 susceptibility to apoptosis in the intrinsic pathway, which utilizes organelles such as the mitochondrion to amplify death signals.

& Bcl-2 family: — Includes proapoptotic and antiapoptotic proteins — Contain BH motifs (BH1–BH4) — Three subclasses.

All Bcl-2 family members have at least one copy of a so-called BH motif (BH; Bcl-2 homolog) of which there are four types (BH1–BH4). On the basis of structural and functional criteria, the Bcl-2 family has been divided into three main subclasses (Figure 17.4):

& Antiapoptotic Bcl-2 members: — Bcl2-2, Bcl-X — Contain BH1–BH4 motifs Proapoptotic Bcl-2 members: — Bak, Bax — Lack BH4 motif — Are kept inactive by binding to antiapoptotic Bcl-2 members.

Antiapoptotic Bcl-2 members (Bcl-2, Bcl-X, Bcl-W, Mcl-1): These proteins harbor BH domains 1 to 4 and a hydrophobic C-terminal tail with which they span the cytosolic surface of various intracellular membranes, such as the outer

Figure 17.4 Domain structure of the Bcl-2 includes the proapoptotic proteins Bax and family. On the basis of functional and Bak which are similar in structure to the Group structural criteria, the Bcl-2 family has been I proteins but lack the N-terminal BH domain. divided into three groups. Group I comprises Group III consists of the “BH3-only proteins,” antiapoptotic proteins characterized by four including Bid, Bad, Nora, and so, on that short, conserved Bcl-2 homology (BH) contain a single BH3 domain and have a domains, known as BH1---BH4. Group II proapoptotic function. 788 17 Apoptosis

mitochondrial membrane. All members of this group have antiapoptotic functions. They serve a principal role of binding and sequestering the BH3-only proteins. In doing so, activation of the proapoptotic Bax and Bak proteins is prevented. Proapoptotic Bcl-2 members (Bak, Bax): The Bak and Bax proteins are similar in structure to the antiapoptotic Bcl-2 members, but lack the N-terminal BH4 domain. Both proteins perform a proapoptotic function in the mitochondrial pathway of apoptosis. In healthy cells, Bak is bound by the antiapoptotic proteins Mcl-1 and Bcl-Xl and kept in an inactive state. To induce apoptosis, this complex must be dissolved, a step that requires participation of the BH3-only proteins in response to an apoptotic stimulus. A similar step is thought to contribute to Bax activation. The activation of Bak/Bax by apoptotic stimuli ﬁnally leads to their oligomerization and insertion into the outer membrane of the mitochondrion. BH3-only proteins: This group consists of large proteins that contain a single BH3 domain [6]. The BH3-only proteins are ascribed the function of upstream sentinels that monitor cellular well-being and are activated by a variety of cellular stresses (Figure 17.5). Once activated, they initiate apoptosis by binding the Bcl-2 antiapoptotic proteins (Bcl-2, Bcl-X) via the BH3 domain. This neutralizes the antiapoptotic action of these proteins and the mitochondrial path of apoptosis is initiated. To avoid unwarranted cell death, BH3-only proteins are restrained by

Figure 17.5 BH3-only proteins monitor typically activated following caspase cleavage, cellular well-being. BH3-only proteins are amplifies the apoptotic response by engaging activated by a variety of cellular stresses. Once the Bcl-2 prosurvival proteins. Bortezomib is a activated, they initiate apoptosis by binding proteasome inhibitor. HDAC, histone and neutralizing Bcl-2 prosurvival proteins via deacetylase. their BH3 domain (red triangle). Bid, which is 17.4 The Mitochondrial Pathway of Apoptosis 789

multiple mechanisms, such as transcriptional control (Bim, Puma and Noxa) and posttranscriptional modiﬁcations (PTMs).

& BH3-only proteins: — Initiate apoptosis — Activated by stress — Contain a single BH3 domain — Neutralize antiapoptotic Bcl-2 proteins — Bind antiapoptotic Bcl-2 proteins via BH3 domain.

17.4 The Mitochondrial Pathway of Apoptosis

The mitochondrial pathway is the major pathway activated in response to cellular stresses such as DNA damage, hypoxia, growth factor deprivation and aberrant oncogene activation. The various stresses elicit the activation of proapoptotic proteins such as the BH3-only proteins and the Bak/Bax proteins, in a pathway that culminates in the permeabilization of the mitochondrial membrane and the release of apoptogenic proteins (Figure 17.6).

17.4.1 Permeabilization of the Mitochondrial Outer Membrane

The crucial event in the mitochondrial pathway of apoptosis is permeabilization of the mitochondrial outer membrane. This occurs suddenly during apoptosis, leading to the release of proteins that promote apoptosis in a caspase-dependent and a caspase-independent fashion. Furthermore, mitochondrial outer membrane permeabilization is accompanied by a loss of mitochondrial functions that are essential for cell survival.

& Mitochondrial pathway of apoptosis: — Involves permeabilization of mitochondrial outer membrane — Initiated by internal signals — Involves activation of BH3-only proteins — Leads to release into cytosol of cytochrome c and other proteins.

In most cases, apoptotic stimuli are funneled into the intrinsic pathway via the BH3-only proteins that counteract the functions of the antiapoptotic Bcl-2/ Bcl-X proteins, allowing the activation of Bax/Bak. Importantly, a crosstalk exists between the intrinsic and extrinsic pathway at this point. In response to activation of the extrinsic pathway, the BH3-only protein Bid is cleaved by activated caspase-8 to yield the truncated tBid protein which then triggers mitochondrial apoptosis. 790 17 Apoptosis

Figure 17.6 The mitochondrial pathway of The initiator procaspase-9 is activated in this apoptosis. Cellular stress (e.g., growth factor complex and triggers the execution phase of deprivation, activation of death receptors, apoptosis, leading finally to cell death. A DNA damage) promotes the release of negative regulation of the mitochondrial cytochrome c from mitochondria in a process pathway occurs at the level of cytochrome c involving death-promoting members of the release and caspase activity. Cytochrome c Bcl-2 family (e.g., Bid, Bax, Bad, Bak). These release can be blocked by antiapoptotic proteins are assumed to translocate to the proteins such as Bcl-2. Mature caspases are mitochondrion or to undergo conformational subject to inhibition by the conserved changes within the outer mitochondrial inhibitors of apoptosis (IAP) family of proteins. membrane, forming a pore-like structure which Other proteins released by the mitochondria facilitates the escape of cytochrome c from the comprise the apoptosis-inducing factor (AIF), mitochondrion. Cytochrome c assembles with endonuclease G (Endo G) and the Smac/ Apaf1 and procaspase-9 to form the Diablo proteins. The latter family of proteins apoptosome, which is composed of seven interferes with the IAP function. MAC, procaspase-9/Apaf1/cytochrome c trimers. mitochondrial apoptosis-induced channel. 17.4 The Mitochondrial Pathway of Apoptosis 791

The mechanisms responsible for permeabilization of the mitochondrial outer membrane have been only partially characterized. In response to apoptotic stimuli, the formation of large channels, named mitochondrial apoptosis-induced channels (MAC) is observed that allow the release of cytochrome c from the intramembrane space to the cytosol. The proapoptotic Bax and Bak proteins cooperate in the formation of MACs. A key role is ascribed to Bax in this process; Bax resides predominantly in the cytosol and translocates to the mitochondrial outer membrane upon apoptotic stimuli. Bak is localized to the mitochondrial outer membrane and cooperates with Bax to form pores that allow the leakage of apoptogenic proteins such as cytochrome c into the cytosol [7]. Among the proteins released, cytochrome c is the main proapoptotic factor that triggers formation of the apoptosome and an initiation of caspase activation. In addition to cytochrome c, other soluble proteins contained in the intermembrane space of the mitochondria are released through the outer membrane and participate in the organized destruction of the cell. Among these proteins are the apoptosis-inducing factor (AIF), endonuclease G, the protease HtrA2/Omi, and the Smac/Diablo proteins.

17.4.2 Formation of the Apoptosome and Triggering of a Caspase Cascade

The release of cytochrome c from the mitochondria promotes the assembly of a multiprotein complex, the apoptosome, which contains cytochrome c, the adapter protein Apaf1, and procaspase-9, an initiator caspase [8]. Formation of the apoptosome leads to an activation of the initiator caspase-9, most likely via a dimerization-mediated process, which then activates procaspase-3, an effector caspase.

& Apoptosome: — Multiprotein complex — Contains seven copies of Apaf1 — Contains seven copies of cytochrome c — Contains procaspase-9 — Organized in a wheel-like structure — Formation is triggered by release of cytochrome c from the mitochondria — Apoptosome assembly triggers the activation of procaspase-9.

The activation of caspase-9 by apoptosome formation sets in motion a cascade of caspase activation events. At the top of this cascade is caspase-3, which cleaves other downstream effector procaspases (caspases-2, -6, -8 and -10) or apoptotic substrates containing the recognition motif DXXD. Activation of this hierarchically structured cascade leads to the proteolysis of multiple substrates, and the cell is committed to death. The cellular infrastructure is destroyed, and changes at the plasma membrane are triggered that promote engulfment by phagocytes. 792 17 Apoptosis

17.4.2.1 Other Apoptogenic Proteins Released from Mitochondria In addition to cytochrome c, other proteins such as AIF, endoG, HtrA2/Omi and Smac/Diablo are released upon mitochondrial outer membrane permeabilization. These proteins are thought to contribute to apoptosis in both caspase-dependent and caspase-independent fashion.

AIF: Release of the apoptosis-inducing factor (AIF) precedes cytochrome c release and caspase activation. Upon receipt of an incoming apoptotic signal, AIF translocates to the nucleus where it mediates chromatin condensation and the large-scale fragmentation of DNA by mechanisms yet to be determined. EndoG: Endonuclease G (endoG) is a rather nonspeciﬁc nuclease that is released from mitochondria in association with AIF. Once liberated, endoG translocates to the nucleus where it participates in the degradation of DNA, and possibly also of RNA. Smac/Diablo: These proteins promote apoptosis by binding to inhibitors of apoptosis (IAP) which relieves their inhibitory activity. Omi/HtrA2: This protease resides in the mitochondrial intermembrane space. Upon apoptosis induction, Omi/HtrA2 is released into the cytosol and promotes cell death by inhibiting the IAP proteins and by degrading cellular proteins.

& Permeabilization of mitochondria triggers the release of various proteins: — AIF — Endo G — Smac/Diablo — Omi/HtrA2

17.5 Death Receptor-Triggered Apoptosis

One major pathway of apoptosis is activated by external ligands that bind to and activate receptor systems known as death receptors (Figure 17.7). The death receptors are transmembrane receptors that belong to the superfamily of TNF receptors (Section 14.3).

& Death receptor-triggered apoptosis is externally activated by ligand binding to transmembrane receptors: Fas/CD95 TNF-R 1 DR4, DR5.

The binding of ligands to the extracellular portion of the trimeric death receptor activates the receptor and leads to recruitment of an intracellular death domain 17.5 Death Receptor-Triggered Apoptosis 793

Figure 17.7 The Fas signaling pathway. to the proteolysis of substrates containing the Binding of Fas ligand to Fas/CD95 triggers DXXD motif. Second, caspase-8 cleaves the formation of the death-inducing signaling Bcl-2 family protein Bid, whose truncated form complex (DISC) composed of Fas ligand, Fas, initiates the mitochondrial pathway of FADD and procaspase-8. The latter is activated apoptosis by triggering cytochrome c release in the DISC to form the mature caspase-8, and apoptosome formation. FADD, Fas- which can transduce the apoptotic signal in associated death domain protein; DD, death two ways. First, caspase-8 produces mature domain; DED, death effector domain. effector caspase-3 from its precursor, leading 794 17 Apoptosis

containing adapter proteins such as the FAS-associated death domain (FADD) and the TNFR-associated death domain (TRADD). Depending on the cellular context, the activated death receptors can transmit proapoptotic, antiapoptotic, anti- inﬂammatory or proinﬂammatory signals. The main features of the proapoptotic signaling pathways mediated by Fas/CD95 will be presented in the following subsections.

17.5.1 The Fas/CD95 Signaling Pathway

Fas/CD95 has a central role in the physiological regulation of programmed cell death in the immune system, where it is used mainly to instruct lymphocytes to die during immune responses. A deﬁciency in Fas/CD95 can result in abnormal lymphoid development and autoimmune diseases. The ligand for the Fas/CD95 receptor (Fas ligand/CD95 ligand) is a homo- trimeric protein that binds to Fas/CD95, causes the clustering and activation of inactive Fas/CD95 complexes, and allows the formation of a death-inducing signaling complex (DISC).

& Fas/CD95 signaling involves: — Binding of ligands to transmembrane receptors Fas/CD95 — Formation of DISC complex.

The Fas-DISC (Figure 17.7) contains the adapter protein FADD and caspases-8 or -10, which can initiate the process of apoptosis. Clustering of the components of Fas-DISC is mediated by homotypic interactions between death domains found on Fas/CD95 and on FADD, and between death effector domains found on FADD and procaspase-8.

& DISC contains: — Activated Fas/CD95 — Adapter FADD — Procaspase-8 or -10.

As a result of Fas ligand-induced clustering of Fas/CD95, FADD, and caspase-8 or caspase-10, these initiator caspases are processed and activated in an autoproteolytic fashion by induced proximity. The processed caspase-8 or -10 are then released from the DISC and activate downstream apoptotic proteins. Depending on the cell type, two different downstream pathways are triggered. In type I cells, processed caspase-8 is produced in large amounts and directly activates a caspase cascade. Among the caspases activated is caspase-3, which cleaves other caspases or vital substrates of the cell and thus paves the way for the execution phase of apoptosis. 17.6 Links of Apoptosis to Cellular Signaling Pathways 795

& DISC formation triggers: — Cleavage and activation of procaspase-8 or -10 and subsequent activation of effector caspase-3 or — Cleavage of Bid and translocation of tBid to the mitochondria and subsequent activation of the mitochondrial pathway.

In type II cells, the correct activation of effector caspases requires amplification via the mitochondrial pathway of apoptosis. Here, smaller amounts of active caspase-3 are produced which cleave the proapoptotic BH3-only protein Bid. The truncated form of Bid, tBid, translocates to the mitochondria where it induces mitochondrial outer membrane permeabilization and the release of proapoptotic proteins such as cytochrome c, Smac/Diablo, Endo G, and AIF (Section 17.5). As a result, effector caspases are activated and caspase-independent apoptosis is triggered which finally directs the cell to death. Of the many regulatory influences that modulate Fas-mediated apoptosis, regulation by FLIP stands out. The cellular FLICE-inhibitory protein (c-FLIP) is a catalytically inactive procaspase-8/-10 homolog that associates with the signaling complex downstream of death receptors, thereby preventing DISC-mediated processing and the release of caspase-8.

17.5.2 Tumor Necrosis Factor-Receptor 1 and Apoptosis

The tumor necrosis factor (TNF) receptor (TNF-R) is another cytokine receptor that can trigger apoptosis. The binding of cognate TNF ligands to TNF-R may activate several signal transduction pathways (Section 13.3), including the activation of caspase-8. Binding of the TNF trimer to TNF-R1 results in receptor clustering and the association of the adapter protein TRADD with the intracellular domain of the receptor. Additional adaptors are then recruited, including FADD, which allows the binding and activation of caspase-8 within the TNF-R1 multiprotein complex.

17.6 Links of Apoptosis to Cellular Signaling Pathways

Like most functions in animal cells, the apoptotic program is regulated by signals from other cells, which can either activate or suppress. In addition to these extracellular controls, the apoptotic program is also controlled by intracellular signaling pathways. At different levels of the apoptotic program, there are links to cell–cell interactions, to growth factor-controlled signaling pathways, to the cell cycle, and to the DNA-damage checkpoint system. As discussed in Chapter 16, the suppression of apoptosis is a crucial step in tumorigenesis, and numerous links exist between the malfunction of apoptotic proteins and tumor formation. 796 17 Apoptosis

Overall, the present knowledge of links to intracellular and extracellular signaling pathways is very incomplete, and any detailed understanding is limited to a few examples, two of which are highlighted below.

17.6.1 PI3 Kinase/Akt Kinase and Apoptosis

The PI3 kinase/Akt kinase pathway (Section 9.4) is an example of a signaling pathway that has a distinct antiapoptotic function and promotes cell survival. It can mediate antiapoptotic signals as well as growth-promoting signals (Figure 17.8). The antiapoptotic signal conduction starts at PI3 kinase to Akt kinase, which is

activated by the messenger substance PtdInsP3 formed by PI3 kinase. Two main routes have been identiﬁed by which activated Akt kinase can inﬂuence the apoptotic program [9].

Figure 17.8 Antiapoptotic signaling by the PI3 kinase also phosphorylates and activates the kinase (PI3-K)/Akt kinase pathway. The PI3 transcription factors NFkB and CREB, which kinase/Akt kinase pathway inhibits apoptosis have the genes for the antiapoptotic proteins and promotes cell survival in several ways. In IAP and Bcl-2 as targets. Members of the one reaction, Akt kinase phosphorylates and forkhead (FKH) family of transcription factors inactivates Bad protein, which is a are inhibited upon phosphorylation by Akt proapoptotic protein. Phosphorylated Bad is kinase, preventing transcription of the bound by 14-3-3 proteins, which makes it proapoptotic genes for Fas ligand and the Bim unavailable for the triggering of apoptosis. Akt protein. 17.6 Links of Apoptosis to Cellular Signaling Pathways 797

In the ﬁrst route, Akt kinase promotes cell survival by directly phosphorylating transcription factors that control the expression of proapoptotic and antiapoptotic genes. As an example, the Akt-catalyzed phosphorylation of proteins of the forkhead (FKH) family of transcription factors changes their subcellular localization, which leads to their export from the nucleus and sequestration in the cytoplasm by binding to 14-3-3 proteins. As a result, the transcription of FKH-controlled proapoptotic proteins is not possible and apoptosisisinhibited.Thisnegativeregulation is contrasted by a positive regulation of the activity of the transcription factor NFkB, which is involved in the regulation of cell proliferation, apoptosis, and survival in response to a wide range of growth factors and cytokines. A large part of the survival- promoting function of NFkB is mediated through its ability to induce prosurvival genes such as the genes for the inhibitor IAP.

& PI3 kinase pathway regulates apoptosis via two routes: — Negative control of the proapoptotic transcriptional function of forkhead proteins — Phosphorylation of proapoptotic proteins such as Bad.

A second route by which Akt kinase controls apoptosis is by Akt kinase directly phosphorylating the key regulators of apoptosis. The best-studied example of this type of control involves the Bad protein, which is a member of the proapoptotic family of BH3-only proteins. The Bad protein is phosphorylated by Akt kinase at Ser residues, and this modiﬁcation promotes translocation of Bad to the cytosol, where it is found complexed with 14-3-3 proteins. By applying this mechanism the proapoptotic effect of Bad can be inhibited; however, the effect on Bad is not universal and is observed only in some cell types.

17.6.2 The Protein p53 and Apoptosis

The tumor suppressor protein p53 has both growth-inhibiting and proapoptotic properties that are essential to its tumor-suppressing activity (Figure 17.9). As outlined in Section 16.8.3, the growth-controlling activity is mediated mainly by the kinase inhibitor p21CIP1, which is regulated by p53 at the level of expression. In addition, p53 can exert a proapoptotic function which is separate from the growth- inhibiting function. Apoptosis induced by p53 is especially important during conditions of DNA damage and stress, and can be categorized as either transcription-dependent and/or transcription-independent reactions.

& p53 promotes apoptosis by: — Induction of proapoptotic genes such as Bax, Puma, Noxa, Fas, DR4, DR5 — Repression of antiapoptotic genes such as Bcl-2 — Direct interaction with Bcl-2. 798 17 Apoptosis

Figure 17.9 Pathways of DNA damage- program and lead to cell death. Furthermore, mediated and p53-mediated apoptosis. The p53 activates transcription of the inhibitor tumor suppressor protein p53 is activated by p21Kip1 leading to cell cycle arrest via inhibition DNA damage, malfunction of signaling of CDKs. Less well-characterized, pathways, and by various stress influences. In transcription-independent pathways (e.g., a transcription-dependent pathway, p53 direct interaction with mitochondria) are also functions as a transcription activator of various known by which p53 can activate the apoptotic proapoptotic genes which trigger the apoptotic program.

17.6.2.1 Apoptotic Genes Activated by p53 The list of genes activated by p53 includes many that are known to be important for apoptosis. Components of both the intrinsic and extrinsic pathways are activated by p53 at the level of transcription. For example, p53 can engage the death receptor pathway through activation of the genes for the death receptors Fas, DR4, and DR5. Apoptosis via the intrinsic pathway can be promoted through p53-dependent transcription of the proapoptotic BH3-only proteins Puma and Noxa. Another essential component of the intrinsic pathway, the proapoptotic Bax protein was the ﬁrst identiﬁed p53-regulated Bcl-2 family member. Other components of the intrinsic pathway regulated by p53 at the transcriptional level include the Apaf-1 and caspase-6 proteins. Furthermore, p53 can negatively regulate cell survival through activation of the lipid phosphatase PTEN. In another mode of transcriptional regulation of apoptosis, p53 also can repress antiapoptotic genes such as the antiapoptotic proteins Bcl-2 and Bcl-X. References 799

17.6.2.2 Transcription-Independent Induction of Apoptosis by p53 Aside from its primary function as a transcription factor, p53 can promote apoptosis independently of transcription [10]. In response to a broad range of apoptotic stimuli, a fraction of the p53 can translocate to the outer membrane of the mitochondrion where it interacts with the antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL and neutralizes their activity. Furthermore, cytosolic p53 has been shown to activate the proapoptotic protein Bax directly, inducing mitochondrial outer membrane permeabilization, with its further consequences.

Questions 17.1. Which types of caspases do you know, and what is their function in the apoptotic program? 17.2. Describe the catalytic properties and mechanism of activation of caspases. 17.3. Which types of death platforms do you know? Which domains are used to assemble these platforms? 17.4. Give examples of caspase substrates. 17.5. Which types of Bcl-2 proteins do you know, and what is their function in apoptosis? 17.6. Describe the characteristics and major protein components of the mitochondrial pathway of apoptosis. 17.7. What are the major proteins to be released from mitochondria in response to an apoptogenic stimulus? 17.8. Describe the sequence of events that allows Fas ligand to trigger apoptosis. 17.9. Describe the linkage of Akt kinase signaling to apoptosis. 17.10. How does p53 induce apoptosis?

References

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299–310. PubMed PMID: 20159550. 8 Park, H.H. (2012) Structural features of Pubmed Central PMCID: 3222298. caspase-activating complexes. Int. J. Mol. 6 Shamas-Din, A., Brahmbhatt, H., Leber, B., Sci., 13 (4), 4807–4818. PubMed PMID: and Andrews, D.W. (2011) BH3-only 22606010. Pubmed Central PMCID: proteins: Orchestrators of apoptosis. 3344246. Biochim. Biophys. Acta, 1813 (4), 508–520. 9 Matheny, R.W., Jr and Adamo, M.L. (2009) PubMed PMID: 21146563. Epub 2010/12/ Current perspectives on Akt Akt-ivation 15. eng. and Akt-ions. Exp. Biol. Med. (Maywood)., 7 Walensky, L.D. and Gavathiotis, E. (2011) 234 (11), 1264–1270. PubMed PMID: BAX unleashed: the biochemical 19596822. transformation of an inactive cytosolic 10 Speidel, D. (2010) Transcription- monomer into a toxic mitochondrial pore. independent p53 apoptosis: an alternative Trends Biochem. Sci., 36 (12), 642–652. route to death. Trends Cell Biol., 20 (1), PubMed PMID: 21978892. Pubmed Central 14–24. PubMed PMID: 19879762. Epub PMCID: 3454508. 2009/11/03. eng. j801

Index

a AMP-activated protein kinase (AMPK) 766 A-kinase anchoring protein (AKAP) 371, 374, anaphase-promoting complex (APC) 76, 684 432ff., 436ff., 454 – mitotic APC 684, 702 – isoforms 438 – nonmitotic APC 684, 702 – signal transduction 437 – substrates 76, 702 Abelson (Abl) tyrosine kinase 513ff. – subunits 684 – activation 515, 517 antagonistic ligand 505 – autoinhibition 516 anti-oncogene 741f. – functions 517f. antigen-presenting cell (APC) 618 – inactivation 517 antigrowth signal 728 – interaction partners 515f. apoptogenic protein 792 – medical importance 517 apoptosis 388, 567, 590, 631, 707, 709, 741, – oncogenic activation 518 744, 769, 777ff. – regulation 517 – evasion 726, 729 – substrate selection 515f. – links to cellular signaling pathways 795ff. – structure 513, 517 – mitochondrial pathway 789ff. acetylome 57 – PI3 kinase/Akt kinase pathway 796 activation function 1 (AF-1 function) 265 apoptosis-inducing factor (AIF) 792 activation function 2 (AF-2 function) 263 apoptosome 785, 791 activation loop 424f., 428, 482, 484, 511, apoptotic pathway 778 676 – extrinsic pathway 779 activation segment 424 – intrinsic pathway 779 adapter protein 24, 497ff. aporeceptor complex 282 adenomatosis polyposis coli (APC) 719, 737, Arg ﬁnger 549 740, 765, 770ff. arrestin 317ff., 363ff. adenylyl cyclase (AC) 352ff., 400 ataxia telangiectasia and Rad3-related (ATR) – formation of cAMP 371ff. sensor kinase 707 – inhibition 355 – functions 709 – regulation 355ff. ataxia telangiectasia mutated (ATM) sensor – stimulation 355 kinase 707ff. – structure 354 – ATM interactions 708 – types 371f. – function 708 adrenaline 12f. atypical protein kinase (aPK) 419 Akt kinase see protein kinase B autocrine signaling 14f. allostery 34, 162 autocrine stimulation 727 alternative reading frame (ARF) protein 570, autoinhibition 428, 484, 492 743, 746 autoinhibitory sequence element 424 alternative splicing 209ff., 215 autophagosomal pathway 64 – regulation 211, 214 autoprocessing 96

Biochemistry of Signal Transduction and Regulation, Fifth Edition. Gerhard Krauss Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 802j Index

b – substrate binding 460 – B-cell receptor (BCR) 531, 618ff. subunits 459 – structure 619f. calcineurin 461, 467ff. – bacterial transcription 133 regulatory function 467 – basal transcription apparatus 139 subunit structure 467 Bcl-2 family 786ff. calmodulin 352, 355, 357, 372, 383, 382, – antiapoptotic Bcl-2 members 787 396ff., 467 – – BH3-only proteins 788f. binding 398 – – domains 787 structure 397 – – proapoptotic Bcl-2 members 787f. substrate activation 398f. – bistability 120 target proteins 399f. bone morphogenetic protein (BMP) 633, 635, cAMP responsive element (CRE) 174f. 637 cancer 718ff. – branching 114 acquired capabilities 726 – bromodomain 185, 193 altered DNA methylation 718f. – altered histone modiﬁcation 720 c – hallmarks 725 – C-helix 423 initiation 723 – c-jun-terminal kinase (JNK) 574f., 582f., 588ff. micro RNAs 720 – input signals 589 cancer gene 721 – – signal entry points 589 drivers 721f. – – substrates 590f. passengers 721f. C-terminal domain (CTD) 132, 142f., capping 210, 215 146ff. carcinogenesis 722 þ Ca2 381ff., 450ff., 563 caretaker gene 741f. – activation of proteins 395 caspase 777, 779ff. – as signal molecule 394ff. – activation 783, 785 – channels 381, 385, 388ff. – caspase cascade 791 – concentration 391 – control by inhibitor proteins 785 – distribution 383 – domains 782 – inﬂux 388f. – effector caspases 780ff. – oscillations 391f. – gatekeeper 786 – – receptors 394 inhibitors 786 – release 381ff., 388f. – initiator caspases 780ff. – removal 390 – maturation 784 – sensor 396, 400 – mechanism 781f. – – storage 381ff., 390 properties 780 þ Ca2 /calmodulin-dependent protein kinase – regulation 783 (CaMK) 455ff. – substrates 786 – function 455 caspase recruitment domain (CARD) – multifunctional CaMK 456 783 – substrates 456 catalytic domain 541 þ Ca2 -binding protein 395f. b-catenin 770ff. þ Ca2 /calmodulin-dependent protein kinase II – activation 772 (CaMKII) 456ff. – downregulation 772 – activation 457ff. Cbl protein 77 – autoinhibition 458 CDK inhibitor (CKI) 666, 668, 671ff., 744 2þ – – Ca /calmodulin binding 458 function 672 CIP1 – deactivation 460 – p21 673f. – KIP1 – holoenzyme 458 p27 672, 677 – – memory function 460f. regulation 672f. – phosphorylation 458 CDK-activating kinase (CAK) 141f. – structure 457ff. CDK-cyclin complex 674 Index j803

– CDK1-cyclin B 700f. – regulation 73 – CDK2-cyclin E 687, 710 – structure 73 – CDK4-cyclin D1 685f – substrate recognition 74 – CDK4/6-cyclin D 685 cyclic ADP-ribose (CADP-ribose) 387 – structures 675 cyclic AMP (cAMP) 352, 371ff., 433ff. CDK8 module 145 – compartmentalization 374 cell cycle 661ff. – degradation 353, 371ff. – checkpoints 665f. – formation 353, 371ff. – control points 663 – targets 372ff. – external control 663 cyclic GMP (cGMP) 375ff. – key elements 666ff. – targets 377f. – phases 661f. cyclin 667ff. – regulation by proteolysis 681 – D-type cyclins 685 – S phase entry 684ff. – degradation 671 cell cycle control 661ff. – expression 671 – antimitogenic signals 665 – functions 670 – cyclin-dependent protein kinases 667 – oncogenic activation 737 – effectors 705 – phosphorylation 671 – external cell cycle control 664f. – regulation 670 – intrinsic control mechanisms 662f. – subcellular distribution 671 – mitogenic signals 664 – types 669 – principles 661ff. cyclin D 685f. cell proliferation 567, 697 – control of translation 696 – dysregulation 730 – regulation 686 cell signaling 1ff. – stability 696 ceramide 402 – transcriptional control 695 – formation 403 cyclin-dependent protein kinase (CDK) 148, – function 403 666ff. chaperone 177, 283 – activation 669 cholera toxin 332f. – activation loop phosphorylation 676 cholesterol membrane anchor 95 – inhibitory phosphorylation 677 chromatin-binding domain 192 – phase-specific activation 680 chromatin modification 151, 159, 177, 182, – phosphorylation 676 215 – regulation 675, 678f., 705 chromatin remodeling 180f. – structural basis of regulation 674 chromatin structure 175ff., 214 – substrates 679ff. chromosomal instability (CIN) 717 cyclin E 687 chronic myelogenous leukemia (CML) 735f. – control 687 – treatment 736 – multiple regulation 688 CIP/KIP family 672 cysteine-rich domain (CRD) 650 citrullination 61f. cytidine methylation 199 coactivator 153, 256, 271ff. cytokine 593ff. conventional protein kinase (ePK) 419 – classification 594 core promoter 137f. – function 595 coregulator 153, 173, 269f. – structure 595 corepressor 153, 256, 274 cytokine-binding homology region (CHR) 597 covered promoter 178f. cytokine receptor 593ff. CRE-binding (CREB) protein 174f., 432 – activation 596ff., 599, 601 CREB-binding protein (CBP) 174f., 636 – classification 596 cross regulation 47 – cytokine binding 599, 601 crosstalk 114, 194f., 279, 300 – domains 597 Cullin-RING ligase 73ff. – downstream effectors 598 – examples 75 – general features 596 804j Index

– isoforms 599 – control 699 – non-signaling subunits 599 – initiation 700 – oligomeric structure 598f. – replication components 699 – shared b-chain receptors 603f. dual lipidation 95 – shared cc-chain receptors 603 dual-specificity phosphatase (DUSP) 581f. – signaling subunits 599 – structure 596ff. e cytokine receptor signaling 598, 615f. 4E-binding protein (4E-BP) 223ff. – regulation 615f. EF-hand 396f. cytoplasmic tyrosine kinase see Non-RTK EGF receptor (EGFR) 481, 504, 513, 555, 614 d – oncogenic activation 734 damage-regulated autophagy modulator – signaling 504f. (DRAM) 766 EGFR kinase 486 Darwinian evolution 722 – activation 486 Dbl homology (DH) domain 541f. electrical signaling 4 death domain (DD) 648ff., 783 b-element 296f. death effector domain (DED) 783 elongation 132, 135, 146ff., 202, 204, 216 death inducing signaling complex (DISC) 784, elongation factor 326 793ff. endocrine cell 13 death platform 783f. endocrine signaling 13f. death receptor-triggered apoptosis 792ff. endocytosis 506 degron 73f. endonuclease G (endoG) 792 denitrosylation 412 energy metabolism 568 desensitization 300, 318, 345f. enhanceosome 159f. – heterologous desensitization 315 epidermal growth factor (EGF) 481 – homologous desensitization 315f. ErbB network 125 deubiquitination 81 ERK pathway 586ff. diacylglycerol (DAG) 357f., 378f., 393, 401, – activation 587 450ff., 503, 563 – input signals 586f. – formation 382, 401 – substrates 587 – function 382, 401f. erythropoietin (EPO) 593f. direct repeat 155 erythropoietin receptor (EPOR) 598, 606 DNA-binding domain 154, 173 estrogen receptor (ER) 252, 264, 276f., 279f., DNA-binding motif 154f. 285f. DNA-binding protein 167ff. – nongenomic functions 287 – activity control 167f. – signal transmission 285 DNA damage 702ff., 711, 762 eukaryotic initiation factor-2 (eIF-2) 226f. – recognition 707 – control by phosphorylation 228 – response 708 eukaryotic transcription 131ff. DNA damage checkpoints 702ff., 741, 777 – basic features 132ff. – components 703ff. – components 131, 134 – G1DNA damage checkpoint 710f. – elementary steps 135f. – organization 703ff. external signal 5, 7, 15, 19 – protein kinases 708 – reception 15 DNA double-strand break (DSB) 706 extracellular messenger 3 DNA element 149, 155, 170 extracellular regulated kinase (ERK) 438, DNA methylation 197ff., 200ff., 718f. 573ff., 577, 588 – biological functions 202f. – types 200 f DNA methyltransferase (DNMT) 200f. farnesylation 93f. DNA replication 699ff. Fas/CD95 signaling pathway 793ff. – checkpoints 702ff. feedback loop 117 Index j805 feedback regulation 504 gap junction 3f. ferritin 220f. GDI dissociation factor (GDF) 545 fibroblast growth factor (FGF) 480 gene expression 129ff., 197 fibroblast growth factor receptor (FGFR) – basic steps 129ff. 480f. – regulation 129ff. fibroblast growth factor receptor substrate general transcription factor (GTF) 131, 134, (FRS) 497 137, 139, 143 focal adhesion kinase (FAK) 626ff. genetic imprinting 203 furin 647 genetic instability 717 genomic signaling 251, 254f., 281 g genotoxic stress 749 Gbc-complex 342ff., 441 geranylation 93f. – effectors 344 glucocorticoid receptor (GR) 260 – function 342 glucocorticoid response element (GRE) 280 – signaling 343f. glutathione 405f., 412ff. – structure 342 glycogen phosphorylase 53 Gbcdimer 441 glycolysis 767 G-domain 325 glycosyl-phosphatidyl-inositol (GPI) anchor G1 phase 661ff., 81ff. 98f. – G1 checkpoints 405 Gp130 receptor 601f. – G1 entry 694 Grb2-associated binder (Gab) 499 – G1 progression 672, 684ff., 694 growth factor 476f., 530 – G1/S transition 667, 672, 674, 677, 679, 687, growth factor receptor 476, 578 697f., 712 growth factor receptor binding protein 497 G2 phase 661, 663, 666, 669 growth hormone (GH) 593f. – G2/M checkpoint 712 growth hormone receptor (GHR) 598 – G2 checkpoints 705 GTP analog 324 – G2/M transition 663, 667, 677, 680, GTP hydrolysis 340ff., 548ff. 400 – by Ras 549 G protein 305, 310, 330 – mechanism 548f. – effectors 352ff. – stimulation by GAPs 548 – membrane association 345 GTPase 23f., 336 – structure 337ff. – crosstalk 543 – switch function 337ff. – functions 321 G protein-coupled receptor (GPCR) 301ff., – inhibition 324 344, 380, 391, 451f., 541, 569 – linkage 543 – activation 302, 310 – superfamily 321, 326 – classification 304 – switch function 321, 323 – desensitization 315, 318 GTPase activating protein (GAP) 117f., 323, – downregulation 315 524, 535ff., 541ff. – interacting proteins 313 – activity 540 – ligand binding 309 – catalytic domains 541 – oligomerization 319 – domain structure 539f. – phosphorylation 314ff. – regulation 540 – regulation 312ff. guanine nucleotide dissociation inhibitor – signaling 311f., 363ff. (GDI) 323, 349,535, 537f., 544f. – structure 305, 307 guanine nucleotide exchange factor – subfamilies 302ff. (GEF) 117f., 120, 320, 323, 351, 374, 535ff., G protein-coupled receptor kinase (GRK) 313, 541ff., 546, 566 316f., 349 – activation of GTPases 543 – membrane attachment 317 – catalytic domain 541ff. G protein-coupled signal transmission – Dbl homology (DH) domain 541f. pathway 291ff. – multivalency 542 806j Index

guanylyl cyclase 375ff. – chemical nature 8ff. – ligands 376 – degradation 7 – soluble guanylyl cyclases 377 – modification 7 guanylyl cyclase receptor 376f. – reception 8 – regulation of protein synthesis 226 h – secretion 8 heat shock protein (Hsp) 283f. – storage 8 Hect domain enzyme 69ff. – transport 8 Hedgehog (Hg) protein 95f. hormone agonist 11ff. heme-regulated eIF-2 kinase (HRI) 227f. hormone analog 11f. hemoglobin (Hb) 410 hormone antagonist 11f. – R-state 410f. hormone binding 288 – T-state 410f. hormone derivative 13 heterotrimeric G protein 326ff. hormone-responsive element (HRE) 251, – activation 328, 333ff. 255f., 258f., 261 – characterization 332 Hub protein 122f. – classification 328ff. – characteristics 124 – conformational changes 339 – functions 124 – functional cycle 334ff. hybrid oncoprotein 736 – Gbc-complex 342ff. hydrophilic messenger 369 – Gbc signaling 343f. hydrophobic messenger 369 – in supramolecular complexes 337 hypermethylation 719 – inactive ground state 334 hypoxia-induced transcription factor – novel G protein cycle 350 (HIF) 738 – receptor-independent functions 350ff. – structure 327f., 337ff. i – subfamilies 329ff. immunophilin 283 histone acetylation 182, 184 immunoreceptor tyrosine activation motif histone acetyltransferase (HAT) 55f., 184, (ITAM) 619f. 191, 693 inhibitor of apoptosis (IAP) 786 – regulation 185 inhibitor protein 35, 435 – substrates 185 initiation factor 326 histone arginine methylation 189 initiation region (INR) 138, 141 histone deacetylase (HDAC) 153, 163f., INK4 protein 674, 678 186 inositol messenger 380 histone deacetylation 185 – formation 381 histone demethylase (HDM) 58ff., 188 – function 381 histone lysine methylation 188 inositol phosphate 378 histone methylation 186, 201 – formation 380 histone methyltransferase (HMT) 58f., 188 inositol phospholipid 378ff. fi histone modi cation 176, 183, 196, 202, inositol trisphosphate (ITP3) 378ff., 3825ff., 720 391 – crosstalk 193ff. inositol trisphosphate receptor 385f. – dynamics 198 insulin 223ff. – epigenetics 197 insulin receptor 423 histone modification code 196 insulin receptor kinase (IRK) 484 histone modification patters 196 insulin receptor substrate (IRS) 495, 498 histone phosphorylation 190f. – regulation 498 histone ubiquitination 192 – signaling 496 histone variant 180 integrin 623ff. hnRNP 214 – downstream signaling 626, 628 hormone 2, 7ff., 170, 251ff. – inside-out signaling 624f. – biosynthesis 7 – integrin signaling 624f., 628 Index j807

– outside-in signaling 624f., 627 ligand binding 262, 309 – structure 624 – activation function-2 263 integrin-linked kinase (ILK) 626f. – promiscuous ligand binding 265 interaction domain 41, 43, 61f., 103f., 490 – switch function 264 – classification 44 linker for activation of T lymphocytes – modification-specific interaction domain 41 (LAT) 500, 623 – regulatory interaction domain 43 lipid anchor 90, 112 – versatility and variability 46 – examples 91 interaction network 122f. – structure 91 – modularity 126 – switch function 96f. – redundancy 126f. local mediator 15 – robustness 126f. long noncoding RNA (lncRNA) 231 intercellular signaling 1ff. lysine acetylation 277 – principal mechanisms 3 – regulatory functions 57 – regulation 7f. lysine acetyltransferase (KAT) 55f., 184 – steps 5ff. lysine demethylase (KDM) 58ff., 188f. – tools 3ff. lysine methylation 58ff., 188, 277 interferon (IFN) 593f., 596 lysine methyltransferase (KMT) 58f., 188 interferon (IFN)-b gene 159f. lysophosphatidic acid (LPA) 403f. interleukin (IL) 593f., 603 lysosomal pathway 64 interleukine-2 (IL-2) 595, 603f. interleukine-2 receptor (IL-2R) 603f., 606ff. m intracellular messenger 4 M phase 661, 666, 669, 680ff., 686f., 699ff. intracellular messenger substance see second – control 701 messenger – progression 702 intracellular pathway 6 major histocompatibility complex (MHC) 618 intracellular signaling 1f., 292, 573ff. MAP/ERK kinase (MEK) 575ff., 579 – aberrant activation 728 MAPK kinase (MAPKK, MAP2K) 575, 577, – basics 15ff. 579 – components 19 MAPK multiprotein complex 575, 583, 585f., – molecular tools 18ff. 590 intronic miRNA 241 MAPK pathways 573ff., 588ff., 626 iron regulatory protein (IRP) 220f. – components 575ff. iron-responsive element (IRE) 220f. – feedback loops 580 – major MAPK pathways of mammals 586 j – organization 575ff. JAK/STAT pathway 608ff., 612, 614f. – p38 pathway 588ff. Janus kinase (Jak kinase) 604, 608f. – regulation by inhibitory proteins 583 – coupling of other receptor types 610 – regulation by MAPK phosphatase 581ff. – kinase regulation 609 – regulation by protein phosphatases and – nuclear functions 610 inhibitors 579ff. – pseudokinase domain 609 – scaffolding 583ff. JNK module 591 MAPK phosphatase (MKP) 581ff. juxtamembrane autoinhibition 484 MAPK/ERK pathway 118, 121, 555, 560 MAPK-activated protein kinase 1 (MAPKAP- k K1) 588 kinase suppressor of Ras (KSR) 560, 584 MAPKK kinase (MAPKKK, MAP3K) 575, 577, kindlin 625f. 579, 591 mediator 134, 139, 143f., 150, 158, 273 l – function 144 layered network 124ff. – structure 144f. leukocyte inhibitory factor (LIF) 595 mediator complex 143ff. leukocyte inhibitory factor receptor (LIFR) 602 – regulatory roles 145 808j Index

MEK kinase (MEKK) 556, 559, 565, 575f. n membrane anchor 345 N-end rule 74 membrane-associated messenger 26 natriuretic peptide receptor (NPR) 376f. messenger ribonucleoprotein (mRNP) natural killer (NK) cells 603 216 negative feedback 117ff., 505, 560, 580 metal ion center 410 nerve growth factor (NGF) 480 methyl CpG-binding protein (MBP) 201 network 115 microRNA (miRNA) 229ff., 233ff. network structure 121 – biogenensis 233, 236, 239 NFkB pathway 85ff., 652 – cancer 243, 720ff. – canonical pathway 87 – functions 240 – noncanonical pathway 87 – mature miRNA 239 nicotinic acid adenine dinucleotide phosphate – processing 235f. (NAADP) 387f. – regulation 238ff., 242, 244 nitric oxide (NO) 375, 404ff., 564 – regulatory functions 240 – reactivity 405f. – targets 241 – signal transduction 405 – transcription 234, 239 – synthesis 406 microsatellite instability (MSI) 717 – targets 410 miRNA-induced silencing complex – toxic action 413 (miRISC) 229 nitrosative stress 413f. – formation 235 nitrosylation 409ff. mitochondrial apoptosis-induced channel – nitrosylation of hemoglobin 410 (MAC) 791 – nitrosylation of metal centers 409 mitogen-activated protein kinase (MAPK) 107, – physiological functions 409 118, 287, 363f., 425, 501, 503, 532, 573ff., NO-sensitive guanylyl cyclase 409 577, 587 NO-synthase (NOS) 404, 406f., 412 – atypical MAPKs 575 – activation 408 – conventional MAPKs 574 – cofactors 407 mitogenic signal 694 – forms 407 moderator of nongenomic activity of ER – eNOS 408 (MNAR) 287 – iNOS 409 modular signaling complex 31 – nNOS 408 – regulation 33 – substrates 407 – signal-directed assembly 33 non-G protein signaling 349 – fi speci city 33 non-genomic signaling 251, 254f., 257 – variability 33 non-genotoxic stress 749, 763 monomeric GTPase 535, 539 non-Smad signaling pathway 636 – GTPase-activating proteins 539ff. nonreceptor protein tyrosine phosphatase – guanine nucleotide exchange factors 541ff. (PTPN) 519, 521, 523 – Ras family 545 – autoinhibition 523 mRNA 131 nonreceptor tyrosine-specific protein kinase mRNA circularization 217f. (non-RTK) 507ff., 593f., 598, 601, 623, 626, fi multisite protein modi cation 42f. 735 multivalent scaffold 436 – activation 604 Myc transcription factor 737ff. – autophosphorylation 604 – cooperation with other transcription – binding of signaling proteins 606 factors 738 – domains 508 – regulation of Myc transcription 739 – function 507ff. – transcriptional targets 739 – oncogenic nonreceptor tyrosin kinases 735 myristic acid anchor 92 – phosphorylation 605f. myristoylation 91f. – structure 507ff. myristoyl-electrostatic switch 97 Notch extracellular truncation (NEXT) 646f. myristoyl-ligand switch 97 Notch intracellular domain (NICD) 647f. Index j809

Notch receptor 642ff. oncogenic receptor tyrosine kinase 734f. – activation 644, 646 – ErbB2/neu receptor 734f. – domain organization 644f. oncogenic stress 749, 762 – downstream signaling 646 oncogenic transcription factors 737 – function 643 oncoprotein 546, 744 – ligands 643f. open promoter 178f. – processing 644 orphan nuclear receptor 257, 268 – structure 643 nuclear factor of activated Tcells (NF-AT) 468f. p nuclear receptor (NR) 251ff. P-tyrosine-binding (PTB) domain 493 – AF-2 function 263 – subgroups 493 – coactivator 271f. p130Cas 500 – coregulator 269f. p38 module 591 – corepressors 274 p38MAPK pathway 589 – cytoplasmic functions 284 – input signals 589 – DNA-binding elements 258, 260f. – signal entry points 589 – domains 270 – substrates 590f. – ligand-binding domain 261ff., 267 palindromic sequence 155 – ligand-dependent translocation 281 palmitic acid anchor 92 – ligands 252ff., 275 palmitoylation 92f. – lipidation 278 paracrine signaling 14f. – long-range actions 279 pathogen-associated molecular pattern – nongenomic functions 284ff. (PAMP) 653f. – principles of signaling by NR 254ff. PDGF receptor (PDGF-R) 489f., 513, 518, 555 – regulation of signaling 274ff. peptidyl arginine deaminase (PADs) 61 – signaling 251ff. peroxisome proliferator activator receptor – speciﬁcity 280 (PPAR) 263, 266 – structure 257ff., 266 peroxynitrite 413 – subcellular localization 280 pertussis toxin (PTX) 333, 361 – sumoylation 278 Philadelphia translocation 735 – transactivation function 265 phosducin 346f. – transcription activation 269 phosphatase and tensin homolog – transcription repression 269 (PTEN) 445f. – transcriptional regulation 268 phosphatidylinositol (PtdIns) 378ff. – ubiquitination 278 phosphatidylinositol-3,4,5-triphosphate nuclear translocation 283 (PtdIns(3,4,5)P3) 392f., 444 nucleosome remodeler 181 phosphodiesterase (PDE) 354, 372, 437 nucleotide exchange 542 phosphoinositide 392ff. phosphoinositide-3 kinase (PI3K) 439ff., 452, o 492, 500, 566, 620 oncogene 715ff., 722, 729ff. – activation 441f. – activation 730ff. – classiﬁcation 440 – functions 733ff. – properties 440 – oncogene products 734 phosphoinositide-dependent protein kinase – structural changes 730 (PDK) 443f., 452f. oncogene Mdm2 756ff. phospholipase 358 – control of Mdm2 function 759 phospholipase A2 588 – control of p53 756 phospholipase C (PLC) 357ff., 380, 401, 566 – downregulation of Mdm2 by ARF 758 – activation 360f. – p53 independent functions of Mdm2 – domains 359f. 759 – PLC-b 358ff., 394 – regulation of Mdm2 757 – PLC-c 362, 394f., 492, 501ff., 620, 623 oncogenic fusion protein 731f. – PLC-d 362 810j Index

– PLC-e 362f. protein arginine methylation 61f. – stimulation 359, 361 protein arginine methyltransferase – types 359ff. (PRMT) 58, 189 phosphorylated protein 52 protein degradation pathway 63f. – interactions 52 protein inhibitor of activated STATs – properties 52 (PIAS) 615ff. phosphorylation 171ff., 190f., 223, 276ff., 300, protein kinase 51, 53, 148, 227, 417 314, 316, 450, 465, 528, 641 – activation 429 – activating phosphorylation 559 – amino acid specificity 418f. – inhibitory phosphorylation 559 – characteristics 417 phosphotyrosine-binding (PTB) domain 348, – classification 417 495 – conserved amino acids 421 PI3K/Akt pathway 439ff.,796f. – control of activity 427, 429 – regulation of apoptosis 797 – inactivation 429 piwi-interacting RNA (piRNA) 230f. – inhibitors 428 PKC-interacting protein 453 – reaction 421f. platelet-deribed growth factor (PDGF) 482, – regulation 420ff. 489 – structural elements 422ff. pleckstrin homology (PH) domain 360 – structure 417, 420ff. poly(A)-binding protein (PABP) 219 – substrate binding 425ff. polyadenylation 210 – substrate specificity 426 poly-ubiquitin linkage 82f., 87 – targeting 431 positive feedback 119f., 504, 581 protein kinase A (PKA) 372f., 422, 431ff. postsynaptic density (PSD) 460 – activation 433 posttranslational modification (PTM) 23, 27, – domains 434 29, 31, 36ff., 51, 58, 95, 103, 108ff., 117, 149, – phosphorylation site 434 152, 165, 170, 176, 272, 274, 276ff., 281, 301, – regulation 373, 431ff., 435f. 464, 490, 502, 536, 579, 608, 640, 739 – structure 432 – allosteric and conformational functions 38 – substrate specificity 432 – antagonistic action 49 – subunits 432f. – chemical nature 37ff. protein kinase B (PKB) 442ff. – classes 37 – activation 443, 445 – convergent recognition 50 – domains 442f. – dynamic nature 38 – inhibition of apoptosis 444 – multisite PTM 47 – phosphorylation 443 – mutually exclusive PTM 48 – promotion of cell survival 444, 446 – recognition of PTMs 38 – regulation of cellular metabolism 445 – regulation of intramolecular interactions 49 – stimulation of protein synthesis 444 – regulatory PTMs 38 – signaling 443ff. PPAR-RXR complex 266f. protein kinase C (PKC) 378f., 381, 395, 401ff., pre-mRNA 209, 212 438, 447ff. pre-mRNA processing 209ff., 215 – activation 449ff. preinitiation complex (PIC) 135, 148, 150, – autoinhibition 450 204, 215 – C1 domain 448ff. prenalytion 94 – C2 domain 448, 450 prenyl anchor 93 – classification 447 procaspase 786 – cofactors 450 promoter 132, 138, 177 – functions 451, 454 – promoter types 178f. – phosphorylation 452 proteasome 78ff. – receptors 453 – activator 80 – regulation 450ff. – structure 79 – stimulation by phorbol esters 449 – substrate delivery 80 – structure 447 Index j811

– subfamilies 447 – isoforms 556 – substrates 454f. – oncogenic activation 561 protein lysine acetylation 55 – phosphorylation 559 – enzymes 56 – regulation 557f., 560 – general aspects 55 – structure 557 – regulation by protein lysine acetylation 55 Raf-1 kinase inhibitory protein (RKIP) 583 – regulatory functions 57f. Ral GTPase 562 protein methylation 58ff. Ran family 570 protein modification 39ff., 45 Rap GEF 562 protein phosphatase (PP) 417ff., 461ff. Rap GTPase 562 – targeting 431 – regulation 563 protein phosphatase 2A (PP2A) 465 Ras exchange motif (REM) 553f. – C-terminal methylation 466 Ras-GAP 552 – subunit structure 466 Ras-GEF 553 protein phosphatase 2B (PP2B) see calcineurin – activation 553 protein phosphatase I (PPI) 464 – regulation 554 protein phosphorylation 51ff., 112, 419, 430 – structure 553 – allosteric functions 53 Ras gene 546 – general aspects 51f. Ras nanocluster 551 – organization of signaling pathways 54 Ras pathway 441 protein S-nitrosylation 410ff. Ras protein 326, 501, 545ff. – regulatory functions 410 – domains 548 – selectivity 410 – downstream effectors 565 – target proteins 412 – effector loop 548 protein tyrosine phosphatase (PTP) 504, – effector molecules 564 519ff., 608, 614, 617 – input signals 562ff. – activity 527 – membrane localization 551 – classification 520 – oncogenic mutations 550f., 567 – functions 519f. – properties 545 – mechanism 520, 522 – reception of signals 562 – negative regulation 526 – structure 547f. – oncogenic functions 526 – transforming mutants 550 – oxidation 528ff. – transmission of signals 562 – positive regulation 526f. Ras signal transduction 552f., 555 – regulation of cell signaling 525ff. – GTPase activating proteins 552 – subcellular localization 532 – Guanine nucleotide exchange factors 553 – structure 520 – Raf kinase 555 – tumor suppression 526f. Ras signaling pathway 566, 737 protein tyrosine phosphatase activator – oncogenic activation 737 (PTPA) 465 Ras superfamily 535ff., 545, 568 proteolysis 78, 528, 681ff., 779ff. – crosstalk among members 538 proto-oncogene 731f. – structural organization 537 PTEN phosphatase 446 reactive oxygen species (ROS) 413, 529, 531, 582 r receptor 15, 19, 252ff. R-Ras protein 561 – classification by signaling activity 632 Rab family 569 – interaction between hormone and Raf kinase 555, 565 receptor 21f. – activation 557f. – intracellular receptor 21 – domains 557 – membrane-bound receptor 20 – formation of heterodimers 559 – transmembrane receptor 21 – formation of homodimers 559 receptor activity 22 – inactive state 558 – regulation 300 812j Index

receptor-activity modifying protein – apoptosis 744 (RAMP) 313 – control by phosphorylation 690 receptor for activated protein kinase C (RACK) – domain structure 689 protein 453f. – functions 689 receptor for 9-cis-retinoic acid (RXR) 262f., 266 – hyperphosphorylated Rb 690 receptor interaction domain (RID) 271 – hypophosphorylated Rb 690 receptor phosphorylation 300 – in cancer 743ff. receptor protein tyrosine phosphatase – inactivation 744 (PTPR) 519, 521ff. – tumor suppressor function 743 receptor recycling 300 retinoid 10 receptor tyrosine kinase (RTK) 474f., 541, 554, Rho/Rac family 568, 589 563, 569, 593, 614 rhodopsin 305f., 337 – activation 479f., 485 – structure 306 – adapter proteins 494 ribosomal S6 kinase (RSK) 588 – autoinhibition 483ff. RING domain enzyme 71ff. – autophosphorylation 478, 483, 487, 494 RNA-dependent protein kinase (PKR) 427 – classification 475ff. RNA-induced silencer complex (RISC) 233, – coupling of effectors 487 237 – dimerization 480 RNA interference (RNAi) 209ff., 233, 243 – disfunction in disease 506 RNA polymerase 135, 137, 146f. – domains 475, 477 RNA processing 209ff. – downstream effectors 494ff., 500f. RNA-recognition motif (RRM) 213 – downstream signaling 485 RNA silencing 229ff., 245 – endocytosis 506 – basics 230ff. – function 474ff. – componenets 232 – heterodimerization 481f. RNA-specific eIF-2 kinase (PKR) 228f. – inhibition 505 ryanodine receptor 381, 385f. – kinase domain 482 – ligand binding 477ff. s – negative regulation 505 S-adenosyl-L-methionine (SAM) 61 – receptor dimerization 477 S6 kinase phosphorylation 226 – signal transmission 474 S-nitroso-glutathione (GSNO) 412, 414 – signaling network 501f. S phase 661, 663f., 669, 684, 699ff. – structure 474ff. scaffold – trafficking 506 – as allosteric regulators 108 – ubiquitination 506 – as signaling enzymes 108 recognition element (RE) 150ff., 156, 161f. – organization of feedback loops 107 – clustering 159 – organization of signaling circuits 105 – selection 161 – organization of signaling complexes 107f. – sequence 161 – targets of regulation 107 recognition sequence 155 scaffold protein 24, 103ff., 314 recoverin 400 – general aspects 103 regulated intermembrane proteolysis Scr homology and collagen (Shc) protein (RIP) 643 499 regulator of G protein signaling (RGS) 314, second messenger 25, 352, 357, 369ff., 541 327, 336, 342, 346ff. – formation 370 regulatory circuit 116 – function 370 regulatory GTPase 320ff., 340 – general properties 369ff. regulatory transcription factor 166, 258 – types 369f. replication stress 707 senescence 741f., 747ff., 766, 769f. repression 275 sequence-specific transcription factor 134, repressor 164, 169, 222 150f., 153, 169, 205 retinoblastoma protein (Rb) 687ff., 743ff. sequential signaling 106 Index j813

Ser/Thr-phosphate-specific protein – regulatory modifications 38 phosphatase (PSP) 461 – unstructured section 30 Ser/Thr-phosphorylation 54, 496f. – variability 18 Ser/Thr-specific protein kinase 417ff., 447, silencing 190, 198 631ff. silencing mediator for retinoic and thyroid Ser/Thr-specific protein phosphatase 461ff. hormone receptors (SMRT) 274 – classification 462 Skp1-Cullin-F-box complex (SCF complex) 75, – function 462f. 682f. – inhibitors 464 – proteolysis 682 – regulation 462, 464 – structure 75 – structure 462 – substrate 75, 683 signal dampening 496f. – subunits 683 signal-directed inhibitory modification 17 Smad anchor for receptor activation signal processing 6 (SARA) 638 – in signaling paths and signaling Smad protein 636ff. networks 108ff. – activation 638 signal recognition particle (SRP) 326 – classification 636 signal-regulated transcription factor 167 – DNA binding 640 signal switching 319 – domains 636 signal transduction network 165 – expression 641 signal transmission 473ff., 535ff. – I-Smad 631, 636f., 639ff. signaling 103ff., 251ff. – phosphorylation 638 – by nuclear receptors 251ff. – R-Smad 631, 634, 636ff., 640f. – organization 103ff. – transcriptional regulation 640 – spatial control 111f. Smad protein signaling 631ff. – specificity 109 – regulation 640 – temporal control 113 small GTPase 535, 537ff., 541f., 565, 568, 570 – via ion channels 292 small interfering RNA (siRNA) 229ff., 237, – via transmembrane receptors 291 243ff. signaling circuit 105f., 116 – applications 246f. signaling complex 109 – functions 246f. – PTM-induced formation 109 – posttranscriptional silencing 246 signaling enzyme 23f., 34ff. – processing 243 signaling modul 27, 30f. – sources 243 – differential use 31 small noncoding RNA (sncRNA) 231 – subtypes 31 small ubiquitin-related modifier (SUMO) 88f. signaling network 108ff., 501 – regulation 89 signaling pathway solenoid 176 – architecture 113 sphingomyelin 402 – malfunction 715ff. sphingomyelinase 402 signaling protein 16, 284, 298 sphingosine 403 – activation 16f. spliceosome 211 – catalytic domain 28 splicing 214 – clustering 112 SR protein 213f. – deactivation 16f. Src homolog 2 (SH2) domain 491ff. – function 29 Src tyrosine kinase 508ff., 608 – interaction domain 28 – activation 511ff. – isoforms 18 – domains 509 – lipidation 90ff. – inactive state 509, 511 – modular structure 27, 29f. – regulation 509f. – mutations 729 – signal transduction pathways 514 – properties 27 – structure 509f. – regulatory domain 28 STAT protein 611 814j Index

– acetylation 615 – activation 152, 161, 187, 256, 268 – activation 612f. – activator 165, 169 – function 612 – coregulators 153 – signaling function 614 – initiation 149 – structure 612 – proteins 133 – unphosphorylated STATs 615 – regulation 129ff., 149ff., 163, 175ff. 203 steroid hormone 9f., 254, 258, 276, 281 – repression 152, 163f., 268 steroid hormone receptor 256, 258, 281f. – steps 132 sumoylation 89, 641 transcription factor 158, 162, 204, 279, 374 suppressor of cytokine signaling (SOCS) 119, – classification 165f. 505, 608, 616f. – control 165ff. synaptic scaffold 105 – modular structure 152 – repression mechanisms 164 t transcription factor E2F 691ff., 745f. T-cell receptor (TCR) 618ff. – activation 691 – activation 621f. – classes 692 – signaling 621 – control by Rb 691f. – structure 618ff. – repression 691 – subunits 619 transcription regulator 170 T3 hormone 251, 253f., 259, 263, 275f., 281, transcriptional activator 172 284, 288f. transducin 331, 338f. talin 625f. transforming growth factor b (TGFb) 632ff. TATA box 138, 140 – function 633 TATA box binding protein (TBP) 138, 140f. – growth regulatory effects 642 TATA box binding protein associated factor – regulation 640 (TAF) 140f. – subfamilies 633 TATA box binding protein-related factor transforming growth factor b (TGFb) (TRF) 141 receptor 631f., 634ff. tetradecanoyl phorbol acetate (TPA) 447, 449, – activation 634f. 451 – domains 635 TFIID 139ff., 204 – phosphorylation 635f. TFIIH 141ff. – structure 635 – enzymatic activities 141f. – regulation of signaling 639 – subunit structure 142 translation thrombin 302, 308 – control 218f., 222f. thyroid hormone receptor-activation protein – initiation 217, 223ff. (TRAP) complex 273, 275 – mRNA-specific control 219f., 222 tissue hormone 15 – regulation 209ff., 217ff. TNF cytokine 651 – regulation via eIF-2 226ff. Toll IL-1 receptor (TIR) 653, 657 – repression 223, 236, 238 Toll-like receptor (TLR) 583 transmembrane adapter protein (TRAP) 623 – activation 655f. transmembrane receptor 291, 441, 473ff., – adapter-mediated downstream 631ff. signaling 657 – aberrant activation 728 – adapters 656 – activation 292 – domains 654 – classification 291 – extracellular TLRs 654 – downstream targets 300 – intracellular TLRs 654 – extracellular domain 294 – ligand binding 655f. – function 299 – signaling 653ff., 658 – intracellular domain 298f. – structure 654 – ligand binding 294 transactivation domain 157f. – signal transmission 473ff. transcription 129ff., 215 – signaling 291, 293 Index j815

– structure 291, 294ff. – negative feedback between p53 and – regulation of activity 300 Mdm2 757 – transmembrane domain 296ff. – p53 recognition element 760 transmembrane signaling 291 – phosphorylation 753, 758 transnitrosylation 412 – posttranscriptional modifications 753f., 760 tumor cell 715ff. – repression 757, 765, 767 – characteristics 715, 723 – selection of target genes 760 – epigenetic changes 717ff. – stabilization 750 – genetic changes 716 – structur 751ff. – insensitivity to antigrowth signals 728 – sumoylation 756 – large-scale changes 717 – targets 763ff. – mutation 715 – transcription-independent induction of – physiologic changes 725ff. apoptosis 799 – self-sufficiency in growth signals 727 – tumorigenic mutations 770 – small-scale changes 717 – ubiquitination 756 tumor evolution 725 tumorigenesis 203, 715ff. tumor necrosis factor (TNF) 87, 594, Tyr-phosphorylation 54, 495 778f. Tyr-specific protein phosphatase SHP-2 501 tumor necrosis factor receptor (TNFR) 589, tyrosine kinase (TK) 299, 473ff., 593 614, 648ff. – activity 473ff. – activation of NFkB pathways 652f. – domains 473, 481f. – apoptosis 795 – biological functions 650 u – domains 649f. ubiquitin 65ff. – downstream signaling 652 – activation 68 – ligands 649f. – degradation 78ff. – receptor activation 652 – transacylation 68 – recruitment of adapter proteins 652 – transfer 68 – structure 650 ubiquitin-binding domain (UBD) 82, 84f., 193 tumor promoter 447, 449 ubiquitin conjugation 66ff., 82f., 85 tumor suppressor 633, 679, 688, 743 – E1 enzyme 66ff. tumor suppressor gene 715ff., 722, 741ff. – E2 enzyme 66ff. – classes 742 – E3 enzyme 66ff., 73 – functions 741ff. – multiplicity 81 tumor suppressor protein 742 – nonproteolytic functions 81ff. tumor suppressor protein p53 747ff. ubiquitin-like protein 88 – aberrant destabilization 770 ubiquitin modification 62ff. – acetylation 755 ubiquitin pathway 671 – activation 747, 750, 762 ubiquitin-protein ligase 69 – apoptotic genes 766, 798 – regulation 69 – apoptosis 797f. – structure 69 – biochemical properties 751ff. ubiquitin receptor 84f. – control by Mdm2 756ff. ubiquitination 66ff., 82, 192, 278, 301, 506, 641 – cousins of p53 751 – domains 752 w – function 749ff. Wnt/b-catenin signaling 770ff. – genes regulated by p53 760 – inactivation 748, 751 x – induction of miRNA 768 X-linked severe combined immunodeficiency – interaction partners 761 disease (X-SCID) 603 – metabolic regulation 767 – methylation 755 z – neddylation 756 zeta-associated protein 70 (ZAP70) 621f.