Molecular mechanisms regulating
phospholipase C‐2 activity
Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulm
vorgelegt von Anja Jasmin Bühler aus Ulm
Universität Ulm Institut für Pharmakologie and Toxikologie
Ulm 2013
Current Dean of the Faculty of Natural Sciences: Prof. Dr. Joachim Ankerhold
First Supervisor: Prof. Dr. Peter Gierschik Second Supervisor: Prof. Dr. Ralf Marienfeld
Day Doctorate Awarded: 24th March 2014 Table of contents
Table of Contents
List of Figures...... v
Abbreviations and Units...... vii
1 Introduction ...... 1
1.1 Signal transduction...... 1
1.2 Phospholipases C...... 2
1.3 Regulation of Phospholipases C...... 5
1.4 Role of PLC2 in inflammatory and autoimmune diseases ...... 14
1.5 Anaplastic lymphoma kinase ...... 17
1.6 Aim of the work...... 23
2 Materials and Methods...... 25
2.1 Materials...... 25
2.2 Microbiology and Molecular Biology...... 40
2.3 Cell culture and DNA transfection...... 48
2.4 Protein Biochemistry ...... 49
2.5 Measurement of phospholipase C activity...... 53
2.6 Thermodynamic calculations...... 55
2.7 Cluster robustness analysis...... 56
2.8 Statistical analysis...... 56
3 Results ...... 58
3.1 Autoinhibitory regulation of PLC2 ...... 58
3.2 Functional reassembly of PLC2 by separately expressed fragments ...... 65
3.3 Phosphorylation induced activation of PLC2...... 69
3.4 Anaplastic lymphoma kinase ...... 73
3.5 Characterization of PLC2 deletion mutants associated with PLAID ...... 84
4 Discussion ...... 110
4.1 Autoinhibitory and phosphorylation-induced regulation of PLC2 activity ...... 111
iii Table of contents
4.2 Molecular mechanisms underlying the human hereditary disease PLAID...... 123
5 Summary ...... 131
6 References...... 133
Acknowledgements ...... 165
Curriculum Vitae...... 166
Publications ...... 167
Appendix ...... 168
Statutory Declaration ...... 169
Sections 2.1.1, 2.6, 2.7, 3.5.1, 3.5.2, 3.5.3,.3.5.4, 3.5.5, 3.5.6, 3.5.7,.3.5.10, 3.5.11, and 4.2 of this dissertation have been used for a publication and are taken from the corresponding manuscript [26].
iv List of Figures
List of Figures
Figure 1-1: Domain organization of mammalian PLC isozymes ...... 4 Figure 1-2: Oncogenic signalling of NPM-ALK ...... 21
Figure 3-1: Role of 2 specific array on the autoinhibitory regulation of PLC2...... 59 G12V Figure 3-2: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH and PLC2SA . 60
Figure 3-3: Inhibitory effect of 2 SH domains on the activity of PLC2SH...... 62
Figure 3-4: Role of the SH2C and SH3 domain in autoinhibitory regulation of PLC2 ...... 64 G12V Figure 3-5: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH mutants...... 65
Figure 3-6: Functional reassembly of truncated PLC2 fragments...... 67 H327A/H372A F897Q Figure 3-7: Functional reassembly of truncated PLC2 fragments with PLC2 and PLC2 ..... 68
Figure 3-8: Effect of mimicking phosphorylation of different tyrosine residues on the activity of PLC2 ...... 71 G12V Figure 3-9: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2 phosphomimetic mutants ...... 73 Figure 3-10: Effect of ALK and its fusion proteins NPM-ALK and EML4-ALK on the activity of PLC isozymes, PLC2 phosphomimetic mutant, and PLC2...... 75 Figure 3-11: Effect of the constitutively active ALK mutants ALKF1174L and ALKR1275Q on the activity of PLC isozymes...... 76 Figure 3-12: Association of ALK and NPM-ALK with PLC isozymes in intact cells ...... 78 Figure 3-13: Effect of ALK and NPM-ALK kinase-dead mutants on the activity of PLC isozymes ...... 79 I310T Figure 3-14: Association of NPM-ALK and NPM-ALK with PLC in intact cells ...... 81 Figure 3-15: Effect of ALKY1604F and NPM-ALKY664F on the activity of PLC isozymes...... 82 Y664F Figure 3-16: Association of NPM-ALK with PLC1 ...... 83
Figure 3-17: Specific activation of PLC219 and PLC220-22 at subphysiological temperatures...... 85
Figure 3-18: Q10 analysis of the activities of wild-type PLC2, PLC219, and PLC220-22...... 86 Figure 3-19: Effect of subphysiological temperatures on cellular inositol phospholipid levels ...... 87
Figure 3-20: Effect of cycloheximide on the activity of PLC219 and PLC220-22 at 31 °C ...... 88
Figure 3-21: The activation of PLC219 and PLC220-22 by subphysiological temperatures is reversible ...... 89
Figure 3-22: Time course of the specific activation and inactivation of PLC219 and PLC220-22 by changes to and from a subphysiological temperatur...... 90 Figure 3-23: Effect of deleting exon 19 to 22 of PLCG2 on the enzymatic activity at 37 °C and 31 °C...... 91
Figure 3-24: Effect of the deletion of PLC1 residues corresponding to exon 19 or exon 20-22 of PLCG2 on the activity of PLC1 at subphysiological temperatures ...... 93
Figure 3-25: Effect of subphysiological temperatures on the activities of PLC2PCI, PLC2SA, PLC2SH and PLC2SH2C...... 94
Figure 3-26: Effect of subphysiological temperatures on the activities of PLC2 deletion mutants...... 96
v List of Figures
Ali5 Ali14 Figure 3-27: Effect of subphysiological temperatures on the activities of PLC2 , PLC2 , and Ali5/Ali14 PLC2 ...... 97
Figure 3-28: Summary and clustering of the effects of the PLC2 mutations...... 98 S707Y Figure 3-29: Effect of subphysiological temperatures on the activity of PLC2 ...... 100
Figure 3-30: Specific activity of PLC2 phosphomimetic mutants at 37 °C and 31 °C...... 101
Figure 3-31: Effect of subphysiological temperatures on the activities of PLC2 phosphomimetic mutants. 102 G12V Figure 3-32: Effect of subphysiological temperatures on the Rac2 -mediated activation of PLC2 and
PLC219 ...... 104 G12V Figure 3-33: Cool temperature shifts the requirement of PLC2 for Rac2 -mediated activation to lower G12V cellular PLC2 expression levels without altering the enzymes sensitivity to Rac2 ...... 105
Figure 3-34: Role of the spPH domain in the specific activation of PLC219 at subphysiological temperatures...... 107
Figure 3-35: Covalent linkage of the split PH domain halves blocks PLC2 sensitivity to cool temperatures ...... 109 Figure 4-1: Temperature dependence of conformational equilibrium constant...... 127
vi Abbreviations and Units
Abbreviations and Units
°C Degree Celcius A Absorbance aa Amino acid Ali Abnormal limb ALCL Anaplastic large cell lymphoma ALK Anaplastic lymphoma kinase
APLAID Autoinflammation and PLC2-associated antibody deficiency and immune dysregulation approx. Approximately ATP Adenosine triphosphate BCR B cell receptor bp Base pair BSA Bovine serum albumin Btk Bruton’s tyrosine kinase BLNK B cell linker (also known as SLP-65) c Concentration C- Carboxy- CD Cluster of differentiation Cdc Cell division cycle cDNA Complementary DNA cf. Confer CNS Central nervous system cpm Counts per minute CSTT Cold stimulation time test CVID Common variable immunodeficiency d Path length Da Dalton DAG Diacylglycerol ddH2O Double-distilled water DLBCL Diffuse large B-cell lymphoma DMEM Dulbecco’s modified Eagle’s medium
vii Abbreviations and Units
DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleoside triphosphate DPPC Dipalmitoylphosphatidylcholine DRG Dorsal root ganglia DTT Dithiothreitol Molar extinction coefficient EDTA Ethylene diamine tetraacetic acid e.g. Example given EGF Epidermal growth factor EML4 Echinoderm microtubule associated protein-like 4 ENU N-ethyl-N-nitrosourea ERK Extracellular-signal regulated kinase FACU Familial atypical cold urticaria FCS Fetal calf serum fp Forward primer g Gram g Gravitational constant Gab2 GRB2-associated-binding protein 2 GAP GTPase-activating protein GDI Guanine nucleotide dissociation inhibitor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GMP Guanosine monophosphate GPCR G-protein-coupled receptor Grb2 Growth factor receptor-bound protein 2 GTP Guanosine triphosphate h hour h (as a prefix) Human Ig Immunoglobulin IGF-1 Insulin-like growth factor 1 IMT Inflammatory myofibroblastic tumor IP Immunoprecipitation
IP3 Inositol 1,4,5-trisphosphate
viii Abbreviations and Units
IR Insulin receptor IRS-1 Insulin receptor substrate 1 ITAM Immunoreceptor tyrosine activation motif Itk Interleukine-2-inducible T cell kinase JAK Janus kinase K Conformational equilibrium constant kb Kilobase kcal Kilo calorie l Liter LAT Linker of activated T cells LB Luria-Bertani LTK Leucocyte tyrosine kinase m Milli- M mol/l MAPK Mitogen-activated protein kinase min Minute MK Midkine mol Mole mRNA Messenger ribonucleic acid N- Amino- NFAT Nuclear factor of activated T-cells NK Natural killer NPM Nucleophosmin NMR Nuclear magnetic resonance NSCLC Non-small cell lung cancer nt Nucleotide OD Optical density PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PBS-T PBS-Tween p130CAS p130 Crk-associated substrate PCA Perchloric acid PCR Polymerase chain reaction PDGF Platelet-derived growth factor
ix Abbreviations and Units
PH Pleckstrin homology PI3K Phosphoinositide 3-kinase
PIP2 Phosphatidylinositol 4,5-bisphosphate
PIP3 Phosphatidylinositol 3,4,5-trisphosphate PKA Protein kinase A PKB Protein kinase B (also known as Akt) PKC Protein kinase C
PLAID Phospholipase C-2-associated antibody deficiency and immune dysregulation PLC Phosphoinositide-specific phospholipase C PMSF Phenylmethanesulphonylfluoride PP Polypropylene PTB Phosphotyrosine-binding PTN Pleiotrophin RA Ras associating RNA Ribonucleic acid rp Reverse primer rpm Rotations per minute rs Reference SNP cluster (rs#) RT Room temperature RTK Receptor tyrosine kinase s Second SA Specific array SCC Squamous cell carcinoma SDS Sodium dodecyl sulfate SEM Standard error of the mean SH Src homology Shp2 Src homology region 2 domain-containing phosphatase-1 SNP Single nucleotide polymorphism SOE Splicing by overlap extension spPH Split pleckstrin homology SPR Surface plasmon resonance STAT Signal transducer and activators of transcription STI Soybean trypsin inhibitor
x Abbreviations and Units
TAE Tris-acetate-EDTA TCR T cell receptor TIM Triosephosphateisomerase TK Tyrosine kinase
Tm Melting temperature Tris Trishydroxymethylaminomethane TRP Transient receptor potential U Enzyme unit UV Ultraviolet v Volume VSMC Vascular smooth muscle cells w Weight WB Western blotting
xi Introduction
1 Introduction
1.1 Signal transduction
Unicellular organisms have to be responsive to physical and chemical changes in their environment in order to ensure their survival. These mechanisms also include the response to the presence of other cells. This communication between cells in turn is essential to organize themselves in a complex community like multicellular organisms. The function of communicating with the environment and also with other cells within an organism is achieved through a number of pathways that receive and process signals originating from the external environment [2]. By this process, referred to as signal transduction, cells are able to convert endocrine signals, e.g. hormones or neurotransmitter, and exocrine signals, e.g. olfactory or acoustic stimuli, for the coordination of physiological functions or cell- cell communication [14]. Depending on the type of the extracellular signal molecule a complementary receptor protein expressed by the target cell, that specifically binds the signal molecule, is required. In general, there are three main types of extracellular signal molecules: hydrophilic molecules, lipophilic molecules, and dissolved gases. Lipophilic signal molecules, e.g. steroid and thyroid hormones are able to diffuse through the plasma membrane and target intracellular receptor proteins which directly regulate the transcription of specific genes. The dissolved gases, e.g. nitric oxide and carbon monoxide, also diffuse through the plasma membrane of the target cell activating the production of cyclic GMP. However, the vast majority of extracellular signal molecules are hydrophilic, cannot pass through the plasma membrane, and therefore require cell surface receptor proteins. The three largest classes of cell surface receptors are ligand-gated ion channels, enzyme-linked cell-surface receptors, like the epidermal growth factor (EGF) receptor, and seven-transmembrane (7TM) receptors, commonly referred to as G-protein-coupled receptors (GPCRs), which constitute the largest group among the cell surface receptors [2].
A wide variety of extracellular stimuli like hormones, neurotransmitter, chemokines, flavour and odour substances, or even photons are transmitted to intracellular pathways via receptor-mediated activation of phosphoinositide specific phospholipases C (PLCs) [204]. PLC isozymes are major effectors of GPCRs [20, 29, 236], whereas the activation of PLC isozymes is of significant importance in hematopoietic cells, in which the 1 Introduction
stimulation of the T cell receptor (TCR) leads to the activation of PLC1, and the engagement of the B cell receptor (BCR) leads to the stimulation of PLC2 activity [289].
In the specific case of PLC2, there are three major mechanisms to control the activity of the enzyme, namely (i) the phosphorylation-induced activation, (ii) the activation by small GTPases, and (iii) the autoinhibitory regulation. All three mechanisms are outlined in sections 1.3.3, 1.3.2, and 1.3.4, respectively.
1.2 Phospholipases C
PLCs are calcium-dependent enzymes located in the cytoplasm and activated upon stimulation of cell-surface receptors. PLCs control the phosphatidylinositol 4,5- bisphosphate (PIP2) levels of cells by catalysing the phosphodiesteratic cleavage of the membrane phospholipid PIP2 to yield the two second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). This catalytic activity of PLCs was first demonstrated in 1975 with impure PLC preparations from Bacillus cereus [171]. IP3 induces the efflux of Ca2+ from the endoplasmatic reticulum into the cytosol [122, 251, 268], whereas DAG is a direct activator of various protein kinases C (PKC) which are in turn regulating several signal cascades involved in cellular processes like development or immune response [186]. Otherwise, PIP2 not only serves as a precursor for intracellular second messenger molecules, but also exerts direct signalling functions. It is involved in processes like the organization of the actin cytoskeleton [229], membrane trafficking [163], the regulation of cytokinesis [113] or cell-cell adhesion by gap junctions [307]. Owing to its regulatory role in diverse important cellular processes, changes in the balance of PIP2 levels are associated with several human diseases [15, 144, 157, 213]. Therefore, a strict regulation of enzymes significantly involved in the homeostasis of PIP2, like PLCs, is of particular importance [70].
1.2.1 Structure, Function, Classification, and Expression
The family of PLCs comprises 13 members which can be further classified due to structural or regulatory analogy. To date cDNAs of 6 different PLC subfamilies were cloned, divided into the PLC-(1-4), -(1,2), -(1,3,4), -,-,and -(1,2) [99, 129, 181,
2 Introduction
225, 254, 255, 313] (Figure 1-1). Although the general similarity between the amino acid sequences of the PLC isoforms is not high, the individual domains exhibit a similarity of 40-50 % [70]. The highly conserved catalytic subdomains X and Y are present in every
PLC subfamily [211]. The crystal structure of PLC1 reveals that both domains are composed of alternating -helices and -strands, which is a typical motif of a triose phosphate isomerase (TIM) barrel. Together, they resemble a distorted but closed TIM barrel which represents the active site of the enzyme [58]. The two halves of the catalytic domains are separated by linkers of different lengths, also called X-Y linker. Exceptions here are the PLC isozymes whose X-Y linker, also referred to as specific array (SA), is composed of a split pleckstrin homology (spPH) domain separated by two Src homology 2 (SH2) domains and one Src homology 3 (SH3) domain. The regulatory function of these X-Y linkers is outlined in section 1.3.4. Other common domains of PLCs are the C2 (calcium dependent lipid-binding) domain and the EF hand motifs. Both domains can bind Ca2+ which is an important co-factor for the catalytic activity of PLC isozymes [58, 182]. The pleckstrin homology (PH) domain is located at the N-terminus of all PLC isozymes, except for PLC, which does not harbour a PH domain at all. Investigations of the PLC1
PH domain demonstrated the high affinity binding of PIP2 to the domain allowing processive hydrolysis of the PLC substrate at the plasma membrane [39, 74, 149, 199, 300]. The N-terminal PH domain of PLC isozymes binds most specifically to membranes containing phosphatidylinositol 3,4,5-trisphosphate (PIP3) [61], whereas the split PH domain of PLC2 was shown to be essential for the activation of the enzyme by RacGTPases (see section 1.3.2 for details) [275]. subunits of heterotrimeric GTP binding (G) proteins bind to the PH domain of PLC2 thereby mediating the activation of the enzyme (see section 1.3.1 for details) [280, 281]. Beside to the so far depicted common structural features of PLCs, the PLC and PLC isoforms harbour additional isozyme specific domains. The PDZ-binding motif (PDZ is an acronym combining the first letters of three proteins - post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) - which were first discovered to share the domain) at the C-terminal extension of PLC isozymes allows the binding of PDZ domain containing proteins potentially involved in the regulation of these PLC isoforms [252, 261]. The known isozyme specific domains of PLC are the Ras-GTPase exchange factor-like domain (RasGEF) at the N-terminus and the two Ras-associating (RA) domains at the C-terminus of the lipase [129].
3 Introduction
Figure 1-1: Domain organization of mammalian PLC isozymes. Common structural features of PLC isozymes are the EF-hand motifs (EF), the catalytic TIM barrel (consisting of the two highly conserved catalytic subdomains X and Y), and the C2-domain (C2). With the exception of PLC, all PLC isozymes harbour a PH domain (PH) at their N-terminus. Isozyme specific domains are the Ras-GTPase exchange factor-like domain (RasGEF) and the two Ras-associating domains (RA) of PLC, as well as the PDZ- binding motif at the C-terminus of PLC. PLC isozymes harbour a unique region, also called specific array, inserted through a flexible loop between the two catalytic domains X and Y. This specific array harbours a second split PH domain separated by two SH2-domains (SH2) and one SH3-domain (SH3).
The tissue distribution of PLC isozymes is often closely linked to their physiological role found in transgenic animals. PLC1, PLC3, and PLC4 are highly expressed in different -/- -/- regions of the brain [210, 253], leading to epilepsy and ataxie in PLC1 and PLC4 null mice, respectively [131]. Mice lacking PLC3 show a decreased opioid sensitivity [297]. Although expressed in the same physiological compartment, these three PLC isoforms seem to have different physiological roles. In contrast, expression of the PLC2 isoform is restricted to the hematopoietic system [30, 120, 193, 237]. The two members of the PLC subfamily possess a rather different expression pattern. PLC1 is expressed ubiquitously, whereas the expression of PLC2, just like PLC2, is limited to cells of the hematopoietic -/- lineage [253]. In accordance with the widespread expression of PLC1, PLC1 mice die at embryonic day 9 [118], whereas PLC2 plays an essential role in the development and function of B cells [81, 277]. PLC1 is abundantly expressed in lung, heart, pancreas,
4 Introduction
skeletal muscle, and kidney [38, 148]. The mRNA of PLC3 is most common in brain, heart, lung, and testis [153]. PLC4 is present at very low concentrations in most tissues compared to other PLCs but shows the strongest expression in testis [146], where it is essential for the acrosome reaction in sperm [71, 72]. The PLC isozyme is mostly expressed in brain, liver, skeletal muscle, and heart [129, 241]. Mice deficient in PLC develop malformations of the heart and display a decreased cardiac function [257, 279]. The expression of both PLC isozymes is found to be neuron-specific in the brain [99, 181, 246], and the expression of PLC is most common in testis and prostate, especially in sperm [225].
1.3 Regulation of Phospholipases C
1.3.1 Regulation by heterotrimeric G proteins
GPCRs constitute the largest group within the family of transmembrane receptors. They were first discovered in the 1970s by Martin Rodbell and Alfred Gilman [154, 160], who received the Nobel prize in 1994 for this pioneering work. Almost 20 years later, in 2012, Robert Lefkowitz and Brian Kobilka received a Nobel prize for their work on GPCRs, again demonstrating the importance of these receptors in today’s medicine. Around 30 % of the currently available drugs target the function of GPCR family members [92, 109]. Within the GPCR signalling, the heterotrimeric G proteins account for the transduction of signals from the receptor to the effector system. G proteins consist of three subunits, designated G, G and G, whereat the two latter subunits form tightly associated G complexes [51]. G proteins are able to bind guanine nucleotides which are of great importance for their function as molecular switches. G proteins can switch between two states: (i) the inactive state where GDP is bound to G, and (ii) the active state where GTP is bound to G. After the binding of GTP, G dissociates from G, and the components transduce the signal to the effector proteins. The termination of the signal is achieved by the intrinsic GTPase catalytic activity of the G subunit, which hydrolyses GTP to GDP, allowing the reassociation of G and G[51, 173]. The switch between the two states of a G protein is tightly regulated by different accessory proteins which can be classified into three main types: GEFs (guanine nucleotide exchange factors) that increase the nucleotide
5 Introduction binding to G; GDIs (guanine nucleotide dissociation inhibitors) that inhibit the dissociation of GDP from G; GAPs (GTPase activating proteins) that accelerate the hydrolysis of GTP bound to G [224, 271].
To date there are 16 different G subunits encoding genes, 5 G subunit genes, and 12 G subunit genes known in human, theoretically allowing almost thousand combinations of heterotrimeric G proteins. However, not every combination is reasonable in signal transduction, and the number of those occurring in tissue and native cells is unproved [51].
The G subunits are classified into four families on the basis of sequence similarity: Gs
(stimulates adenylyl cyclase), Gi (inhibits adenylyl cyclase), Gq/11 (stimulates phospholipase C-), and G12/13 (regulates Rho GTPase signalling through Rho GEF activation) [136, 183, 250]. G subunits were shown to be not only negative regulators of G-dependent signalling but also bind to and activate downstream effector proteins upon stimulation of GPCRs, including adenylyl cyclases [260], ion channels [156, 305], PLC isoforms [308], protein kinases D [111], and phosphoinositide-3 kinase (PI3K) [130]. Furthermore, they are involved in the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway [45] and in chemotaxis [36].
1.3.1.1 PLC
PLC isoforms are activated by both, Gq/11 and G subunits [29, 30, 34, 126, 237]. All four members of the Gq/11 subfamily (Gq, G11, G14, G16) directly activate PLC isoforms, but not PLC and PLC isoforms [236, 262, 273]. It was previously assumed that this isozyme specific effect is mediated by the carboxy-terminal extension of the phospholipase which is uniquely present in PLC isozymes [147, 193, 227, 293]. However, a more recent study by the Harden group revealed that the absence of the carboxy-terminal extension of PLC is not affecting the high affinity binding of PLC to
Gq. The crystal structure of an activated complex of Gq with PLC3 exhibits an in PLC isozymes highly conserved helix-turn helix motif, which follows immediately the C2 domain, as the Gq-binding interface [274]. The activation of PLC by members of the
Gq/11 subfamily is declining in the order PLC1 ≥ PLC3 >> PLC2, which is in contrast to the order observed for stimulation by G, PLC3 > PLC2 > PLC1 [192]. Despite their common capacity to activate PLC, G subunits target other structures within the
6 Introduction
lipase than Gq. The interaction site between PLC and G was mapped to both, the catalytic domain Y and the PH domain [223, 280, 281], whereas the presence of the catalytic domains X and Y seems to be sufficient for -dimer stimulation [102].
1.3.1.2 PLC2 and PLC
In addition to PLC isozymes, there is also evidence that PLC2 and PLC are direct downstream effectors of GPCRs. PLC2 is directly activated by G but not by G subunits, which was observed in intact cells as well as with purified proteins. This regulation of PLC2 seems to be an isozyme specific effect since there is no proof that
PLC1 is an effector molecule for G proteins. In opposition to PLC, the PH domain of
PLC2 appears to be dispensable for the activation by G [312, 313]. The last known
PLC to be stimulated by G proteins is PLC, which is selectively activated by both, G12 [158] and G [290] subunits. However, the mechanisms of activation by G proteins for both, PLC2 and PLC, have to be clarified yet.
1.3.2 Regulation by Ras superfamily GTPases
The Ras superfamily of small, monomeric GTPases comprises more than 150 members which can be classified into five subfamilies due to structural and functional similarity: Ras, Rho, Arf/Sar, Ran, and Rab [218]. Similar to heterotrimeric G proteins, monomeric GTPases act as molecular switches cycling between an active, GTP-bound state and an inactive, GDP-bound state. This GTPase cycle is controlled by (i) guanine nucleotide exchange factors (GEFs) that activate the GTPase by catalysing the exchange between GDP and GTP, (ii) GTPase activating proteins (GAPs) that inactivate the GTPase by stimulating the intrinsic GTPase activity, and (iii) guanine nucleotide dissociation inhibitors (GDIs) that keep the inactive GTPase in the cytosol and prevent GDP-GTP exchange [110]. Currently, four PLC isozymes are known to be directly regulated by monomeric GTP-binding proteins, namely PLC2 [100, 101], PLC2 [203], PLC[129,
241, 291], and PLC1 [231].
7 Introduction
1.3.2.1 PLC2 In the late 1990s, the Gierschik group demonstrated that bovine neutrophils contain a factor that stimulates PLC2 in a GTP dependent manner even in the absence of the carboxy-terminal region of PLC2 which is known to be essential for the activation of the lipase by Gq subunits [100]. Later on the factor was identified as a complex between a
RhoGTPase, Cdc42 and/or Rac1, and a Rho GDI, LyGDI. Thereby, PLC2 was the first mammalian target shown to be regulated by both, monomeric and heterotrimeric GTP- binding proteins [101]. Other GTPases that directly regulate PLC2 are Rac2 [102] and
Rac3 [238], and stimulation of PLC2 by Rac1 and Rac2 is much more pronounced than that by Cdc42. PLC isozymes are stimulated by Rho GTPases in the ranking of
PLC2 > PLC3 ≥ PLC1, which is quiet different from the activation by -dimers.
However, the regulation of PLC2 by dimers and Rho GTPases appears to be independent of each other, since the stimulation of the lipase by both regulators is additive [102].
The binding site between Rac and PLC2 was first investigated by Illenberger and coworkers, who replaced the N-terminal PH domain of PLC2 by the analogous region of
PLC1, showing that this chimeric protein is no longer activated by Rho GTPases [102]. The absolute requirement of the PH domain for Rac stimulation was further approved by surface plasmon resonance (SPR) experiments [238] and finally by a crystal structure displaying Rac1 bound to PLC2 [117]. The crystal structure clearly identifies the PH domain of PLC2 as the major effector site for Rac1. Comparison of the crystal structure of
PLC2 unbound and bound to Rac1 revealed that there are no conformational changes upon binding of Rac, indicating that the activation of PLC2 by Rac is solely mediated by localizing and orientating the lipase at membranes [83]. The translocation of PLC2 to the plasma membrane upon Rac stimulation was also demonstrated in intact cells by confocal fluorescence microscopy [103].
1.3.2.2 PLC2
Our group was able to demonstrate that PLC2, but not PLC1, is activated by constitutively active mutants of Rac1, Rac2, and Rac3, but not by Cdc42 or RhoA in intact cells as well as in a cell free system. The activation by Rac2G12V induced the translocation 8 Introduction of the lipase from the soluble to the particulate fraction. This indicates that, like it was shown for PLC2, the recruitment of the lipase to the substrate membrane is part of the activation of PLC2 by Rac GTPases. Furthermore, the mechanism of PLC2 activation by Rac GTPases is independent of tyrosine phosphorylation since replacement of all four tyrosine residues, known to be involved in phosphorylation-induced activation of PLC2, G12V by phenyalanins had no effect on the activation by Rac2 [203]. In contrast to PLC2, the N-terminal PH domain of PLC2 is not required for Rac-mediated stimulation. Instead, both halves of the spPH domain are necessary and sufficient for the isoform-dependent regulation by Rac2. The direct binding of Rac2 to full length PLC2 as well as to the isolated spPH domain was shown in a surface plasmon resonance approach, whereas for full-length PLC1 and the isolated PLC1 spPH domain no binding was observed. Nuclear magnetic resonance (NMR) spectroscopy data revealed that the two halves of the spPH domain form a canonical PH domain architecture with seven -strands and one -helix.
Despite the low sequence similarity (29 %) between the PLC1 and PLC2 spPH domains, the structures of both domains are quite comparable. Given this fact, three amino acid residues (K862, V893, F897), not conserved in PLC1, where identified to be important for
Rac-mediated activation of PLC2. Since mutation of all three residues (K862I, V893Q, F897Q) did not affect the correct folding of the spPH domain, it is likely that these residues directly affect Rac binding [275]. The crystal structure of Rac2 bound to the spPH domain of PLC2 revealed that the interaction of the spPH domain with Rac2 is mediated by the switch I and switch II domains of Rac2 [28].
1.3.2.3 PLC
The PLC isozyme contains two RA domains at the C-terminus, suggesting that it is maybe regulated by Ras. Indeed it was shown that PLC is directly regulated by H-Ras [129, 241] and Rap1a [241], as well as by RhoA, RhoB, and RhoC [291]. Kelley et al. demonstrated that H-Ras binds in a GTP-dependent manner to the RA2 domain, but not to the RA1 domain of PLC and subsequently activates the lipase in intact cells [129]. Upon activation by H-Ras, PLC is translocated from the cytosol to the plasma membrane [241]. RhoA, RhoB, and RhoC but not other Rho family members like Rac and Cdc42, stimulated PLC in intact cells. However, truncated PLC, missing the two RA domains, still responded to activation by Rho. Also, deletion of the RasGEF and the PH domain did not
9 Introduction affect responsiveness of PLC to Rho stimulation. By comparative sequence analysis a region within the catalytic core of PLC was identified, not present in other PLC subfamilies. Mutational analysis revealed that this region is indeed essential for the activation of PLC by RhoA, RhoB, and RhoC [291].
1.3.2.4 PLC1
The small GTPases RalA and RalB bind to and activate PLC1 in vitro as well as in vivo.
The activation is achieved by abrogating the inhibition of PLC by Calmodulin [231].
Furthermore, the RhoGAP p122, was also shown to activate the PLC1 isoform [89].
1.3.3 Regulation by phosphorylation
Phosphorylation of proteins is one of the major regulatory mechanisms in a wide range of cellular processes. Regulation of the activity of PLC enzymes by phosphorylation is known for PLC, PLC and PLC isozymes. The regulation of PLC1 and PLC2 by receptor or non-receptor tyrosine kinases is studied most extensively and will be described more precisely in sections 1.3.3.1 and 1.3.3.2. PLC2 and PLC3 isoforms are serine- phosphorylated by cAMP-dependent kinase (PKA) and/or PKC which was shown to attenuate their activation by heterotrimeric G proteins [155, 302, 303]. ERK phosphorylates PLC1, thereby mediating the mitogenic activity of insulin-like growth factor 1 (IGF-1) [298]. PLC1 binds to PKC via its PH domain, leading to the phosphorylation and subsequent activation of the lipase [68].
1.3.3.1 Regulation of PLC isoforms by receptor tyrosine kinases
Receptor tyrosine kinases (RTK) are cell surface receptors that are of great significance for many fundamental cellular processes like proliferation, differentiation, cell survival, metabolism and cell cycle control. Their molecular architecture is composed of a ligand binding extracellular part, a single transmembrane helix, and a cytoplasmic part containing the protein tyrosine kinase (TK). Binding of an appropriate ligand induces the dimerization of two receptor molecules giving rise to the mutual phosphorylation of the two constituents at specific tyrosine residues. The resulting phosphotyrosines serve as binding sites for 10 Introduction effector molecules that contain SH2 or PTB (phosphotyrosine-binding) domains, such as PLC or PI3K. The recruited proteins are in turn phosphorylated at specific tyrosine residues by the RTK [150, 210]. PLC1 is phosphorylated in intact cells upon stimulation of e.g. epidermal growth factor (EGF) receptor kinase or platelet-derived growth factor (PDGF) receptor kinase on tyrosine residues 771, 783, and 1254 [132, 133, 185, 272]. Mutational analysis revealed that at least phosphorylation of Tyr783 is essential for the activation of PLC1 in response to RTK engagement. The function of phosphorylation on Tyr771 and Tyr1254 are currently unknown [132]. In contrast, the in literature existing in vitro data for the phosphorylation induced activation of PLC1 are controversial [133, 185].
The interaction of PLC1 with activated growth factor receptors, such as the EGF and
PDGF receptor, is mediated by the amino-terminal SH2 domain (SH2N) of PLC1 [35, 119,
206], whereas the C-terminal SH2 domain (SH2C) was shown to associate intramolecularly with phosphorylated Tyr783 [207]. Most interestingly, Tyr771 and Tyr783 are located within the PLC SA region, more precisely in the linker between the SH2C and the SH3 domain. Since the SA is involved in the autoinhibitory regulation of the lipase, Gresset et al. postulated a mechanism for the phosphorylation induced activation of PLC in which tyrosine phosphorylation of the SA leads to large conformational rearrangements, that cause the release of autoinhibition and thus activation of the enzyme [77]. Furthermore, the second PLC isoform, PLC2, is tyrosine phosphorylated and activated by PDGF stimulation in rat fibroblasts overexpressing PLC2 [256]. These results were confirmed in rabbit vascular smooth muscle cells (VSMCs), endogenously expressing PLC2, which represents a more natural surrounding [90].
1.3.3.2 Regulation of PLC isoforms by non‐receptor tyrosine kinases
Non-receptor tyrosine kinases are, in contrast to RTK, cytoplasmic proteins that play important roles in a large number of signal cascades, especially in the immune system, where they are critical constituents in the signal transduction of activated B and T cells. In consequence of the abundant expression of PLC2 in B cells, and the predominant expression of PLC1 in T cells, the activation of these two proteins has been studied most extensively downstream of the B cell receptor (BCR) and the T cell receptor (TCR), respectively.
11 Introduction
The BCR complex consists of an antigen-binding membrane immunoglobulin (mIg) associated with Ig/Ig heterodimers, containing immunoreceptor tyrosine activation motifs (ITAMs). While the mIg is responsible for the binding of antigens, the Ig/Ig heterodimers permit the transmission of the signal to the interior of the B cell. Upon BCR engagement, ITAMs are phosphorylated by the Src family kinase Lyn, triggering the formation of the signalosome composed of tyrosine kinases such as the spleen tyrosine kinase (Syk) and Bruton´s tyrosine kinase (Btk), of adaptor molecules like B cell linker
(BLNK) and cluster of differentiation (CD)19, and of signalling enzymes such as PLC2, Vav, or PI3K. Signals emitted from the signalosome initiate Ca2+ signalling in which
PLC2 was shown to play a critical role [80, 205]. Studies with chicken DT40 B cells deficient in Lyn, Syk or Btk revealed that at least Btk phosphorylates PLC2 upon BCR stimulation [64, 258, 259]. However, it is hard to prove, if PLC2 is a substrate for Lyn and
Syk kinases as well. Btk phosphorylates PLC2 in vitro as well as in intact B cells on tyrosine residues 753, 759, 1197, and 1217, whereas solely Tyr753 and Tyr759 are important for regulating the activity of the lipase [97, 135, 191, 217, 283]. The phosphorylation of PLC2 by Btk is facilitated by means of the adaptor molecule BLNK, which is accountable for bringing both molecules into close proximity. Upon BCR activation BLNK is phosphorylated by Syk kinase, thus providing a docking site for SH2 domains of PLC2 and Btk [67, 107, 140, 217].
Activation of the TCR results in the phosphorylation and subsequent activation of the
PLC1 isoform. The activation and formation of the TCR signaling complex is quite similar to those observed in B cells. Stimulation of the antigen-specific CD3/T-cell receptor complex leads to the phosphorylation of ITAMs, located at the cytoplasmic part of the TCR, by lymphocyte protein tyrosine kinase (Lck). The phosphotyrosines serve as binding sites for the SH2 domains of Zap-70 (-chain associated protein kinase of 70 kDa) which is phosphorylated and activated, and in turn phosphorylates downstream adaptor proteins such as LAT (linker of activated T cells) and SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa). Phosphorylation of LAT and SLP-76 results in the recruitment of several adaptor and signalling proteins to form a multi protein complex, the so called LAT signalosome. Important proteins that assemble this complex, beside LAT and SLP-76, are PLC1, growth factor receptor-bound protein 2 (Grb2), Vav1, and interleukine-2-inducible T cell kinase (Itk). The three major pathways addressed by the 2+ LAT signalosome are Ca , MAPK, and nuclear factor B (NF-B) signalling [25]. PLC1 12 Introduction is phosphorylated and activated in response to TCR engagement and the major tyrosine phosphorylation sites are the same as observed in cells treated with EGF and PDGF (see section 1.3.3.1) [195, 285]. The recruitment of PLC1 by phosphorylated LAT is mediated via the SH2N of PLC1 and represents an essential step in the phosphorylation induced activation of the lipase upon TCR stimulation [232, 247, 310]. The kinases, however, responsible for the phosphorylation of PLC1 in T cells are still unknown, but possible candidates are ZAP-70, Itk, or Lck. It is worth mentioning that PLC1 is also serine phosphorylated in response to both TCR and EGF stimulation. The phosphorylation of serine 1248 is mediated by PKA and PKC and is likely to exert an inhibitory effect on the lipase [8, 134, 194, 195]. Nevertheless, the role of serine phosphorylation of PLC1 is widely unknown so far.
1.3.4 Autoinhibitory regulation
All previously described regulatory mechanisms exert a positive activating influence on PLC isozymes. However, it is known that PLCs exhibit a specific activity (20-30 µmol/min/mg protein) even in the absence of stimulators like G proteins. Due to the fact that the amount of PIP2 in the brain is on average 2-14 nmol/mg protein and the total amount of PLCs in the brain is 2µg/mg protein, the entire PIP2 would be hydrolysed within 2-20 sec, even without receptor stimulation. The consequences would be a needless Ca2+ signal and waste of energy, as the resynthesis of PIP2 out of DAG and inositol requires 5 equivalents of ATP [212]. Since the human organism is directed very economically, there has to be an inhibitory repressor of PLC activity in unstimulated cells. In the 1990s a general model for the inhibition of PLC activity by autoinhibitory elements within the linker between the two halves of the catalytic domain, also called X-Y linker, was proposed. This includes, among others, activation of the enzyme by limited proteolysis, reassembly of separately expressed X and Y polypeptides and the influence of deletion mutants on the activity of the lipase. Limited protease digestion of PLC1 [39, 55], PLC1
[62], and PLC2 [226] within their X-Y linkers as well as the deletion of the SH domain tandem of PLC1 [93] resulted in elevated PLC activity. Furthermore, recombinant expressed SH2SH2SH3 fragments of PLC1 and PLC2 inhibited PLC1, PLC2, PLC1, and PLC1 in a cell free system [91] and the functional reassembly of independently expressed N- and C-terminal halves of PLC1 [94] and PLC2 [309], missing the X-Y 13 Introduction linker, resulted in an increase in basal PLC activity. Taken together, these findings point to a conserved role of the X-Y linker in the autoinhibitory regulation of PLC isozymes. A general model for the autoinhibitory regulation of PLC isozymes was postulated by Hicks et al., where they demonstrated for PLC2, PLC1, and PLC isozymes, that the deletion of a small part of the X-Y linker is sufficient to activate the enzyme [83]. The crystal structure of PLC2 finally revealed the occlusion of its active site by the X-Y linker. Upon engagement of PLC2 by Rac, the lipase is recruited to the plasma membrane where the highly negatively charged X-Y linker is repelled from the also negatively charged plasma membrane. By this mechanism, the access to the active site of PLC2 is opened and the enzyme is able to catalyze the hydrolysis of its substrate [83]. Since PLC isozymes harbour a structurally very distinct, multidomain X-Y linker (for details see section 1.2.1), it is likely that there is a different mechanism for the autoinhibitory regulation than it was shown for the other PLCs. A model for the intramolecular regulation of PLC1 activity was proposed by De Bell et al., in which they state that the SH2C domain inhibits the enzyme in its basal activity and that the availability of this domain is regulated by the N-terminal half of the spPH domain. Furthermore, the SH2N domain seems to participate in the release of the SH2C domain from its inhibitory constraints [48]. It was additionally demonstrated that phosphorylation of PLC1 within its X-Y linker leads to conformational rearrangements of the SH2C domain with respect to the catalytic domain, resulting in the release of autoinhibition [77]. On the other hand, there is the possibility, that the activation by Rac2 and/or the proximity of PLC2 to the plasma membrane induces the liberation of the enzyme from its autoinhibitory constraints [59].
1.4 Role of PLC2 in inflammatory and autoimmune diseases
In the last decade, several mutations in PLC2 had been associated with inflammatory and autoimmune diseases, in mouse models as well as in humans. A genome-wide N-ethyl-N- nitrosourea (ENU) mutagenesis approach was chosen to identify genetic alterations in genes involved in inflammatory and autoimmune diseases in the mouse. Two of the generated mouse strains exhibited point mutations in the PLCG2 gene, named Ali5 [301]
14 Introduction
(Abnormal limb) and Ali14 [1]. The phenotype of Ali5 mice is characterised by chronic inflammation of the paws and severe eye deformation which is triggered by proliferation of cells in the epithelium and a thinning of the sclera. The Ali5 point mutation is localized in the catalytic subdomain Y, where the aspartic acid on position 993 is substituted by a Ali5 glycine [301]. The biochemical characterisation of PLC2 revealed that the mutation has no impact on the basal activity of the lipase, neither in vitro, nor in intact cells. However, in B the cells Ali5-type mutation caused an increase in sustained Ca2+ entry in response to antigen receptor stimulation. The mechanistic background of this observation is thought to Ali5 be an increased stability and penetrability of PLC2 at the plasma membrane [60, 301]. Ali5 2+ The same effect was observed for platelets, where PLC2 leads to enhanced Ca mobilization, integrin activation and granule secretion and ultimately to a prothrombotic phenotype in mice [56]. The phenotype of Ali14 mice is quite similar to this of Ali5 mice, also suffering, amongst other things, from spontaneous swelling and inflammation of the hind paws. Furthermore, cultured B cells from these mice show an enhanced calcium Ali14 influx. The amino acid residue mutated in PLC2 is located within the N-terminal split Ali5 PH domain, where tyrosine 495 is replaced by a cystein [1]. As it was shown for PLC2 , also the Ali14-type mutation had no impact on the catalytic activity of the enzyme, but their combination resulted in a constitutively active PLC2 enzyme [60]. The importance of a tight regulation of PLC2 activity has become increasingly clear as the loss of autoinhibitory regulation, by either point mutations [311] or larger deletions [190] in the autoinhibitory region of PLC2 was associated with severe inflammatory phenotypes in humans. Just recently Ombrello et al. identified a novel disease pattern characterized by cold-induced urticaria, immunodeficiency, and autoimmunity. Since this unique phenotype could be linked to deletions in the human PLCG2 gene within the autoinhibitory region between the two catalytic subdomains X and Y, the syndrome was designated PLAID
(phospholipase C-2-associated antibody deficiency and immune dysregulation) [190]. PLAID patients suffer, amongst other things, from a previously undescribed form of cold- induced urticaria, the so called familial atypical cold urticaria (FACU) [73]. Cold urticaria, in general, belongs to the physical urticarias triggered by a cold stimulus, provoking whealing and itching of the cold exposed skin [162]. Affected individuals had lifelong symptoms from early childhood on, including pruritis, erythema, and urticaria after cold exposure like cold atmosphere, aquatic activities, handling of cold objects, and ingestion of cold food or beverages. Skin biopsy after a cold challenge revealed an increase in mast cell degranulation compared to healthy controls. However, the processes leading to mast cell 15 Introduction degranulation are poorly understood. In contrast to the other cold urticarial forms, FACU is limited to the exposed skin without a generalized and systemic reaction [73]. Beside this cold-induced unique inflammatory phenotype, PLAID patients also display various other manifestations like recurrent sinopulmonary infections, antibody deficiency, symptomatic autoimmune and allergic disease, and common variable immunodeficiency. Furthermore, the levels of circulating CD19+ B cells, IgA+ and IgG+ class-switched memory B cells, and natural killer cells are diminished in these patients. The characterisation of PLAID B cells exhibited a reduced in vitro class switched antibody production and poor in vitro expansion. As mentioned above, the described phenotype could be linked to two different exon deletions in the PLCG2 gene. In two of the three affected families heterozygous deletions of exon 19 (19) were identified, whereas in the third family exons 20 through 22 (20-22) were deleted on one allele [190]. Both deletions affect the SH domain tandem of PLC2, more precisely the C-terminal SH2 domain and the SH3 domain, which were shown to be important for the autoinhibitory regulation of the enzyme [59]. Indeed, both deletion mutants showed enhanced basal activity in transfected COS-7 cells compared to wild-type PLC2. In spite of this gain in enzymatic activity, downstream signalling in stimulated PLAID B cells was diminished. However, unstimulated B cells from those subjects showed a clear increase in cytosolic Ca2+ levels with decreasing temperatures and mast cells transfected with PLC219 or PLC220-22 displayed spontaneous degranulation at 20 °C but not at 37 °C [190]. Whole exon sequencing of another family revealed a hypermorphic missense mutation in PLCG2 that causes a dominantly inherited autoinflammatory disease with immunodeficiency [311]. Most interestingly, these patients do not suffer from cold urticaria. The described disease is characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. The detected missense mutation is localized in the C-terminal SH2 domain were serine 707 is replaced by tyrosine. To distinguish the described phenotype, caused by a single amino acid substitution, from
PLAID, caused by genomic deletions in PLCG2, the term APLAID (autoinflammation and
PLC2-associated antibody deficiency and immune dysregulation) was proposed [232]. Zhou et al. [311] and unpublished data from our group showed that the basal activity of S707Y PLC2 is elevated compared to wild-type PLC2. Consequently, but in contrast to
PLAID, PLC2-dependent signalling was elevated in APLAID patients, which was 2+ confirmed by elevated levels of IP3 production, intracellular Ca release, and ERK phosphorylation. However, the assumption that the substitution of serine for tyrosine 16 Introduction creates a novel phosphorylation site, which compromises autoinhibition and thereby leads to an increase in basal activity [311], could be rebuted by unpublished experiments from our group.
1.5 Anaplastic lymphoma kinase
1.5.1 Structure, Expression, and Function
Anaplastic lymphoma kinase (ALK) was originally described in 1994 as an oncogene activated in anaplastic large cell lymphomas (ALCL) with the chromosomal translocation t(2;5) [230], causing the fusion of the kinase domain of ALK to the amino terminal half of nucleophosmin (NPM), generating a fusion gene, NPM-ALK (see section 1.5.3.1) [174]. The molecular characteristics of ALK itself were not elucidated until 1997. ALK displays the classical structural features of a RTK containing an extracellular ligand-binding domain, a membrane spanning domain and the intracellular kinase domain. Based on homology ALK constitutes a subgroup together with LTK (leucocyte tyrosine kinase) within the IR (insulin receptor) superfamily [108, 175] and is highly conserved across species [209]. The human ALK gene encodes a protein of 1620 amino acids with a molecular weight of approx. 180 kDa. Posttranslational modifications such as N-linked glycosylation are generating a mature ALK with a molecular weight of approx. 220 kDa [108, 175]. The expression of murine ALK mRNA was preliminary found in the central and peripheral nervous system during embryonic development but at considerably lower concentrations in the adult nervous system [108, 175]. Its expression is also observed in the developing sensory organs, reproductive organs, skin and stomach [266]. The fading expression of ALK mRNA from developmental to adult stages in mice was also found in the developing central nervous system (CNS) of chicken, where ALK localizes to a subset of DRG (dorsal root ganglia) [98]. A similar expression pattern was also found in a subset of DRG neurons in the rat [50]. Furthermore, ALK mRNA was as well detected in the developing CNS in Drosophila [159]. Mice homozygous for a deletion of ALK have normal appearance and no obvious tissue abnormalities but they revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioural tests [16]. The role of ALK in neuronal development is further supported by in vitro studies where it was shown that stimulation of human neuroblastoma (SK-N-SH) cells,
17 Introduction endogenously expressing ALK, by an agonist monoclonal antibody induces activation of ALK which subsequently leads to the activation of the MAP kinase signaling cascade. The activation of this signaling cascade is driving the cells to proliferation and neurite outgrowth [177, 242]. Thus, ALK RTK appears to have an important role in the normal development and function of the central and peripheral nervous system. Nonetheless its physiological role is still enigmatic.
The control of the activity of RTKs can be achieved by expression levels, binding of ligands, or mutations that inhibit or enhance constitutive receptor activity [286]. So far, two activating ligands for ALK were identified, namely pleiotrophin (PTN) [248] and midkine (MK) [249]. Binding of the ligands induces homodimerization of ALK, leading to trans-phosphorylation and activation of the kinase [248]. Both ligands are heparin-binding growth factors and form an own family within the heparin-binding growth factor families. They are involved in processes such as neural development, cell migration and angiogenesis [123, 141, 180]. The expression of both ligands is similar to that of ALK during embryonic development [208]. Just recently it was shown by Allouche that ALK is a novel dependence receptor, meaning that in the absence of ligands the ALK receptor is inactive and its expression is leading to apoptosis, whereas activation of ALK by ligands or as a result of ALK fusion proteins, decreases apoptosis [3, 178].
1.5.2 ALK in disease
Overexpression and constitutive or excessive activation of ALK can lead to the deregulation of cell proliferation and survival resulting in various human cancers. Since the important first description of the ALK fusion protein NPM-ALK in ALCL [174, 230], many other ALK chimeras have been described in diseases like inflammatory myofibroblastic tumors (IMTs) [78], diffuse large B-cell lymphoma (DLBCL) [6], non- small cell lung cancer (NSCLC) [216, 239], esophageal squamous cell carcinoma (SCC) [53, 115], colon, and breast carcinoma [152]. The expression of ALK was also documented in a variety of cancer cell lines where it was shown that overexpression or gain of function mutants of ALK can lead to tumorigenesis (reviewed in [11])
18 Introduction
1.5.2.1 Point mutations, overexpression, and amplification of full‐ length ALK in neuroblastoma
Activating ALK mutations have been reported to be associated with sporadic and familial cases of neuroblastoma [32, 37, 114, 176], which was first described in 2000 [143]. All together there are 12 different residues known to be mutated in neuroblastoma. However, the two master mutation hotspots were found to be F1174L and R1275Q [7]. These mutations were observed in 49 % and 34.7 % of mutated cases, respectively [24]. Both mutations lead to an increase in the catalytic activity of the enzyme and their oncogenic potential was demonstrated by transformation of NIH-3T3 fibroblasts and tumor formation in nude mice [37, 145]. Furthermore, it was shown that both mutations are associated with a defect in N-linked glycosylation of the kinase and that the constitutive activation leads to impaired receptor trafficking resulting in the retainment of the kinase in the endoplasmic reticulum/Golgi compartments [165]. The just recently elucidated crystal structure of ALK in its inactive conformation revealed the autoinhibition of ALK by its C helix. Most interestingly, the majority of the ALK mutations found in neuroblastoma, including F1174L and R1275Q, are located in or near by the area of this autoinhibitory loop. Therefore, these activating mutations are thought to release ALK from its autoinhibitory constraint by allowing the unrestrained mobility of the C helix [145]. Furthermore, these mutations were shown to be associated with constitutive phosphorylation of ALK and its downstream targets [32, 37, 114, 176] and enhanced ATP binding affinity of the kinase [145]. Beside the described gain of function mutations there is also evidence for the amplification of the ALK gene leading to neuroblastoma in about 2 % of neuroblastoma cases [24]. In addition to ALK mutations and amplification of the ALK gene, overexpression of the wild-type ALK protein was also reported to lead to oncogenic activity in neuroblastoma cells. It seems, that a critical threshold of ALK wild-type expression is necessary to achieve this oncogenic activation [198]. However, this mutation- independent overexpression of full-length ALK is still uncharacterized at the molecular level.
19 Introduction
1.5.3 ALK fusion proteins
1.5.3.1 NPM‐ALK
Among the known ALK fusion proteins NPM-ALK is the most studied and the most frequent fusion protein (80 % of translocations), which is found in 40 % to 60 % of ALCL patients [5]. It is created via the recurrent chromosomal translocation t(2;5)(p23;q35) which was found in the late eighties by several groups [12, 63, 124, 164]. Only four years later it was demonstrated that this translocation creates the fusion gene between a so far unknown tyrosine kinase on chromosome 2, and the NPM gene on chromosome 5 [174, 230]. Owing to its association with ALCL the kinase was termed ALK and its expression in ALCL led to the clinically distinct entity ALK+ ALCL [201]. NPM-ALK is a 80 kDa fusion protein containing the first 117 amino acid residues of NPM fused to the cytoplasmic part of ALK starting from residue 1058 [174]. Most interestingly, all so far identified ALK fusion proteins contain exactly the same portion of ALK [54]. NPM itself plays multiple roles in cell growth and cell proliferation and is localized mainly at the nucleolus [189]. There it serves as a bidirectional shuttle for proteins between the nucleus and the cytoplasm [19]. The amino terminal half of NPM, which is fused to the kinase domain of ALK, harbours an oligomerization domain and two nuclear export signals [189]. It was shown that the NPM segment leads to the oligomerization of NPM-ALK resulting in the autophosphorylation of the kinase which is absolutely required for the kinase activity of the chimera [17]. Even more important, NPM-ALK lacking the NPM segment is no longer able to transform NIH 3T3 cells, implying that the NPM segment is essential for the oncogenic potential of NPM-ALK [69]. The presence of a dimerization/oligomerization domain is also a common feature of other ALK fusion partners [201]. In contrast to full- length ALK, NPM-ALK is ectopically expressed in lymphoid cells and localized in the cytoplasm and in the nuclear compartments [17].
1.5.3.1.1 Oncogenic signalling of NPM‐ALK
To date four pathways were identified to be activated by NPM-ALK in ALCL, designated as the PLC, PI3K/Akt, Ras/MAPK, and JAK/STAT pathways (Figure 1-2). STAT (signal transducer and activators of transcription) family members are activated by tyrosine phosphorylation which is in particular mediated by Janus Kinases (JAKs). The subsequent dimerization of STATs allows their translocation to the nucleolus and the activation of transcription of genes involved in proliferation, cell survival, and immune response. The
20 Introduction
JAK/STAT pathway is activated by interferon and cytokine receptor signalling [245]. It was shown that ALK+ ALCL exhibit activated STATs and in turn that STATs are required for the transforming activity of NPM-ALK in mouse embryo fibroblasts. In agreement, inactivation of NPM-ALK with small molecule inhibitors leads to a decreased phosphorylation of STATs. This activation of STATs by NPM-ALK was shown to be a result of the interaction between JAKs and NPM-ALK [201].
Figure 1-2: Oncogenic signalling of NPM-ALK. The so far known pathways addressed by NPM-ALK are the PI3K/AKT, the JAK/STAT, the MEK/ERK and the PLC pathway. For further details see main text.
PI3K was identified as a further downstream signalling molecule of NPM-ALK. It was shown that PI3K and its downstream effector, serine/threonine kinase (Akt), are activated by NPM-ALK and that this activation is induced by the recruitment of the C-terminal SH2 domain of PI3K by NPM-ALK. The activation of this antiapoptotic signalling pathway is important for the transforming potential of NPM-ALK, since the inhibition of PI3K or Akt by inhibitors or dominant-negative mutants is leading to growth arrest or even apoptosis of NPM-ALK+ cells [10, 234].
21 Introduction
The third known pathway addressed by NPM-ALK is the MEK/ERK pathway. This pathway is part of a kinase cascade, the MAPK pathway, which is activated by growth factor receptors. Major cellular processes such as differentiation, cell survival, and proliferation are controlled by this signalling cascade [169]. Co-immunoprecipitation studies showed that NPM-ALK interacts with insulin receptor substrate 1 (IRS-1), Shc, and growth-factor-receptor-bound protein 2 (Grb2) [9, 69]. All three mentioned molecules are responsible for the transmission of signals from growth factor receptors to the MEK/ERK pathway [169]. However, NPM-ALK mutants deficient in interacting with IRS-1 and Shc retain their ability to transform NIH 3T3 cells [69]. In line with these results, there is also evidence that NPM-ALK directly interacts with MEK kinases [46, 295].
The most interesting finding for this work was the link between the PLC pathway and NPM-ALK. Bai et al. already showed in 1998 with pull-down assays that NPM-ALK binds to both SH2 domains of PLC, at which the strongest binding was observed with the N-terminal SH2 domain. The interaction of NPM-ALK and PLC leads to the phosphorylation of the lipase and thereby, most presumably, to the activation of the enzyme. Ba/F3 cells overexpressing NPM-ALK also showed an enhanced inositol phosphate production, pointing again to an activation of PLC by NPM-ALK. Furthermore, Bai et al. identified Tyr664 in NPM-ALK to be essential for the complex formation of the kinase and PLC. In contrast to wild-type NPM-ALK, NPM-ALKY664F did not lead to the transformation of Rat-1 fibroblasts. Taken together these results imply that PLC is an important downstream target of NPM-ALK which accounts for the mitogenic potential of the constitutively active kinase [9]. Somehow conflictive are the results published by Dirks et al.. They indeed reported the phosphorylation of PLC in ALK+ ALCL-derived cell lines, but they mentioned that in these cell lines PLC proteins are even phosphorylated in serum-starved ALCL-cells and that there is no direct proof that NPM- ALK provokes this phosphorylation [52]. The possibility that the activation of PLC by NPM-ALK is cell type specific was further approved by Piccinini et al. [202]. Moreover,
PLC1 was identified in a mass spectrometry approach to be a NPM-ALK interacting protein [46]. Beside this potentially direct interaction between NPM-ALK and PLC there is also evidence that NPM-ALK can activate Rac1 GTPase [42], which is in turn a direct activator of PLC2 [203]. Vav3 is one of the GEFs involved in the activation of Rac1 [42]. The importance of Rac activation in NPM-ALK driven disease progression and metastasis was demonstrated by Rac inhibitors [43]. 22 Introduction
1.5.3.2 EML4‐ALK
Lung cancer is the worldwide second frequent cancer after prostate cancer and breast cancer for men and women, respectively. However, the mortality of lung cancer is the highest among the various cancer forms for both sexes [116]. The fusion of ALK to the echinoderm microtubule associated protein-like 4 (EML4) was only recently described in non-small cell lung cancer (NSCLC) [216, 239]. The basis of this fusion is a small inversion within chromosome 2p (Inv (2) (p21p23)) that joins the N-terminal part of EML4 to the exons 20-29 of ALK, representing the intracellular kinase domain. As it was demonstrated for NPM-ALK, the EML4 fusion partner is mediating ligand-independent dimerization of ALK, leading to the constitutive activation of EML4-ALK. The oncogenic potential of EML4-ALK was demonstrated in cell culture systems and in nude mice [239]. EML4-ALK transgenic mice, expressing the fusion protein specifically in lung alveolar epithelial cells, develop adenocarcinoma a few weeks after birth. Administration of small molecule inhibitors of the kinase activity of EML4-ALK can stop the progression of the tumours and even result in a complete disappearance of tumour tissue [240]. Thus, EML4- ALK seems to play a crucial role in the development of NSCLC and could therefore be a promising target for drug development.
1.6 Aim of the work
The regulatory network of PLC2 consists of three main mechanisms, (i) the activation by RhoGTPases, (ii) the phosphorylation-induced activation, and (iii) the autoinhibitory regulation of the lipase. In the last decade it has become abundantly clear that misregulations of the activity of PLC2 can lead to severe phenotypes in mice as well as in human. A first aim of the present doctoral thesis was to further investigate the molecular mechanisms underlying the autoinhibitory and phosphorylation-dependent regulation of
PLC2, and to identify the structural elements involved in these regulatory processes. The association of exon deletions in the PLCG2 gene with a novel disease pattern, named
PLAID (phospholipase C-2-associated antibody deficiency and immune dysregulation), characterized by cold-induced urticaria, immunodeficiency, and autoimmunity was just recently described. A further aim of this thesis was to investigate the consequences of cold 23 Introduction
temperatures on the activity of the PLC2 mutants associated with PLAID and to identifiy structural elements that mediate the temperature sensitivity of the enzyme.
24 Materials and Methods
2 Materials and Methods
2.1 Materials
2.1.1 Chemicals
Acrylamide 4K 30 % (w/v) (37.5:1 mix) AppliChem, Darmstadt Agar Serva, Heidelberg Agarose Sigma, Deisenhofen Albumine bovine fraction V (BSA) Serva, Heidelberg Ammonium formate Fluka, Taufkirchen Ammonium persulphate Serva, Heidelberg Ampicillin, sodium salt Sigma, Deisenhofen Aprotinine (Trasylol®) Bayer, Leverkusen Bacto Trypton Becton Dickinson, Heidelberg Benzamidine Sigma, Deisenhofen Bromphenol blue Serva, Heidelberg Crystal violet Sigma, Deisenhofen Dithiothreitol (DTT) Serva, Heidelberg DMEM Medium (# 41965-039) Gibco Life Technologies, Karlsruhe Deoxynucleoside triphosphate (dNTP) mix MBI Fermentas, St. Leon-Rot Dimethyl sulfoxide (DMSO) Roth, Karlsruhe - Dowex resin (1x8, 100-200 mesh, Cl form) Sigma, Deisenhofen Dulbecco´s PBS (1x) (# H15-002) PAA, Cölbe ECL™ Western Blotting Detection Reagent Amersham, Freiburg ECL Western Blotting Substrate Thermo Scientific, Schwerte Ethylene diamine tetraacetic acid (EDTA), disodium salt Merck, Darmstadt Ethidium bromide (EtBr) AppliChem, Darmstadt Fetal calf serum (FCS, used in mammalian and avian cell culture) Gibco Life technologies, Karlsruhe
25 Materials and Methods
Formic acid Roth, Karlsruhe L-Glutamine PAA, Cölbe Glycerine Roth, Karlsruhe Glycine AppliChem, Darmstadt 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HEPES (for cell culture) PAA, Cölbe High molecular weight protein marker (HMW) Sigma, Deisenhofen Ink Pelikan, Hannover Kanamycin Serva, Heidelberg Lambda DNA/EcoRI+HindIII Marker, 3 MBI Fermentas, St. Leon-Rot Leupeptine Serva, Heidelberg TM Lipofectamine 2000 Transfection reagent Invitrogen, Karlsruhe Lithium chloride Sigma, Deisenhofen Loading dye (6x) MBI Fermentas, St. Leon-Rot jetPRIME® Polyplus Transfection, Peqlab, Erlangen β-Mercaptoethanol Roth, Karlsruhe Milk powder Fluka, Neu-Ulm Milk powder Roth, Karlsruhe OptiMEM I Medium Gibco Life technologies, Karlsruhe PageRulerTM Broad Range Unstained Protein Ladder Thermo Scientific, Schwerte Penicillin/Streptomycin PAA, Cölbe Pepstatin A Serva, Heidelberg Phosphatase Inhibitor Cocktail 2 Sigma, Deisenhofen Phenylmethylsulphonyl fluoride (PMSF) Sigma, Deisenhofen Polyoxyethylene-sorbitan monolaurate (Tween 20) Sigma, Deisenhofen Protein A-Agarose Roche Applied Science, Mannheim Pyronin Y Sigma, Deisenhofen Quicksafe A Scintillation Fluid Zinsser, Frankfurt RTU 60 Developer Adefo, Dietzenbach RTU 60 Fixer Adefo, Dietzenbach
26 Materials and Methods
Sodium chloride AppliChem, Darmstadt Sodiumdodecyl sulfate (SDS) Roth, Karlsruhe Sodium formate Fluka, Neu-Ulm Sodium pyruvate PAA, Cölbe Sodium tetraborate decahydrate (Borax) Sigma, Deisenhofen Soybean trypsin inhibitor (STI) Fluka, Neu-Ulm N,N,N',N'-Tetramethyl-ethane-1,2-diamine (TEMED) Serva, Heidelberg Thimerosal Sigma, Deisenhofen Trishydroxymethylaminomethane (Tris) Roth, Karlsruhe Trypan blue Merck, Darmstadt Trypsin-EDTA solution (# L11-004) PAA, Cölbe Tween 20 Sigma, Deisenhofen Water for chromatography (LiChrosolv®) Merck, Darmstadt Yeast Extract Servabacter Serva, Heidelberg
All chemicals and reagents used but not listed were obtained in p.a. quality from the following companies: Merck (Darmstadt), Riedel-de Haen (Seelze), or Roth (Karlsruhe).
2.1.2 Radiochemicals myo-[2-3H]Inositol with PT6-271 stabilizer PerkinElmer, Rodgau-Jügesheim
2.1.3 Enzymes and Kits
Antarctic Phosphatase New England Biolabs, Frankfurt Big Dye™ Terminator Ready Reaction Mix PE Biosystems, Wertigheim ® Nucleobond AX PC-Kit-100 Macherey-Nagel, Düren ® Nucleospin Kit Macherey-Nagel, Düren QIAquick Gel Extraction Kit Qiagen, Hilden ® QuikChange II XL Site-Directed Mutagenesis Kit Agilent Technologies, Waldbronn
27 Materials and Methods
™ PfuUltra High-Fidelity DNA Polymerase Agilent Technologies, Waldbronn ™ Phusion High-Fidelity DNA Polymerase Thermo Scientific, Schwerte T4 DNA Ligase Fermentas, St. Leon-Rot ® Zero Blunt PCR Cloning Kit Invitrogen, Karlsruhe
All restriction endonucleases were purchased from Fermentas, St. Leon-Rot or New England Biolabs, Frankfurt.
2.1.4 Consumables 6-well-plate Renner, Dannstadt 24-well-plate Renner, Dannstadt AGFA Cronex 5, Medical X-Ray Film AGFA, Mortsel, Belgium Cell culture dish, 100 mm Renner, Dannstadt Cell culture flask, 75 cm2 Renner, Dannstadt Cell scraper 25 cm Sarstedt, Nümbrecht Chromatography paper 3MM Chr Schleicher & Schuell, Dassel CryoPure tube 1.8 ml Sarstedt, Nümbrecht Micro tube (1.5 ml, PP) Sarstedt, Nümbrecht Microfuge® tube Polyallomer BeckmannCoulter, Brea, USA Multiply®-Pro PCR tubes 0.5 ml Sarstedt, Nümbrecht Nitrocellulose membrane disc filter Sartorius, Göttingen (0.45 µm pore size) Petri dish (100 mm) Sarstedt, Nümbrecht Protran® Nitrocellulose transfer membrane Schleicher & Schuell, Dassel Sterile surcical blade BBraun Aesculap, Tuttlingen Scintillation tube (6 ml) Perkin-Elmer, Rodgau-Jügesheim Scintillation tube (20 ml) Perkin-Elmer, Rodgau-Jügesheim Syringe (1 ml, 5 ml, 20 ml) Becton Dickinson, Heidelberg Syringe filter Filtropur S 0.2 (0.22 µm) Merck, Bruchsal Tube (13 ml, 100x16 mm, PP) Sarstedt, Nümbrecht Tube (15 ml, 120x17 mm, PP) Sarstedt, Nümbrecht Tube (50 ml, 114x28 mm, PP) Sarstedt, Nümbrecht
28 Materials and Methods
2.1.5 Bacterial and Mammalian Cells African green monkey kidney (COS-7) cells Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig Escherichia coli DH5α Clontech, Heidelberg
2.1.6 Plasmids
2.1.6.1 Commercial Plasmids pcDNA3.1(+) Invitrogen, Karlsruhe pcDNA3.1(–) Invitrogen, Karlsruhe pCR®-Blunt Invitrogen, Karlsruhe
2.1.6.2 Recombinant In‐House Plasmids pcDNA3.1(+)-Rac2 C. Walliser (accession number CAG30441.1) G12V pcDNA3.1(+)-Rac2 C. Walliser pcDNA3.1(-)-PLCβ2 B. Möpps (accession number NP_004564) pcDNA3.1(-)-myc-hPLCγ1 C. Walliser (accession number ABB84466) pcDNA3.1(+)-myc-hPLCγ2 C. Walliser (accession number NP_002652) pcDNA3.1(+)-myc-hPLC2PCI L. Becker F897Q pcDNA3.1(+)-myc-hPLC2 C. Walliser pcDNA3.1(+)-myc-hPLC2 C. Walliser Y733E pcDNA3.1(+)-myc-hPLCγ2 L. Becker Y753E pcDNA3.1(+)-myc-hPLCγ2 L. Becker Y759E pcDNA3.1(+)-myc-hPLCγ2 L. Becker Y753E/Y759E pcDNA3.1(+)-myc-hPLCγ2 L. Becker Y733E/Y753E/Y759E pcDNA3.1(+)-myc-hPLCγ2 L. Becker pcDNA3.1(+)-myc-hPLC2-212 M. Retlich
29 Materials and Methods
pcDNA3.1(+)-myc-hPLC2-221 M. Retlich pcDNA3.1(+)-myc-hPLC2-211 M. Retlich Ali5 pcDNA3.1(+)-myc-hPLC2 J. Haas Ali14 pcDNA3.1(+)-myc-hPLC2 J. Haas Ali5/Ali14 pcDNA3.1(+)-myc-hPLC2 J. Haas S707Y pcDNA3.1(+)-myc-hPLC2 J. Haas
2.1.6.3 Recombinant Plasmids received from other institutes pcDNA3-hALK S. Turner, Cambridge (accession number NM_004304.4) pcDNA3-hALKF1174L S. Turner, Cambridge pcDNA3-hALKR1275Q S. Turner, Cambridge pcDNA3-hNPM-ALK S. Turner, Cambridge (accession number U04946.1) pcDNA3-hEML4-ALK S. Turner, Cambridge (accession number AB663645.1)
2.1.7 Oligonucleotides
All oligonucleotides used for polymerase chain reactions or for sequencing purposes were synthesised by Thermo Fisher Scientific, Ulm or Sigma-Aldrich, Steinheim.
2.1.7.1 Sequencing Primers
2.1.7.1.1 General Plasmid Sequencing Primers
T7 Promotor (for sequencing the 5´ ends of pcDNA3.1 inserts) 5´-TAA TAC GAC TCA CTA TAG GGA GA-3´ 5´-TAA TAC GAC TCA CTA TAG GG-3´ (GATC Biotech, Konstanz)
BGH Reverse Primer (for sequencing the 3´ ends of pcDNA3.1 inserts) 5´-TAG AAG GCA CAG TCG AGG-3´
30 Materials and Methods
Reverse Primer (for sequencing the 5´ ends of pCR®-Blunt inserts) 5´-CAG GAA ACA GCT ATG AC-3´
Universal Primer (for sequencing the 3´ ends of pCR®-Blunt inserts) 5´-GTA AAA CGA CGG CCA G-3´
2.1.7.1.2 Human PLC1 cDNA Sequencing Primers hPLCg1_S1 5´-TCT ACT CCA AGA AGT CGC-3´ hPLCg1_S2 5´-GAT ACA TTG CAG GCA CCC AC-3´ hPLCg1_S3 5´-ATG TAC AGC GCC CAG AAG-3´ hPLCg1_S4 5´-GAC ACC ATG AAC AAC CCT C-3´ hPLCg1_S5 5´-CAG AGA AAC ATG GCC CAA TAC-3´ hPLCg1_S6 5´-ACC AGC AGC AAG ATC TAC-3´ hPLCg1_S6b 5´-CAA CGA GGA TGA GGA GGA G-3´ hPLCg1_S7 5´-CCC CAA GTT CTT CTT GAC AGA C-3´ hPLCg1_S8 5´-CAG ACA GTG ATG CTA GGG AAC-3´ hPLCg1_S9 5´-CCA GAA TGT GGA GAA GCA AG-3´ hPLCg1_S10 5´-GTG AAA AAG ATC CGT GAA G-3´ hPLCg1_S11 5´-TTG TGG CCC TCA ACT TCC-3´ hPLCg1_S12 5´-GAG TTT GTG GTG GAC AAT GG-3´
2.1.7.1.3 Human PLC2 cDNA Sequencing Primers PLCg2_S1 5´-GGG CTT CTT GGA TAT CAT GG-3´ PLCg2_S2 5´-CTC CGA GAG TTG AAG ACC ATC-3´ PLCg2_S3 5´-TGA ACA AAG TCC GTG AGC-3´ PLCg2_S4b 5´-CAT TGA ACT GGA CTG CTG-3´ PLCg2_S5 5´-ATG TCA ACA TGG AGG ACA AG-3´ PLCg2_S5b 5´-CCA GCT GCG GGA GAA GAT C-3´ PLCg2_S6 5´-AGA CCT TCC CCA ATG ACT ACA C-3´ PLCg2_S7 5´-ATG CTG ATG AGG ATT CCC-3´ PLCg2_S8 5´-GAT CCC AGT GAA ATC AAT CCG-3´ PLCg2_S9c 5´-AGT TTG CCA CAG ACA GGG TG-3´ PLCg2_S10 5´-CTG ACA GCA TCA TCA GAC-3´ PLCg2_S11 5´-TCA AGG TTC TCG GTG CTC-3´ 31 Materials and Methods
2.1.7.1.4 Human ALK cDNA Sequencing Primers
ALK_S1 5´-TGC ATG ACC TCA GGA ACC-3´ ALK_S2 5´-GTG CTA GTG GAG AAC AAA AC-3´ ALK_S3 5´-AGA GAG ACT GGA GAA TAA C-3´ For sequencing the whole ALK cDNA the sequencing primers NPM-ALK_S2 to NPM-ALK_S7 were additionally used.
2.1.7.1.5 Human NPM‐ALK cDNA Sequencing Primers
NPM-ALK_S1 5´-AAG GAT GAG TTG CAC ATT G-3´ NPM-ALK_S2 5´-TGA GTA CAA GCT GAG CAA G-3´ NPM-ALK_S3 5´-CAG GAC GAA CTG GAT TTC-3´ NPM-ALK_S4 5´-GGA AAA CCA CTT CAT CCA C-3´ NPM-ALK_S5 5´-TGG ATA TAT GCC ATA CCC-3´ NPM-ALK_S6 5´-AGG AAG AGA AAG TGC CTG-3´ NPM-ALK_S7 5´-TAC GGC TCC TGG TTT ACA G-3´
2.1.7.2 PCR Primers
2.1.7.2.1 Primers for the amplification of PLC2 SH domains
All Primers contain an AvrII (CCTAGG) restriction site.
SH2N (aa 515-638)
AB063 (fp) 5´-GTCCTAGGGGAGGAAGTGCCCC-3´
AB080 (rp) 5´-CCTAGGGGGGTTGGGCACAG-3´
SH2C (aa 637-747)
AB066 (fp) 5´-GTCCTAGGGAACCCCAACCCC-3´
AB081 (rp) 5´-GGCCTAGGTCTTTCCATATTGTAGCGC-3´
SH3 (aa 767-839)
AB077 (fp) 5´-GTCCTAGGGCCGTCCATGCC-3´
AB048 (rp) 5´-GGGCCTAGGAATAATCTGCTTTTCTAGCTC-3´
32 Materials and Methods
YY-SH3 (aa 746-839)
AB078 (fp) 5´-GGGCCTAGGGAGAGATATAAACTCCCTCTAC-3´
AB048 (rp) Sequence see SH3
SH2N-SH2C (aa 515-747)
AB063 (fp) 5´-GTCCTAGGGGAGGAAGTGCCCC-3´
AB081 (rp) Sequence see SH2C
SH2C-SH3 (aa 637-839)
AB079 (fp) 5´-GTCCTAGGGAACCCCAACCCC-3´
AB048 (rp) Sequence see SH3
2.1.7.2.2 Primers for the generation of the two truncated PLC2 halves,
PLC2‐X and PLC2‐Y All Primers contain an XbaI (TCTAGA) restriction site.
PLC2-X (aa 1-514)
AB038 (fp) 5´-AGTTCTAGAGCCGCCATGTCCACCACGGT-3´
AB091 (rp) 5´-GGTCTAGACTACAGGTCCTCCTCGGAGATCAGCTTCTGC TCCTCCATAGTCTGTTCAATGTCATCAC-3´ contains a myc-tag
PLC2-Y (aa 842-1265)
AB092 (fp) 5´-GCTCTAGAGCCGCCATGGACAATCCCTTAGGGTCTCTTTG-3´
AB037 (rp) 5´-CCCTCTAGACTACTACAGGTCCTCCTCGG-3´
33 Materials and Methods
2.1.7.2.3 Mutagenesis Primers
PLC2SH with artificial linker containing an AvrII restriction site (GCCCTAGG) and introducing five additional residues in positions 515-519 of the protein (ALG_SH domains_PR) (plasmid for cloning)
AB073 (fp) 5´-GAACAGACTATGGAGGCCCTAGGGAAGACAATCCCTTAGG-3´
AB074 (rp) 5´-CCTAAGGGATTGTCTTCCCTAGGGCCTCCATAGTCTGTTC-3´
PLC2SH with artificial linker containing an AvrII restriction site (GCCCTAGG) and introducing five additional residues in positions 515-519 of the protein (...ALGPR...) (control plasmid)
AB075 (fp) 5´-GAACAGACTATGGAGGCCCTAGGGCCTAGGGAAGACAATCCC TTAGG-3´
AB076 (rp) 5´-CCTAAGGGATTGTCTTCCCTAGGCCCTAGGGCCTCCATAGTC TGTTC-3´
Y1197E PLC2 (T3589G/C3591G)
AB087 (fp) 5´-GAGCGAAGAGGAACTTGAGTCCTCCTGTCGCCAGC-3´
AB088 (rp) 5´-GCTGGCGACAGGAGGACTCAAGTTCCTCTTCGCTC-3´
Y1217E PLC2 (T3650G/T3652G)
AB089 (fp) 5´-CAACCAGCTCTTTCTGGAGGACACACACCAGAACTTG-3´
AB090 (rp) 5´-CAAGTTCTGGTGTGTGTCCTCCAGAAAGAGCTGGTTG-3´
ALKI1250T (T3749C) and NPM-ALKI310T (T929C)
AB094 (fp) 5´-CACTTCATCCACCGAGACACTGCTGCCAGAAACTGCCTC-3´
AB095 (rp) 5´-GAGGCAGTTTCTGGCAGCAGTGTCTCGGTGGATGAAGTG-3´
PLC219 (646-685), PLC2-21219, PLC2-22119, PLC2-21119
AB096 (fp) 5´-CCACGAGTCCAAGCCGGCTAGGGGCAAGGTAAAGC-3´
AB097 (rp) 5´-GCTTTACCTTGCCCCTAGCCGGCTTGGACTCGTGG-3´
34 Materials and Methods
ALKY1604F (A4811T) and NPM-ALKY664F (A1991T)
AB100 (fp) 5´-CCTGGAGCTGGTCATTTCGAGGATACCATTCTG-3´
AB101 (rp) 5´-CAGAATGGTATCCTCGAAATGACCAGCTCCAGG-3´
PLC1668-707 (PLC1“19“)
AB102 (fp) 5´-CGCCCACGAGAGCAAAGAGGCTGAGGGCAAGATCAAGC-3´
AB103 (rp) 5´-GCTTGATCTTGCCCTCAGCCTCTTTGCTCTCGTGGGCG-3´
PLC219-22 (template PLC220-22)
AB108 (fp) 5´-CCCCACGAGTCCAAGCCGTGGAAAGGAGACTATGGAACCAG-3´
AB109 (rp) 5´-CCTGGTTCCATAGTCTCCTTTCCACGGCTTGGACTCGTGGGG-3´
W899A PLC219 (template PLC219) AB110 (fp) 5´-GGAGCTCTTTGAGGCGTTTCAGAGCATCCGAG-3´
AB111 (rp) 5´-CTCGGATGCTCTGAAACGCCTCAAAGAGCTCC-3´
Y509A/F510A PLC2-21119 (template PLC2-21119) AB112 (fp) 5´-GAATGGTATCCCCACGCCGCTGTTCTGACCAGCAG-3´
AB113 (rp) 5´-CTGCTGGTCAGAACAGCGGCGTGGGGATACCATTC-3´
2.1.7.2.4 SOE‐Primers
PLC2SA (476-908)
AB038 (fp A) 5´-AGTTCTAGAGCCGCCATGTCCACCACGGT-3´ contains a XbaI restriction site
AB031 (rp A) 5´-GTACTTCATGTTGTTCTCCTTGGTGTCAATCTTGT GTTCGTCCTTCTTGTCCTCCATGTTGACA-3´
AB030 (fp B) 5´-TGTCAACATGGAGGACAAGAAGGACGAACACAAG ATTGACACCAAGGAGAACAACATGAAGTAC-3´
AB037 (rp B) 5´-CCCTCTAGACTACTACAGGTCCTCCTCGG-3´ contains a XbaI restriction site
35 Materials and Methods
PLC2SH (515-840)
AB038 (fp A) Sequence see PLC2SA
AB033 (rp A) 5´-CTCTGCAAAGAGACCCTAAGGGATTGTCTTCCTCCAT AGTCTGTTCAATGTCATCACTGAAGG-3´
AB032 (fp B) 5´-CCTTCAGTGATGACATTGAACAGACTATGGAGGAAGA CAATCCCTTAGGGTCTCTTTGCAGAG-3´
AB037 (rp B) Sequence see PLC2SA
PLC2SH2C (639-766)
AB038 (fp A) Sequence see PLC2SA
AB067 (rp A) 5´-CTCTGAGGCATGGACGGGGGGTTGGGCACAGGG-3´
AB068 (fp B) 5´-CCCTGTGCCCAACCCCCCGTCCATGCCTCAGAG-3´
AB037 (rp B) Sequence see PLC2SA
PLC220-22 (686-806)
AB038 (fp A) Sequence see PLC2SA
AB098 (rp A) 5´-GCTGGATCCTGGTTCCATAGTCTCCTTTCCACCTGAA GGTGATGGCATAGGAGTCGCTCC-3´
AB099 (fp B) 5´-GGAGCGACTCCTATGCCATCACCTTCAGGTGGAAAG GAGACTATGGAACCAGGATCCAGC-3´
AB037 (rp B) Sequence see PLC2SA
PLC1708-828 (PLC1”20-22”)
PLCg1_Sense 5´-TTTGGATCCGCCGCCATGGCGGGCGCCGCGTC-3´ (fp A) contains a BamHI (GGATCC) restriction site (C. Walliser)
AB105 (rp A) 5´-CTTCCCTCCGTAGTCCCCTCGCCACCGGAAAGAGATGG CATATGAGTTGGGTTC-3´
AB107 (fp B) 5´-GAACCCAACTCATATGCCATCTCTTTCCGGTGGCGAGGG GACTACGGAGGGAAG-3´
36 Materials and Methods
AB106 (rp B) 5´-CCGAATTCCTACTACAGGTCCTCCTCGGAGATC-3´
contains an EcoRI (GAATTC) restriction site
2.1.8 Antibodies
2.1.8.1 Primary Antibodies
Myc-Tag (# 2276, 9B11) (mouse monoclonal IgG) Cell Signaling Technology, Danvers, MA, USA
PLC2 (# sc-407, Q-20) (rabbit polyclonal IgG) Santa Cruz, Heidelberg
PLC2 (404) (rabbit polyclonal IgG) Peter J. Parker, London, UK ALK (# 3791, 31F12) (WB) (mouse monoclonal IgG) Cell Signaling Technology, Danvers, MA, USA ALK (# 3333 ,C26G7) (IP) (rabbit monoclonal IgG) Cell Signaling Technology, Danvers, MA, USA
2.1.8.2 Secondary Antibodies
Anti-Mouse IgG-Peroxidase (# A5278) Sigma, Deisenhofen (goat IgG) Anti-Rabbit IgG-Peroxidase (#A6154) Sigma, Deisenhofen (goat IgG)
2.1.9 Instruments ABI Prism™ 310 Genetic Analyzer Perkin-Elmer, Weiterstadt Axiovert 25 Microscope Zeiss, Oberkochen Bench centrifuge 5430 Eppendorf, Hamburg Bench centrifuge 5415 D Eppendorf, Hamburg Bench centrifuge 5417 R Eppendorf, Hamburg Electrophoresis chamber (DNA) MBT, Giessen 37 Materials and Methods
Electrophoresis chamber (big) (SDS-PAGE) Amersham Pharmacia, Freiburg Electrophoresis Power Supply EPS 301 Amersham, Freiburg Electrophoresis Power Supply EPS 600 Amersham, Freiburg Elix® 5 Millipore, Eschborn Heater TechneDRI-BLOCK® DB3A Labtech, Burkhadtshausen HERA Safe Laminar Flow Workbench Heraeus, Fellbach Immunoblot Transfer Chamber Renner GmbH, Darmstadt Incubator CB 210 Binder, Tuttlingen Incubator Certomat H Braun, Melsungen J2-HS Centrifuge Beckman Instruments, München Liquid Scintillator Analyzer, Tricarb 288TR Packard, Canberra Magnetic hot plate stirrer RCTbasic IKA, Staufen Milli-Q® Millipore, Eschborn Optima™ TLX Ultracentrifuge Beckman Instruments, München Orbital shaker Unimax 2010 Heidolph, Kehlheim Overhead shaker Reax 2 Heidolph, Kehlheim pH meter 765 calimatic Knick, Berlin PTC 200 Peltier Thermal Cycler MJ Research, Oldendorf Rocking platform shaker Duomax 1030 Heidolph, Kehlheim Shaker Certomat R Braun, Melsungen Shaker Thermomixer comfort Eppendorf, Hamburg Shaker Thermomixer compact Eppendorf, Hamburg Shaker Titramax 100 Heidolph, Kehlheim Spectrophotometer GeneQuant Pro Amersham, Freiburg UV transilluminator IL 200M (312 nm) Bachofen, Reutlingen Videodocumentation system CF8/1 DX Kappa, Gleichen Vortex mixer Reax control Heidolph, Kelheim Vortex Whirlmix 34524 Cenco Instrumenten, Breda, Netherlands
2.1.10 Construction of individual incubation chambers
Chambers allowing the temperature-controlled incubation of individual 24-well tissue culture plates (92424; TPP, Switzerland) were custom assembled using 15 x 12 x 23 cm
38 Materials and Methods
Styrofoam containers (Schaumaplast, Reilingen, Germany) equipped with circuits made up of one Velleman VM148 thermostat control module (190655-62) and one Dallas DS18S20-55 temperature sensors with digital output (176168-62) to control two serially connected heating foils (532878-62; all from Conrad Electronic, http://www.conrad.de) to be placed on either side of the tissue culture plate during incubation. To allow for additional external, analogue control of the temperature, a 10 mm whole was drilled into the wall of the container and the tissue culture plate to allow insertion of a thermometer into one well. Power supply to up to 8 individual chambers was through a TDK-Lambda LS75-12 AC/DC converter unit (511823-62; Conrad). A circuit diagram can be found in the appendix.
2.1.11 Software
Adobe® Photoshop® CS3 Extended 10.0 Adobe Systems GmbH, München Chromas version 2.33 Technelysium Pty Ltd, Tewantin, Australia Clustal Omega Sievers et al. [233] GraphPad Prism® 4 GraphPad Software, Inc., San Diego, USA Micrografx Designer 9.0 Micrografx Inc., München Microsoft Office 2003 Microsoft Corp., Washington, USA Microsoft Office 2010 Microsoft Corp., Washington, USA NEBcutter V 2.0 Vincze et al. [267] OMIGA 2.0 Oxford Molecular Ltd. 1996-1999, Oxford, UK PPSP: prediction of PK-specifc phosphorylation site Xue et al. [299] Zotero 4.0.11 Center for history and new media, George Mason University, Fairfax, USA, http://zotero.org
39 Materials and Methods
2.2 Microbiology and Molecular Biology
2.2.1 Preparation of Luria‐Bertani (LB) Medium and Agar Plates
E. coli bacteria were grown in sterile Luria-Bertani (LB) medium. LB medium was autoclaved and then stored at room temperature (RT). For the selection of bacteria harbouring a plasmid with the antibiotic resistance gene the medium was supplemented with the appropriate antibiotic. Ampicillin and Kanamycin were prepared as stock solutions and diluted 1:1000 in LB medium. LB agar plates were prepared by adding 1.5 % (w/v) agar to the LB medium before autoclaving. The solution was then allowed to cool to hand-warm temperature and after adding the appropriate antibiotics the solution was poured in sterile Petri dishes. After solidifying at RT the plates were stored at 4 °C.
LB-Medium: 1 % (w/v) Bacto Trypton 0.5 % (w/v) Yeast Extract Servabacter 1 % (w/v) NaCl
Ampicillin stock solution: 50 mg/ml in ddH2O, sterile filtered through a nitrocellulose membrane disc filter (0.22 μm pore size)
Kanamycin stock solution: 30 mg/ml in ddH2O, sterile filtered through a nitrocellulose membrane disc filter (0.22 μm pore size)
2.2.2 Preparation of Competent Bacteria
The Inoue method [104] was used to prepare competent bacteria. A single colony of E. coli DH5 bacteria streaked on an agar plate without antibiotics was picked with a sterile tip and introduced into a sterile tube containing 25 ml of LB medium. The bacterial suspension was grown for 6-8 h in an incubator (37 °C, 200 rpm). Of this preculture, 2 ml, 4 ml, and 6 ml were added to 250 ml of SOB medium in a sterile flask, and allowed to grow overnight (20 °C, 200 rpm) to an optical density at 600 nm (OD600) of 0.55. The culture reaching this density first was centrifuged at 2500 x g at 4 °C for 10 min. The pellet was resuspended in 80 ml of ice-cold Inoue buffer. The suspension was again centrifuged at 2500 x g at 4 °C for 10 min and the pellet was resuspended in 20 ml of ice-cold Inoue buffer. The suspension was supplemented with 1.5 ml DMSO and incubated in an ice 40 Materials and Methods
water bath for 10 min, after which 50 μl aliquots were snap-frozen in liquid N2 and the competent bacteria stored at -80 °C.
Inoue buffer: 55 mM MnCl2
15 mM CaCl2 250 mM KCl 10 mM PIPES/KOH (0.5 M, pH 6.7)
SOB medium: 2 % (w/v) Bacto Trypton 0.5 % (w/v) Yeast Extract Servabacter 10 mM NaCl 2.5 mM KCl
10 mM MgCl2
10 mM MgSO4, pH 7.0
2.2.3 Transformation of competent bacteria
An aliquot of competent bacteria was thawed on ice (50 µl for transformation of bacteria with a plasmid from a ligation reaction, 25 µl for re-transformation of bacteria with a plasmid). The appropriate plasmid (10 ng in a volume of ≥ 2µl), ligation reaction (2 µl) or PCR reaction (2 µl) was added and the bacterial suspension was incubated on ice for 30 min, followed by a heat-shocking for 90 sec at 42 °C in a water bath. Immediately after the heat shock the bacteria were placed on ice for 2 min and then supplemented with 500 µl of prewarmed (37 °C) LB medium, and incubated at 37 °C for 1 h on a rotary shaker (220 rpm). Fifty µl of a re-transformation reaction or 400 µl of the suspension was then plated on a LB agar plate containing appropriate antibiotics and incubated at 37 °C over night.
2.2.4 Plasmid DNA preparation
For small or medium scale preparations of plasmid DNA the Nucleospin® Plasmid Kit and the Nucleo Bond® PC 100 Kit (both from Macherey-Nagel) were used, respectively. Therefore LB medium (3 ml for small scale and 50 ml for medium scale preparations) containing the appropriate antibiotics were inoculated with single colonies picked from the respective LB plates and grown over night in an incubator (37 °C, 200 rpm). The
41 Materials and Methods preparations were processed according to the manufacturer’s protocols. DNA was eluted in ddH2O and stored at -20°C.
2.2.5 Photometric Quantification of DNA
Nucleic acids absorb UV light with an absorption peak at 260 nm. This feature allows the photometric measurement of DNA concentrations. To relate the absorption at 260 nm with the DNA concentration the Beer-Lambert law A = ε × d × c, with A, absorbance, ε, molar extinction coefficient, d, path length, c, concentration, was used. The average molar -1 -1 extinction coefficient for double-stranded DNA is ~50 (μg/ml) cm at 260 nm. The purity of the DNA solution was determined by measuring the ratio of the absorbance at 260 nm and 280 nm (OD260/280). Pure DNA solutions resulted in values between 1.8 and 2.0. Lower values indicate contaminations with proteins or phenol.
2.2.6 Agarose Gel Electrophoresis
Agarose gel electrophoresis is a method used to separate DNA molecules by size. The negatively charged nucleic acid molecules migrate through an agarose matrix when an electric field is applied. Short DNA molecules migrate faster and therefore farther than long ones. The Agarose gel was prepared by dissolving 1 % (w/v) agarose in TAE buffer by boiling the solution in a microwave. The solution was allowed to cool to ~ 50 °C before pouring into a gel rack. The DNA samples were supplemented with DNA sample buffer at the rate of 5:1 (v/v) and loaded into the agarose gel wells. A voltage of 100 V was applied for 1 hour. To visualize the DNA the agarose gel was stained for 10 min in a TAE bath containing ~ 0.0003 % (w/v) ethidium bromide. After a short destaining (~ 1 min) in H2O the stained DNA was visualized on a UV transilluminator and images obtained were printed out for documentation.
1 x TAE buffer: 40 mM Tris/HCl, pH 8.0 1 mM EDTA 0.1 % (v/v) acetic acid 6 x DNA sample buffer: 60 % (v/v) Glycerin 0.25 % (w/v) bromphenol blue 60 mM EDTA, pH 8.0
42 Materials and Methods
To avoid a possible damage of DNA by UV light which may impair reactions like the ligation of DNA fragments, a more gentle staining method was applied. DNA stained with crystal violet is already visible at ambient light which renders irradiation of DNA with UV light unnecessary. Therefore 30 μl of crystal violet solution (10 mg/ml (w/v)) were added to 30 ml of dissolved, cooled agarose before pouring the gel. The DNA samples were mixed with crystal violet sample buffer instead of DNA sample buffer.
6 x crystal violet sample buffer: 30 % (v/v) glycerine 0.3 mg/ml crystal violet
2.2.7 Gel Purification of DNA Fragments
® DNA fragments to be ligated were extracted from agarose gels using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s protocol except that the DNA was eluted in 30 µl of ddH2O.
2.2.8 DNA Restriction Digest
Restriction endonucleases cut double-stranded DNA at specific recognition sites which turns them into an important tool to analyse or prepare DNA fragments, e.g. for ligation. For analytical or preparative DNA restriction digests, the following reactions were prepared:
analytical preparative Plasmid DNA 1 µg 5 µg
Specific buffer (10x) 1,5 µl 5 µl
Restriction endonuclease 5 U 25 U ad H O 15 µl 50 µl 2
The reactions were incubated for 1h at the temperature specific for the used restriction endonuclease. Digests involving more than one enzyme were performed according to the manufacturer’s suggestions either as one reaction or with an intermittent step involving the isolation of digested DNA by agarose gel electrophoresis and gel extraction.
43 Materials and Methods
2.2.9 Dephosphorylation of DNA Fragments
To prevent re-ligation of DNA plasmids cut at a single restriction site in a later ligation reaction the linearized plasmid DNA had to be dephosphorylated. Antarctic phosphatase (NEB) was used according to the manufacturer’s protocol to remove the 5' phosphate groups of DNA molecules and thereby preventing re-ligation. Products from preparative restriction digests were directly used for dephosphorylation after agarose gel electrophoresis and gel purification of the desired DNA fragments. After heat inactivation of antarctic phosphatase according to the manufacturer’s protocol the DNA molecules were directly used for ligation reaction, omitting any purification step.
2.2.10 DNA Ligation
DNA Ligation is used to generate recombinant plasmid DNA by inserting DNA fragments into plasmid vectors. For this purpose T4 DNA ligase, which forms covalent phosphodiester bonds in an ATP-dependent manner between the 3' hydroxyl group and the 5' phosphate group in double-stranded DNA or RNA molecules, was used. For ligation of two DNA fragments the following reaction was prepared:
25 ng Linear vector DNA 5:1 Insert DNA molar ratio over vector 1,5 µl 10 x T4 DNA ligase buffer 0,5 µl T4 DNA ligase (5 U/µl)
15 µl ad H2O
The ligation reaction was incubated at 22 °C for 1 hour before transformation of competent bacteria. The leftovers of this reaction were incubated at 16 °C over night which allows a second transformation in case the first transformation failed.
2.2.11 Polymerase Chain Reaction (PCR)
Hardly any invention has changed biological sciences in a way polymerase chain reaction (PCR) did [179]. By means of this method it is possible to amplify marginal amounts of DNA very rapid, which is of great interest for molecular biology as well as for clinical 44 Materials and Methods approaches. Kary Mullis, the innovator of this method, received the Nobel prize in chemistry in 1993 for this pioneering work. To specifically amplify defined DNA sequences DNA oligonucleotides, highly complementary to the DNA region targeted for amplification, so called primers have to be designed. The following 50 µl standard PCR reaction and the cycling parameters were used, unless specified otherwise:
1 µl DNA template (50 ng) 1 µl dNTP mix (10 mM each dNTP) 10 µl 5 x Phusion® HF buffer 1 µl Forward primer (100 µM) 1 µl Reverse primer (100 µM) 0.5 µl Phusion® high-fidelity DNA polymerase
50 µl ad H2O
Table 2-1: Standard PCR cycling conditions
2-step 3-step
Step Temperature Time Temperature Time Cycles
Initial denaturation 98 °C 2 min 98 °C 2 min 1
Denaturation 98 °C 20 s 98 °C 20 s
Primer annealing - - X °C 20 s 30
Extension 72 °C 30 s/kb 72 °C 30 s/kb
Final extension 72 °C 7 min 72 °C 7 min 1
Melting temperatures (Tm) of the primers were calculated with the nearest-neighbour method [23]. The annealing temperature was calculated dependent on the melting temperature (Tm) of the primers. Annealing was always performed at + 3°C of the lower
Tm primer, except for primers with Tm ≥ 72 °C where the 2-step PCR protocol was used.
45 Materials and Methods
The PCR products were separated by agarose gel electrophoresis and the desired bands were extracted from the gel and subcloned into the vector pCR-Blunt.
2.2.12 Site‐directed Mutagenesis
The QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) was used to insert point mutations or deletions into plasmid DNA. This method relies on the fact that DNA produced in a PCR reaction is resistant against digestion by DpnI endonuclease since DpnI endonuclease specifically digests methylated and hemi-methylated DNA. The whole plasmid is amplified in vitro by the help of oligonucleotides containing the desired mutation, followed by digest of the template DNA by DpnI. The generated mutated plasmid DNA is directly introduced into competent bacteria. For introducing point mutations or deletions into plasmid DNA the reaction was performed according to the manufacturer’s protocol except that 100 ng template DNA was used. For the deletion of multiple amino acids annealing of the primers was performed at lower temperatures. To generate the PLC, PLC119, PLC19-22 constructs an annealing temperature of 55 °C was used. In both cases transformation of competent DH5 bacteria was performed as described in section 2.2.3.
2.2.13 Splicing by Overlap Extension (SOE)
Splicing by overlap extension is a technique used to recombine two DNA fragments at precise junctions and thereby e.g. deleting large fragments within a gene [95]. Therefore two DNA fragments containing overlapping sequences are produced in two separate PCR reactions, PCR A and PCR B. In a third PCR reaction, PCR C, these PCR products are recombined. Thereby the overlapping sequence of both DNA fragments constitutes an origin for DNA polymerase like primers do in a standard PCR reaction. Extension of this overlap effectively „splices“ the original molecules together. SOE was used to generate the
PLC2SA, PLC2SH, PLC2SH2C, PLC220-22, and PLC120-22 constructs. For PCR A and PCR B the standard PCR protocol was used (Table 2-1). The resulting PCR products were separated by agarose gel electrophoresis and the particular bands were extracted from the gel. The reaction mix for PCR C was prepared as in the standard PCR reaction adding the same amount of the two extracted products from PCR A and PCR B (usually 1µl of the higher concentrated PCR product and the appropriate higher volume of
46 Materials and Methods the lower concentrated PCR product) instead of regular primers and template. The cycling conditions are specified in Table 2-2 and Table 2-3. After 10 cycles 1 μl each of forward primer A and reverse primer B were added to the reaction mix and cycling continued for additional 30 cycles.
Table 2-2: PCR cycling conditions for splicing by overlap extension
Step Temperature Time Cycles
Initial denaturation 98 °C 2 min 1
Denaturation 98 °C 20 s
Primer annealing X °C X s 10
Extension 72 °C 30 s/kb
Addition of forward primer A and reverse Primer B
Denaturation 98 °C 20 s
Primer annealing X °C 20 s 30
Extension 72 °C X s
Final extension 72 °C 7 min 1
Table 2-3: Annealing temperatures and extension times for splicing by overlap extension
Annealing Construct Extension time temperature
PLC2SA 68 °C 1 min 30 s
PLC2SH 68 °C 1 min 30 s
PLC2SH2C 70 °C 1 min 30 s
PLC220-22 68 °C 2 min
PLC120-22 72 °C 3 min
The PCR products were separated by agarose gel electrophoresis and the desired bands were extracted from the gel and subcloned into the vector pCR-Blunt.
47 Materials and Methods
2.2.14 DNA Sequencing
DNA sequences were either determined by GATC Biotech Konstanz or by the use of ABI PRISM® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits, Version 2.0, Applied Biosystems.
2.3 Cell culture and DNA transfection
2.3.1 Cell counting
For counting, cells were diluted 1:4 in trypan blue and counted in a Neubauer counting chamber. Viable cells in all four big squares were counted and the number of cells per ml was determined by multiplying the number of counted cells with 104.
Trypan blue solution: 0.5 % (w/v) trypan blue 0.85 % (w/v) NaCl
2.3.2 COS‐7 cell culture
The COS-7 cell line is a fibroblast-like cell line derived from the kidney of African green monkeys. The COS-7 cells are cultured in culture dishes (Ø 100 mm) in adherent culture at
37 °C in a humidified atmosphere of 90 % air and 10 % CO2 in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Before reaching confluence (every three or four days) the cells were passaged. Therefore the old medium was aspirated and the cells were washed with 5 ml of Dulbecco’s PBS (PAA). Thereafter the cells were incubated with 2.5 ml trypsin/EDTA at 37 °C for ~10 min to detach them from the cell culture dish. After the incubation the cells were rinsed from the cell culture dish with 5 ml of medium by pipetting up and down. The resulting single cell suspension was centrifuged (300 x g, 5 min, RT) and the pellet was resuspended in an appropriate volume of medium to dilute by a factor of eight for subculture.
48 Materials and Methods
2.3.3 Transient transfection of COS‐7 cells
To transiently transfect COS-7 cells, the cells were seeded 24 h prior transfection in culture dishes (Ø 100 mm), 6-well plates, or 24-well plates at a density of 2.5 x 106, 0.4 x 106, or 0.075 x 106 cells per well in a volume of 10 ml, 2 ml/well, or 0.5 ml/well, respectively. At least 1 h before transfection the medium was replaced in each case with 10 ml, 2 ml/well, or 0.5 ml/well of fresh medium. For transfection LipofectamineTM 2000 (Invitrogen) or jetPRIME® (Polyplus transfectionTM) was used according the manufacturer’s manual. The total amount of DNA was kept constant within the samples of one assay by adding empty pcDNA3.1(+) vector. After transfection with LipofectamineTM 2000 the cells were incubated for 24 h in the same medium. In case of transfection with jetPRIME® the medium was changed 4 h after transfection.
2.4 Protein Biochemistry
2.4.1 Protein Quantification
The determination of protein concentrations in crude cell extracts was performed using the Bradford method [21]. This method is based on the binding of the dye Coomassie Brilliant Blue to proteins and the resulting shift of the absorption peak of the dye from 465 nm to 595 nm. A standard curve was prepared by adding water to 0, 10, 20, 30, 40, and 50 μl of the protein standard (0.34 mg/ml bovine IgG) to a total volume of 800 μl. Protein samples were diluted and an appropriate volume (20 to 40 µl) was added to separate tubes and supplemented with water to a total volume of 800 µl. After addition of 200 µl Bradford reagent the tubes were vortexed and the absorbance was measured at 595 nm. The protein concentration was determined by linear regression of the standard curve by means of the computer program PG Prot. All values were determined by double or triple determination.
Bradford reagent: 0.04 % (w/v) Coomassie Brilliant Blue G 250 21 % (v/v) ethanol 42.5 % (v/v) phosphoric acid
49 Materials and Methods
2.4.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE)
SDS-PAGE is a method according to Laemmli [142] in which denatured proteins are separated according to their Stokes radius, and thus according to their molecular mass. The separation is achieved through the molecular sieve effect of a porous polyacrylamide matrix. The running gel was poured between two glass plates, covered with water and allowed to polymerize, so that a vertical gel is formed. In order to achieve a sharper band separation, a stacking gel was poured on top of the running gel after removing the water. Each of the protein samples were mixed with an equal volume of (+)-sample buffer and boiled for 5 min at 95 °C. The samples were loaded and the gel was run in tank buffer at 45 mA. Empty lanes were loaded with a mix of equal volumes of (-)-sample buffer and water.
Table 2-4: SDS-PAGE gel preparation
Running gel Stacking gel
7 % 10 % 6%
Monomer solution 7 ml 10 ml 2 ml
Running buffer 7.5 ml 7.5 ml -
Stacking buffer - - 2.5 ml
H2O 15 ml 12 ml 5.3 ml
10 % (w/v) SDS 300 µl 300 µl 100 µl
10% (w/v) APS 150 µl 150 µl 50 µl
TEMED 10 µl 10 µl 5 µl
Monomer solution: 30 % (w/v) acrylamide 4K solution mix 37.5:1 (292.2 g/l acrylamide 4K, 7.8 g/l bisacrylamide 4K) Running buffer: 1.5 M Tris/HCl, pH 8.8
90.75 g Tris were dissolved in 400 ml ddH2O and pH was adjusted to 8.8 with approximately 16 ml of conc. HCl, filled
up with ddH2O to 500 ml and the solution was filtered through a 0.45 µm cellulose nitrate filter.
50 Materials and Methods
Stacking buffer: 0.5 M Tris/HCl, pH 6.8
12 g Tris were dissolved in 160 ml ddH2O and pH was adjusted to 6.8 with approximately 9 ml of conc. HCl, filled
up with ddH2O to 200 ml and the solution was filtered through a 0.45 µm cellulose nitrate filter. 2 x (+)-sample buffer: 125 mM Tris/HCl, pH 6.8 4 % (w/v) SDS 20 % (v/v) glycerol 10 % (v/v) β-mercaptoethanol 0.002 % (m/v) pyronineY 2 x (-)-sample buffer: 125 mM Tris/HCl pH 6.8 4 % (w/v) SDS 20 % (v/v) glycerol 10 % (v/v) β-mercaptoethanol Tank buffer: 25 mM Tris/HCl, pH 8.6 190 mM glycine 0.1 % (w/v) SDS
2.4.3 Western Blotting of SDS‐Polyacrylamide Gels
In the Western blotting method proteins are transferred form a SDS gel to a nitrocellulose membrane. Therefore the SDS gel and a piece of nitrocellulose membrane (Protran®) were soaked in transfer buffer and sandwiched between two sponge layers and two layers of blot paper in a blot chamber filled with transfer buffer. The proteins were then electroblotted with a current of 125 mA for 15 h for PLCs or 18 h for larger proteins like ALK. After blotting, the marker lane was separately stained with ink (3.3 % (v/v) blue ink, 0.3 % (v/v) Tween-20).
For the immunodetection of the transferred proteins the membrane was first blocked with 5 % (w/v) dried milk in PBS-T for 1 h. After blocking the membrane was incubated with the primary antibody diluted in PBS-T for 2 h. After washing the membrane three times for 7 min in PBS-T the membrane was incubated with the horseradish peroxidase-labelled secondary antibody diluted 1:1000 in PBS-T for 30 min. After washing as described above
51 Materials and Methods
and briefly rinsing the membrane with H2O the immunoreactive proteins were detected using the ECL Western blotting detection system according to the manufacturer’s instructions. The chemiluminescence was visualized by blackening and developing of an x- ray film. All incubation steps were performed at room temperature on a vertical shaker.
Transfer buffer: 25 mM Tris/HCl, pH 8.6 190 mM glycine 20 % (v/v) methanol
PBS-T: 80 mM Na2HPO4
20 mM NaH2PO4, pH 7.5 100 mM NaCl 0.5 % (v/v) Tween-20
2.4.4 Immunoprecipitation (IP)
COS-7 cells were grown on culture dishes (Ø 100 mm) and transiently transfected as described in section 2.3.3. Twenty-four h after transfection the medium was replaced with 10 ml of fresh medium. After another 20 h of incubation the cells were washed once with 5 ml of Dulbeccos’s PBS (PAA), scraped into 500 µl of ice cold lysis buffer and transferred into 1.5 ml centrifuge tubes (Beckman coulter). After 30 min of incubation on ice the lysates were precleared by ultracentrifugation (100,000 x g, 4 °C, 1 h). To couple antibodies reactive against the c-myc epitope or ALK to Protein A-Agarose beads (Roche), 30 µl of beads were washed once with 50 µl of 1 x PBS and equilibrated in 15 µl of 1 x PBS (50 % (v/v) slurry). The equilibrated beads were mixed with 500 µl of antibody stock diluted 1:1000 in 1 x PBS. The mixture was incubated by end-over-end rotation at 4 °C for 1 h and afterwards washed two times with 500 µl of lysis buffer. The precleared lysate was added to the antibody-coated Protein A-Agarose beads and the samples were incubated for 2 h at 4 °C by end-over end rotation. The beads were collected by centrifugation, washed three times with 500 µl of lysis buffer, resuspendend in 60 µl of 1 x SDS-PAGE sample buffer and boiled at 95 °C for 5 min. The interaction between the proteins was analyzed by western blot using antibodies reactive against the c-myc epitope and ALK.
52 Materials and Methods
Lysis buffer (according to [9]): 10 mM Tris/HCl, pH 7.5 130 mM NaCl 5 mM EDTA 0.5 % (v/v) Triton X-100 0.1 % (w/v) BSA 1 mM PMSF 10 µM Leupeptine 2 µM Pepstatin A 2 µg/ml Soybean trypsin inhibitor (STI) 1 µg/ml Aprotinin 3 mM Benzamidine 1 mM DTT 10 µl/ml Phosphatase Inhibitor Cocktail 2 (Sigma)
1 x PBS: 80 mM Na2HPO4
20 mM NaH2PO4 100 mM NaCl, pH 7.5
2.5 Measurement of phospholipase C activity
2.5.1 Analysis of Inositol Phosphate Formation in Intact COS‐7 Cells
Cells were seeded in 24-well plates and transfected as described in section 2.3.3. Twenty- four hours after transfection the medium was removed by aspiration and the cells were washed once with 300 µl/well of Dulbecco’s PBS and the cells were incubated for 20 h in 200 µl per well of DMEM with supplements as specified in section 2.3.2, 2.5 µCi/ml myo- [2-3H]inositol, and 10 mM LiCl [188]. In case the cells were incubated in the absence of
10 % CO2 atmosphere the medium was additionally supplemented with 25 mM Hepes and 2 mM sodium pyruvate to maintain pH [243]. The cells were then lysed with 200 µl/well of 10 mM ice-cold formic acid at 4 °C. After 30 min the lysis was stopped by transferring the supernatants from the wells into reaction tubes filled with 300 µl of 10 mM NH4OH 53 Materials and Methods
[188]. To remove cell debris the samples were centrifuged at 20,000 x g for 5 min at 4 °C. The supernatant was loaded onto a column containing 0.25 ml of Dowex® 1x 8-200 ion exchange resin that had been converted to the formate form and equilibrated with H2O as described [31]. The columns were washed once with 3 ml of H2O and then twice with 3.5 ml of 60 mM sodium formate and 5 mM sodium tetraborate, and inositol phosphates were eluted with 3 ml of 1 M ammonium formate and 100 mM formic acid [294]. The eluate was supplemented with 15 ml of Quicksafe A liquid scintillator (Zinsser Analytic) and the radioactivity was quantified by liquid scintillation counting. The columns were reused after regeneration as described [31].
2.5.2 Lipid extraction
To investigate the uptake of myo-[2-3H]inositol and the subsequent conversion of this 3 reagent into [ H]PIP2 in COS-7 cells when incubating the cells at different sub- physiological temperatures the lipid extraction protocol [60] was performed. COS-7 cells were grown on 6-well plates with a density of 0.25 x 106 cells/well and transiently transfected with empty pcDNA3.1(+) vector as described in section 2.3.3. Twenty-four hours after transfection the medium was replaced with 1.5 ml/well of fresh medium supplemented with 2.5 µCi/ml myo-[2-3H]inositol, and 10 mM LiCl. In order to maintain pH in the absence of 10 % CO2 atmosphere the medium was additionally supplemented with 25 mM Hepes and 2 mM sodium pyruvate [243]. After another 20 h of incubation in individual incubation chambers (see section 2.1.10) at different temperatures (25 °C, 27 °C, 29 °C, 31 °C, 33 °C, 35 °C, and 37 °C) the cells were washed once with 1.5 ml of Dulbecco’s PBS and lysed with 1.2 ml of 4.5 % (v/v) perchloric acid (PCA) for 30 min at 4 °C. To determine the amount of myo-[2-3H]inositol taken up by the cells 10 µl aliquots of the labling mix before and after incubation of the cells at different temperatures were transferred into a scintillation vial. Three ml of Quicksafe A liquid scintillator (Zinsser Analytic) were added and total radioactivity was determined by liquid scintillation counting. After lysis the cells were scraped into PCA, transferred into a 1.5 ml reaction tube and centrifuged at 3700 x g for 20 min at 4 °C. The PCA pellets were resuspended in 100 µl of water and 375 µl of chloroform:methanol:HCl, 100:200:15 (ratio differs from [60]) were added. The tubes were vortexed and 125 µl of chloroform plus 125 µl of 0.1 M HCl were added. The mixtures were vortexed and centrifuged at 700 x g for 10 min at RT.
54 Materials and Methods
Fifty µl of the chlorophormic phase (lower one), which contains the phospholipids was subjected to liquid scintillation counting as described above.
2.6 Thermodynamic calculations
According to [85], the 10-degree temperature coefficient, Q10 , of an enzyme reaction can be calculated for an arbitrary temperature intervall T from
T 10 QT (Q10 ) (1)
Ai Using T Ti Tref and QT , where Ti and Tref are the individual and an Aref arbitrarily chosen reference temperatures and Ai and Aref are the individual and the reference activities, this equation can be linearized to
A log i 0.1(T T )log (Q ) (2) 10 i ref 10 10 Aref
A log i 0.1log (Q )T 0.1log (Q )T (3) 10 10 10 i 10 10 ref Aref
A Upon plotting the T vs. the log i , the Q value(s) can be calculated from the i 10 10 Aref slopes of the linear portions of the resultant graphs.
The molar heat capacity CP of PLC219 and PLC220-22 was estimated according to the protocol developed by Clapham and Miller [41] for the thermodynamic analysis of temperature-sensitive transient receptor potential (TRP) channels, using the following working relationship:
55 Materials and Methods
ln K(T ) S0 R CP 1 T0 T ln(T0 T) R (4) where K(T) is the equilibrium constant of a protein in equilibrium between two
conformations, S0 the standard state entropy, R is the gas constant, CP is the molar
heat capacity, and T0 is the temperature of minimum K. To estimate S0 , we chose the
o minimal K determined experimentally at 39 C, 0.003, as K 0 , and set H 0 to zero, using equation 9 of [41]:
S0 ln K 0 R (5)
CP and T0 were then estimated by non-linear least squares curve fitting to equation (4) and K(T ) PA (T ) (1 PA )(T ) , where PA is the probability of PLC2 be in the fully active conformation A, using the global curve fitting procedure contained in GraphPad Prism, version 4.03 (GraphPad Software, San Diego California USA). To this end, the extra sum of squares F-test was used to determine whether the best-fit values of the two parameters differed between the two data sets. The simpler model was selected unless had a P value less than 0.05.
2.7 Cluster robustness analysis
An exhaustive cluster robustness analysis was performed using model-based clustering of infinite mixture multivariate Gaussians for k = 2-5 clusters. First, all leave-out-one subsets of the dataset were clustered. Then all cluster assignments were compared pair-wise using the maximum cluster assignment (MCA) index [139]. Finally, the MCA indices were compared to random baseline clustering using the Wilcoxon rank sum test. From this comparison, k = 3 (corresponding to three clusters) was chosen as the one with the largest mean difference in the MCA index (Figure 3-28).
2.8 Statistical analysis
The experiments described in this thesis were replicated at least three times. The results of representative experiments are shown in the form of mean ± SEM of triplicates. The
56 Materials and Methods significance of differences between means ± standard errors was assessed by using the unpaired t test with two-tailed P values contained in GraphPadInStat®, version 3.10 (www.graphpad.com). To account for propagation of errors, the standard errors of quantities corresponding to sums or differences of measured quantities were calculated as the sums in quadrature of the absolute standarderrors of the measured quantities [170]. Two effects are said to be synergistic, if the effect of two components tested in combination is statistically significantly higher than the sum of the individual effects [76].
57 Results
3 Results
3.1 Autoinhibitory regulation of PLC2
Besides the mechanisms of activating PLC isozymes by upstream regulators, like Rho GTPases (see section 1.3.2) or receptor and non-receptor tyrosine kinases (see section 1.3.3), there is also abundant evidence in the literature that PLCs are autoinhibited in their basal state by structural elements within their X/Y linker. Protease cleavage of PLC1,
PLC1, and PLC2 within the X/Y linker region resulted in an increase in basal PLC activity [39, 55, 62, 226]. Furthermore, the deletion of the SH2SH2SH3 tandem in PLC1 led to an enhanced PLC activity [93]. The analysis of the crystal structure of PLC2 and subsequent biochemical analyses revealed the occlusion of the active site of the enzyme by its X/Y linker [83]. Data from our group show, that also PLC2 is regulated by autoinhibitory elements within its specific array [59, 275]. All these findings point to a conserved role of the X/Y linker in autoinhibitory regulation of PLC isozymes. In the last years it became increasingly clear, that the loss of the autoinhibitory regulation of PLC2 can lead to severe phenotypes in mouse models as well as in humans [1, 56, 190, 301, 311]. Therefore it is of great importance to get a better understanding of the molecular mechanisms that lie behind this kind of intramolecular regulation.
3.1.1 PLC2 is autoinhibited by its specific array.
To analyse the role of the PLC2 specific array in autoinhibition of the enzyme, two deletion mutants were established, in which either the whole specific array (PLC2SA, aa 476-908) or only the SH2SH2SH3 tandem (PLC2SH, aa 515-840) was deleted. In intact, transiently transfected COS-7 cells both mutants showed a marked increase in basal activity compared to wild-type PLC2 (Figure 3-1, left panel), whereas the expression of all three proteins was similar (Figure 3-1, inset). These data suggest that PLC2 is subject to autoinhibitory regulation by its specific array. In order to further characterize and quantify the enhanced basal activities of the deletion mutants, COS-7 cells were transfected with increasing amounts of plasmid DNA encoding wild-type PLC2, PLC2SA, and
PLC2SH (Figure 3-1, right panel). The activity of both mutants increased with 58 Results increasing amounts of vector used for transfection. Specifically, transfected cells showed a 40-fold increase in PLC activity, for both deletion mutants, compared to cells expressing wild-type PLC2 when 50 ng each of the encoding vectors were used for transfection. An octamer peptide, referred to as the phospholipase C inhibitory (PCI) peptide, is located at the C-terminal end of the SH2C domain (aa 726-733) and was reported to be inhibitory when added to PLC enzymes in vitro and in intact cells [87, 91]. Furthermore, the deletion of this octamer was shown to be sufficient to overcome autoinhibition of the PLC2 isozyme [59]. However, the basal activity of the PLC2PCI mutant was considerably lower compared to PLC2SA and PLC2SH (Figure 3-1, right panel). COS-7 cells transfected with 50 ng vector encoding PLC2PCI only showed a 3.7-fold increase in basal activity compared to wild-type PLC2. This implies that deletion of the PCI region is not sufficient to fully negotiate autoinhibition.
Figure 3-1: Role of 2 specific array on the autoinhibitory regulation of PLC2. Left panel, COS-7 cells were transfected as indicated at the abscissa with 500 ng of either empty vector (Co.), vector encoding PLC2
(PLC2), PLC2SA (SA), or PLC2SH (SH). Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. Inset, COS-7 cells were transfected as described above with empty vector (lane 1), vector encoding PLC2 (lane 2), PLC2SA (lane 3), or PLC2SH (lane 4). Cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope. Right panel, COS-7 cells were transfected with 500 ng per well of vector encoding PLC2 (PLC2, □) or increasing amounts
59 Results
(1 ng, 2 ng, 5 ng, 10 ng, 20 ng, and 50 ng) of vector encoding PLC2SA (SA, ▼), PLC2SH (SH, ), or PLC2PCI (PCI, ○). The total amount of DNA was maintained constant at 500 ng for each transfection by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined.
3.1.2 The spPH domain but not the SH domain tandem is required
for Rac2‐mediated activation of PLC2.
Experiments from our group showed that the spPH domain of PLC2 is both necessary and sufficient for the Rac2-mediated activation of the enzyme [275]. To examine the effect of
Rac2 on the deletion mutants PLC2SH and PLC2SA, COS-7 cells were cotransfected with decreasing amounts of DNA encoding PLC2SH and PLC2SA together with Rac2-DNA or DNA encoding constitutively active Rac2G12V-DNA (Figure 3-2). In line with the previously obtained results, PLC2SA lacking both halves of the spPH domain is G12V not activated by constitutively active Rac2 , whereas PLC2SH, still harbouring the G12V spPH domain, is clearly activated by Rac2 . Surprisingly PLC2SH was also activated by wild-type Rac2, even though to a lower extend than by Rac2G12V. Furthermore, it is obvious that the SH domain tandem is not necessary for the Rac2-mediated activation of
PLC2 and that the autoinhibitory regulation and the activation by Rho GTPases seem to be independent of each other.
G12V Figure 3-2: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH and
PLC2SA. COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty vector
(Co.), 500 ng of vector encoding PLC2 (wt), or decreasing amounts (10 ng, 5 ng, 2 ng, 1 ng, and 0.5 ng) of 60 Results
vector encoding PLC2SH (SH, left panel) or PLC2SA (SA, right panel), together with 25 ng of either empty vector (Control), vector encoding Rac2 (Rac2), or vector encoding Rac2G12V (Rac2G12V). The total amount of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined.
3.1.3 The C‐terminal SH2 domain and the SH3 domain in combination, but not alone, exhibit the strongest
autoinhibitory effect on PLC2.
The results obtained so far identified the SH domain tandem as the autoinhibitory element inhibiting PLC2 in its basal activity. Since the SH domain tandem is a multi-domain complex, consisting of two SH2 domains and one SH3 domain, it is possible that the single domains contribute with diverse strength to the inhibitory regulation of the lipase. To assess this hypothesis, single SH domains or combinations of two SH domains were inserted into PLC2SH, to identify the domains executing the strongest inhibitory effect on PLC2SH. The insertion of the 2 SH domain sequences into the PLC2SH cDNA was accomplished by introducing a linker containing an AvrII restriction site in the deletion site of PLC2SH. The SH domains (SH2N, aa 515-638; SH2C, aa 637-747; SH3, aa 767-839; SH2N-SH2C, aa 515-747; SH2C-SH3, aa 637-839) were amplified with primers containing an AvrII restriction site on either end and inserted into PLC2SH by restriction and ligation. The linker introduced five additional residues in positions 515-519 of the protein. However, there were no functional differences between the PLC2SH mutants with and without these residues (data not shown). The system was further verified by introducing the whole SH domain tandem in PLC2SH, thereby generating a reconstituted
PLC2 (PLC2SH+SH). The reconstituted lipase behaved similar to wild-type PLC2, in terms of the basal activity and the stimulation by Rac2G12V (Figure 3-3A, upper panel). Furthermore, the expression of both enzymes was similar (Figure 3-3A, lower panel). The insertion of each SH domain alone reduced the basal activity of PLC2SH by 46.7 % for the insertion of SH2N, by 32.9 % for the insertion of SH2C, and by 39.4 % for the insertion of the SH3 domain (Figure 3-3B, upper panel). Since important regulatory tyrosine phosphorylation sites (Y753 and Y759) are located in the linker between the SH2C and the
SH3 domain, an extended version of the SH3 domain (YY-SH3), including the SH2C-SH3 linker (aa 746-766), was also checked for its ability to inhibit PLC2SH. As shown in the 61 Results upper panel of Figure 3-3B, the additional insertion of the linker containing the tyrosine phosphorylation sites did not cause a further inhibition (24 % inhibition of PLC2SH activity) compared to the insertion of SH3 alone. The same results were observed by the simultaneous insertion of two SH domains, at which SH2N together with SH2C and SH2N together with SH3 caused a 33.8 % and 57.3 % inhibition of PLC2SH, respectively.
However, the strongest inhibitory effect was detected when the SH2C domain together with the SH3 domain were introduced into PLC2SH. Both domains in combination reduced the basal activity of the deletion mutant by 87.4 % in this experiment, indicating that both domains in combination but not alone are the major components inhibiting PLC2 in its basal activity. But it should also be noted, that the other domains also account, albeit with lower intensity than SH2C-SH3, for the autoinhibitory regulation of the lipase. This was also confirmed by the observation, that only the insertion of the whole SH domain tandem was able to fully inhibit PLC2SH.
Figure 3-3: Inhibitory effect of 2 SH domains on the activity of PLC2SH. A, upper panel, COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty vector (Co.), 500 ng of vector encoding PLC2 (wt), or 500 ng of vector encoding PLC2SH+SH (SH+SH), or PLC2SH (SH) together with 25 ng of either empty vector (Control), Rac2 (Rac2), or Rac2G12V (Rac2G12V). The total amount
62 Results of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. A, lower panel, COS-7 cells were transfected as described above and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope. B, upper panel, COS-7 cells were transfected as indicated at the abscissa with 50 ng of either empty vector (Co.), vector encoding PLC2 (wt), PLC2SH (SH), PLC2SH+SH2N (+SH2N), PLC2SH+SH2C
(+SH2C), PLC2SH+SH3 (+SH3), PLC2SH+YY-SH3 (+YY-SH3), PLC2SH+SH2NSH2C (+SH2N-
SH2C), PLC2SH+SH2CSH3 (+SH2C-SH3), or PLC2SH+SH2N-SH3 (+SH2N-SH3). The total amount of DNA was maintained constant at 500 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, lower panel, COS-7 cells were transfected with 500 ng of the encoding vectors described above and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
The amounts of encoding vectors used for transfection in Figure 3-3B had to be quite small (50 ng of plasmid DNA/well) to clearly see the differences in the activities of the mutants. Due to the small amounts of protein expressed, we were not able to check for expression differences via immunoblotting in this case. To get at least a rough idea of the expression, COS-7 cells were transfected with 500 ng of encoding vectors and the expression was demonstrated via immunoblotting as shown in the lower panel of Figure 3-3B. The expression levels of the insertion mutants were similar or even higher than the expression level observed for PLC2SH in this experiment, indicating that the lower activities of the insertion mutants compared to PLC2SH are not due to expression differences.
The role of the SH2C and SH3 domain in the autoinhibitory regulation of PLC2 was further approved by a more detailed analysis of the activity of the PLC2SH+SH2C,
PLC2SH+SH3, and PLC2SH+SH2C-SH3 mutants. Therefore, COS-7 cells were transfected with increasing amounts of the encoding vectors. The activity of all three mutants increased with increasing amounts of vector used for transfection (Figure 3-4A).
However, cells transfected with PLC2SH+SH2C-SH3 showed an on average about 6.8-fold lower activity than the mutants in which each domain alone was inserted. The expression levels of all three mutants were similar, with the tendency of an even higher expression of the PLC2SH+SH2C-SH3 mutant compared to the other two mutants (Figure 3-4B). 63 Results
Figure 3-4: Role of the SH2C and SH3 domain in autoinhibitory regulation of PLC2. A, COS-7 cells were transfected with 500 ng of vector encoding PLC2 (PLC2, □) or increasing amounts (5 ng, 15 ng,
50 ng, 150 ng, and 500 ng) of vector encoding PLC2SH+SH2C (SH+SH2C, ▼), PLC2SH+SH3
(SH+SH3, ▲), or PLC2SH+SH2C-SH3 (SH+SH2C-SH3, ○). The total amount of DNA was maintained constant at 500 ng in each transfection by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
3.1.4 PLC2SH mutants are more sensitive to stimulation by Rac2.
The activation of the PLC2SH insertions mutants, described above, by Rac2 was assessed by cotransfection of COS-7 cells with the encoding vectors for wild-type Rac2 or constitutively active Rac2G12V. To detect a possible stimulation of the mutants by Rac2G12V, it was important to adjust a low basal activity of the mutants. This was obtained by using only 1 ng of lipase encoding vector for transfection. As shown in Figure 3-5,
PLC2SH as well as all of the insertion mutants are responsive to the stimulation by G12V G12V Rac2 . In contrast, wild-type PLC2 was only activated by Rac2 when 500 ng instead of 1 ng of encoding vector was used for transfection. This demonstrates the increased susceptibility of PLC2SH and its insertion mutants to the activation by Rho GTPases like Rac2. 64 Results
G12V Figure 3-5: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH mutants. COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty vector (Co.), 500 ng or 1 ng of vector encoding PLC2 (wt), or 1 ng of vector encoding PLC2SH (SH), PLC2SH+SH2N
(+SH2N), PLC2SH+SH2C (+SH2C), PLC2SH+SH3 (+SH3), PLC2SH+YY-SH3 (+YY-SH3),
PLC2SH+SH2NSH2C (+SH2NSH2C), PLC2SH+SH2CSH3 (+SH2CSH3), or PLC2SHSH2N-SH3
(SH2N-SH3) together with 25 ng of either empty vector (Control), vector encoding Rac2 (Rac2), or vector encoding Rac2G12V (Rac2G12V). The total amount of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined.
3.2 Functional reassembly of PLC2 by separately expressed fragments
The functional reassembly of PLC isozymes by separately expressed halves was already demonstrated for PLC1 [94] and PLC2 isozymes [309]. Furthermore, coexpression of the N- and C-terminal halves of these enzymes led to an enhanced PLC activity compared to the holoenzymes, again demonstrating the importance of the X-Y linkers in autoinhibitory regulation of the lipases. To further exploit the utility of assembling PLC2 fragments for functional analysis, we constructed two truncated versions of PLC2. One represents the 65 Results
N-terminal part of the enzyme from the N-terminus up to the end of spPHN (PLC2-X, aa 1-514) whereas the other truncated PLC2 starts at the beginning of spPHC and ends at the C-terminus of the enzyme (PLC2-Y, aa 842-1265). The assembly of these two parts results in a PLC2 variant lacking the SH2SH2SH3 tandem (Figure 3-6A). As expected, both mutants did not show any activity in intact cells by themselves, since both lack important catalytic residues (Figure 3-6B, left panel). However, coexpression of the two halves led to catalytic activity, which was markedly (approximately 20-fold) enhanced compared to wild-type PLC2, whereas the expression of the proteins was similar (Figure
3-6C, left panel). These results indicate (i) that the two halves of PLC2 reassemble to an intact enzyme which is able to catalyze the hydrolysis of PIP2 and (ii) that the absence of the SH2SH2SH3 tandem relieves the enzyme from autoinhibitory control.
Experiments from our group showed that both spPH domains are necessary for the Rac2- mediated activation of PLC2 [275]. Furthermore, the three-dimensional structure of the
PLC1 spPH domain revealed that the two halves of the spPH domain fold into a canonical PH domain and that the SH2SH2SH3 domain tandem is not required for the correct folding of the PH domain [288]. This led us to the assumption that the reassembly of the two
PLC2 fragments not only results in the formation of an intact catalytic domain, but potentially also in the formation of an intact PH domain. Indeed, coexpression of the G12V PLC2-X and PLC2-Y together with constitutively active Rac2 led to an about 4.7-fold increase in PLC activity compared to the control activity, whereas both halves alone were not stimulated by Rac2G12V (Figure 3-6B, right panel). Intriguingly, the effect of stimulation by wild-type Rac2, which was previously observed for PLC2SH (Figure
3-2), was also detected for the reassembled PLC2 enzyme.
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Figure 3-6: Functional reassembly of truncated PLC2 fragments. A, Domain organisation of PLC2 and the truncated PLC2 mutants PLC2-X and PLC2-Y. For details see main text. B, left panel, COS-7 cells were transfected as indicated at the abscissa with 500 ng of vector encoding PLC2 (wt), truncated PLC2 mutants PLC2-X (X) and PLC2-Y (Y) or cotransfected with 250 ng of vector encoding PLC2-X together with 250 ng of vector encoding PLC2-Y (X + Y). Right panel, COS-7 cells were cotransfected as indicated at the abscissa with 500 ng of vector encoding PLC2 (wt), truncated PLC2 mutants PLC2-X (X) and PLC2-Y
(Y), or 250 ng of vector encoding PLC2-X and 75 ng of vector encoding PLC2-Y (X+Y) in combination, together with either empty vector (Control) or 25 ng of vector encoding wild-type Rac2 (Rac2) or constitutively active Rac2G12V (Rac2G12V). The total amount of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. C, cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
The formation of the intact catalytic domain as well as the PH domain out of separately expressed halves was further investigated by the use of two distinct and well characterized
PLC2 mutants. The replacement of histidines 327 and 372, which are located in the
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H327A/H372A catalytic domain X, by alanins (PLC2 ) abrogates the catalytic activity of the enzyme [235]. As expected, no stimulation by Rac2G12V was observable in intact cells expressing the catalytically inactive PLC2 mutant (Figure 3-7). However, coexpression together with PLC2-X, harbouring an intact X domain, produced a PLC2 enzyme that was catalytically active, which was indicated by the sensitivity to Rac2-mediated activation. A complementary experiment regarding the spPH domain was carried out using F897Q the PLC2 mutant, which is insusceptible to stimulation by Rac2, but still exhibits its catalytic activity [275]. Intriguingly, the Rac2 sensitivity was regained by coexpression with PLC2-Y. The presented results impressively demonstrate the in vivo association of full-length PLC2 mutants with the appropriate PLC2 halves, which can replace the respective mutational inactivated domains. It is also striking that no elevated basal activity was observed for coexpression of the truncated PLC2 halves with the full-length mutant enzyme, indicating that the presence of the SH domain tandem restores the autoinhibitory control of the lipase.
H327A/H372A F897Q Figure 3-7: Functional reassembly of truncated PLC2 fragments with PLC2 and PLC2 . A, COS-7 cells were cotransfected as indicated at the abscissa with 250 ng of either empty vector (Co.), 68 Results
H327A/H372A vector encoding PLC2 (wt), truncated PLC2 mutants PLC2-X (X) and PLC2-Y (Y), PLC2 F897Q (H327A/H372A), or PLC2 (F897Q) together with 25 ng of either empty vector (Control) or vector encoding wild-type Rac2 (Rac2) or constitutively active Rac2G12V (Rac2G12V). The total amount of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
3.3 Phosphorylation induced activation of PLC2
PLC2 is activated by receptor and non-receptor protein tyrosine kinases, which plays an important role in B-cell receptor signalling. Upon receptor stimulation, PLC2 gets phosphorylated at tyrosine residues 753 and 759 [135], which are located in the SH domain tandem, more precisely in the linker between the C-terminal SH2 domain (SH2C) and the SH3 domain (Figure 3-8A). In addition to these two phosphorylation sites, Watanabe et al. identified two additional phosphorylation sites in the C-terminal region of PLC2, Y1197 and Y1217 [283]. However, the regulatory impact of phosphorylating PLC2 on these residues still remains ambiguous.
3.3.1 Tyrosine 733 plays a crucial role in the phosphorylation‐
induced activation of PLC2.
Since protein tyrosine phosphorylation in cells is a dynamic, rapidly reversible event and the modification itself is inherently labile, we established phosphomimetic mutants of
PLC2 by replacing the appropriate tyrosine residues by glutamic acid, which carries a terminal carboxyl group mimicking the negative charge of the phosphate. These phosphomimetic mutants of PLC2 can then be used for biochemical analysis both in intact cells and in crude cell extracts. To examine the effect of mimicking phosphorylation of different tyrosine residues in PLC2, COS-7 cells were transfected with the encoding vectors and inositol phosphate formation was determined. Figure 3-8B shows that
69 Results replacing tyrosine residues 753 or 759 by glutamic acid had no effect on the activity of
PLC2 when compared to the activity of the wild-type enzyme. Also, the double Y753E/Y759E phosphomimetic mutant PLC2 (2YE) did not show any differences in basal activity. All further mutants were generated in the setting of the 2YE mutant, as indicated at the abscissa in Figure 3-8B. Combining 2YE with Y1197E resulted in a 12.2-fold stimulation of PLC2 activity compared to the wild-type enzyme, whereas the triple phosphomimetic mutant 2YE/Y1217E only showed a slight activation (4.7 fold). Mimicking phosphorylation of all four well known phosphorylation sites
(Y753E/Y759E/Y1197E/Y1217E) stimulated PLC2 about 13-fold, which is comparable as it was observed for the triple phosphomimetic 2YE/Y1197E. Interestingly, Rikova et al. recently identified a novel phosphorylation site in PLC2 which was found in non-small cell lung cancer (NSCLC) tissue [216]. This novel phosphorylation site, Y733, lies immediately upstream of the two established phosphorylation sites, Y753 and Y759 in an area within the SH2C domain, which was shown to be an important regulatory element for the autoinhibitory regulation of PLC2. This element, also called PCI (phospholipase C inhibiting peptide), consists of 8 amino acids (726YRKMRLRY733), and its deletion results in the loss of autoinhibition, thereby generating a constitutively active PLC2 enzyme [59]. Furthermore, the PCI peptide was reported to be inhibitory when added to PLC enzymes in vitro [91] as well as in intact cells [87]. We therefore extended our set of phosphomimetic
PLC2 mutants by including the mutation Y733E. Like the previously tested phosphomimetic PLC2 mutations, the mutation Y733E by itself had no effect on the activity of PLC2 and the combination with 2YE had an effect on the basal activity that is comparable to the activity of the triple phosphomimetic mutant 2YE/Y1197E. However, mimicking phosphorylation of Y733 together with the both so far most actively mutants 2YE/Y1197E and 2YE/Y1197E/Y1217E gave raise to the most pronounced increase in phospholipase activity, 81-fold and 85.9- fold compared to PLC2, respectively. These results showed for the first time the activation of PLC2 using a phosphomimetic approach and furthermore demonstrated arrestingly the constitutive activation of PLC2 by phosphorylation of tyrosine 733.
The expression levels of the most active phosphomimetic mutants 2YE/Y733E/Y1197E and the quintuple mutant 2YE/Y733E/Y1197E/Y1217E are elevated compared to the wild- type enzyme (Figure 3-8C), but not in an intensity that could explain the 81-fold and
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85.9-fold activation of both mutants, respectively, meaning, that the obtained PLC activities are not due to the observed differences in expression of the lipases.
Figure 3-8: Effect of mimicking phosphorylation of different tyrosine residues on the activity of PLC2.
A, Domain organisation and localisation of regulatory tyrosine phosphorylation sites in PLC2. For details see main text. B, COS-7 cells were transfected as indicated at the abscissa with 500 ng of empty vector (control Y733E Y753E activity), vector encoding PLC2 (PLC2) or PLC2 phosphomimetic mutants PLC2 (733), PLC2 Y759E Y753E/Y759E Y733E/Y753E/Y759E (753), PLC2 (759), PLC2 (753/759), PLC2 (733/753/759), Y753E/Y759E/Y1197E Y753E/Y759E/Y1217E Y753E/Y759E/Y1197E/Y1217E PLC2 (753/759/1197), PLC2 (753/759/1217), PLC2 Y733E/Y753E/Y759E/Y1197E Y733E/Y753E/Y759E/Y1197E/Y1217E (753/759/1197/1217), PLC2 (733/753/759/1197), PLC2 (733/753/759/1197/1217). Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. C, COS-7 cells were transfected as described above, and cells from one well were lysed in 100
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µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
3.3.2 Activation of PLC2 mediated by Rac2 is independent of the phosphorylation induced activation.
Our group was previously able to show that PLC2, but not PLC1, is activated in intact cells by active mutants of the Rho GTPases Rac1, Rac2, and Rac3, but not by Cdc42 and RhoA, and that this activation is mediated by the two halves of the spPH domain [203,
275]. However, the activation of PLC2 by phosphorylation or Rho GTPases seems to be independent of each other. The mechanisms of both activating processes and their relation to each other still remain unclear. To examine the effect of Rac2 on PLC2, already activated by phosphorylation, all phosphomimetic mutants were expressed in COS-7 cells with or without Rac2 or constitutively active Rac2G12V (Figure 3-9A). Coexpression of G12V Rac2 and PLC2 increased PLC2 activity about 32-fold (Figure 3-9A, left panel) and 16-fold (Figure 3-9A, right panel) in the experiments shown. All phosphomimetic mutants are activated by constitutively active Rac2G12V in a manner that is comparable or even G12V stronger compared to the Rac2 -mediated activation of PLC2. Phosphomimetic mutants that showed enhanced phospholipase activity are also activated by wild-type Rac2, as it was already observed for other constitutively active PLC2 mutants, e.g. PLC2SH (Figure 3-2). The expression levels of the lipases alone, as well as together with Rac2 or Rac2G12V were similar (Figure 3-9B), indicating, that the observed stimulations by Rac2G12V as well as wild-type Rac2 are not due to expression differences.
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G12V Figure 3-9: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2 phosphomimetic mutants. A, COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty vector Y733E Y753E (Co.), 500 ng of vector encoding PLC2 (wt), PLC2 phosphomimetic mutants PLC2 (733), PLC2 Y759E Y753E/Y759E Y733E/Y753E/Y759E (753), PLC2 (759), PLC2 (753/759), PLC2 (733/753/759), Y753E/Y759E/Y1197E Y753E/Y759E/Y1217E Y753E/Y759E/Y1197E/Y1217E PLC2 (753/759/1197), PLC2 (753/759/1217), PLC2 Y733E/Y753E/Y759E/Y1197E (753/759/1197/1217), or 100 ng of vector encoding PLC2 (733/753/759/1197) and Y733E/Y753E/Y759E/Y1197E/Y1217E PLC2 (733/753/759/1197/1217) together with 25 ng of either empty vector (Control), vector encoding Rac2 (Rac2), or vector encoding Rac2G12V (Rac2G12V). The total amount of DNA was maintained constant at 525 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
3.4 Anaplastic lymphoma kinase
The interesting finding, that tyrosin 733 seems to play a crucial role in the phosphorylation-induced activation of PLC2 (see section 3.3.1), raised the question as to which protein kinase is involved in phosphorylating PLC2 at this important regulatory tyrosine residue. In silico prediction of protein phosphorylation sites and the correponding 73 Results kinases using the PPSP algorithm (prediction of protein kinase specific phosphorylation sites) [299], identified anaplastic lymphoma kinase (ALK) as a protein kinase potentially phosphorylating PLC2 on position Y733. Interestingly, (i) a novel, constitutively active ALK fusion protein, EML4-ALK, was identified in NSCLC, in the same phosphoproteomic approach that also detected Y733 as a novel phosphorylation site in
PLC2 [216], and (ii) Bai et al. [9] showed that another activated ALK fusion protein
(NPM-ALK) interacts with the SH2 domains of PLC2.
3.4.1 Isozyme specific activation of PLC isozymes by ALK and NPM‐ALK
In order to investigate the effect of ALK and its fusion proteins NPM-ALK and EML4- ALK on the activity of different PLC isozymes, COS-7 cells were transfected with the encoding vectors and inositol phosphate formation was measured (Figure 3-10A).
Coexpression of ALK and PLC1 or PLC2 resulted in a 17-fold and 5-fold stimulation of phospholipase activity, respectively. Surprisingly, NPM-ALK stimulated PLC1 (5.7-fold) but failed to stimulate PLC2 in transfected COS-7 cells. The ALK fusion protein EML4- ALK, recently discovered in NSCLC patients, had neither an impact on the activity of
PLC1, nor on the activity of PLC2. In contrast to the PLC isozymes, coexpression of
PLC2 together with NPM-ALK, or EML4-ALK did not lead to an enhanced, or in the case of ALK only resulted in a slightly enhanced inositol phosphate formation compared to the control activity. The initial hypothesis, that ALK, and maybe also its fusion proteins, phosphorylate PLC2 on tyrosine residue 733 and thereby activate the lipase, was refuted Y733E/Y753E/Y759E/Y1197E/Y1217E 5YE as the quintuple phosphomimetic mutant PLC2 (PLC2 ) was markedly activated by ALK (9.5-fold). As it had been observed before for wild-type 5YE PLC2, NPM-ALK and EML4-ALK failed to activate PLC2 . The same holds true for all other PLC2 phosphomimetic mutants (data not shown). These results present the identification of novel tyrosine kinases activating PLC isozymes at least in transfected COS-7 cells.
As shown in Figure 3-10B, coexpression of PLC isozymes with ALK or ALK fusion proteins, led to an obvious increase in the expression of the lipases compared to cells transfected with lipases only (Control). However, this fact is also true for PLC2,
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coexpression of PLCs together with EML4-ALK, as well as for coexpression of PLC2 together with NPM-ALK, which all together did not show an enhanced inositol phosphate formation. Therefore, the stimulation of PLC1 by ALK and NPM-ALK, and the 5YE stimulation of PLC2 and PLC2 by ALK, is likely not to be due to the enhanced protein levels of the PLC isozymes.
Figure 3-10: Effect of ALK and its fusion proteins NPM-ALK and EML4-ALK on the activity of PLC isozymes, PLC2 phosphomimetic mutant, and PLC2. A, COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty vector (Co.), 300 ng of vector encoding PLC1 (PLC1), 500 ng of Y733E/Y753E/Y759E/Y1197E/Y1217E 5YE vector encoding PLC2 (PLC2), 150 ng of vector encoding PLC2 (PLC2 ),
300 ng of vector encoding PLC2 (PLC2) together with 250 ng of either empty vector (Control), vector encoding ALK (ALK), NPM-ALK (NPM-ALK), or EML4-ALK (EML4-ALK). The total amount of DNA was maintained constant at 750 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1, PLC2, and 5YE PLC2 , an antibody reactive against PLC2 for PLC2, and an antibody reactive against ALK for ALK, NPM-ALK, and EML4-ALK. 75 Results
3.4.2 Wild‐type ALK as well as constitutively active ALK mutants are able to activate PLC isozymes.
ALKF1174L and ALKR1275Q were found to be mutated in sporadic and familial cases of neuroblastoma, creating a constitutively active ALK, and thereby gaining their oncogenic R1275Q potential [7, 37, 145]. In Figure 3-11A it is depicted that ALK activates PLC1 to a similar extend as wild-type ALK (both about 9-fold compared to control activity), whereas F1174L ALK exhibits the strongest stimulatory effect on PLC1 (14.9-fold). Both ALK variants as well as the wild-type enzyme had more or less comparable effects on the activity of PLC2. We observed a 4.6- and 4.7-fold stimulation for ALK and its F1174L R1275Q variant, whereas ALK only led to a 3.2-fold stimulation of PLC2 in this experiment.
On the other hand, PLC2 is also only weakly stimulated by the constitutively active mutants of ALK (1.7-fold compared to control activity). The expression levels of ALK and its variants were similar, whereas the expression levels of all lipases were again upregulated in the presence of ALK, as it had been elucidated before (Figure 3-11B).
Figure 3-11: Effect of the constitutively active ALK mutants ALKF1174L and ALKR1275Q on the activity of PLC isozymes. A, COS-7 cells were cotransfected as indicated at the abscissa with either 500 ng of empty
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vector (Co.), 300 ng of vector encoding PLC1 (PLC1), 500 ng of vector encoding PLC2 (PLC2), or 300 ng of vector encoding PLC2 (PLC2) together with 250 ng of either empty vector (Control), vector encoding ALK (ALK), vector encoding ALKF1174L (F1174L), or vector encoding ALKR1275Q (R1275Q). The total amount of DNA was maintained constant at 750 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2,5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1 and PLC2, an antibody reactive against PLC2 for PLC2, and an antibody reactive against ALK for ALK, ALKF1174L, and ALKR1275Q.
3.4.3 NPM‐ALK associates with PLC1 but not with PLC2.
To further study the observed activation of PLC isozymes by ALK and NPM-ALK, immunoprecipitation studies were performed. Therefore, COS-7 cells were cotransfected with cDNAs encoding PLC1 or PLC2 together with vectors encoding ALK or NPM-ALK and immunoprecipitations using an antibody reactive against the c-myc epitope of PLC isozymes and an antibody reactive against ALK and NPM-ALK were performed. PLC1 and PLC2 were efficiently precipitated using an anti-myc antibody (Figure 3-12A and C, left panels). There was no coprecipitation observed for ALK neither with PLC1 nor with
PLC2 (Figure 3-12A, right panel). However, the inverse experiment in which ALK was precipitated with an anti-ALK antibody, a coprecipitation of both PLC isozymes was detectable (Figure 3-12B). These results are in line with the observed activation of PLC1 and PLC2 by ALK (section 3.4.2). NPM-ALK clearly coprecipitated with PLC1 whereas there was only a weak association observable between NPM-ALK and PLC2 (Figure 3-12C). The same results were obtained by precipitating NPM-ALK with an anti-ALK antibody. There was again a strong association observeable for NPM-ALK and PLC1 whereas the detectable signal for coprecipitation of PLC2 was again only very negligible (Figure 3-12D), which is in accordance with the results observed for the isozyme specific activation of PLC isozymes by NPM-ALK in transfected COS-7 cells (section 3.4.1).
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Figure 3-12: Association of ALK and NPM-ALK with PLC isozymes in intact cells. COS-7 cells were transfected with 15 µg per well of either empty vector (Control), 10 µg per well of vector encoding PLC1
(PLC1) and PLC2 (PLC2), or 5 µg per well of vector encoding ALK (ALK) or NPM-ALK (NPM-ALK), or cotransfected with 10 µg per well of vector encoding PLC1 or PLC2 together with 5 µg per well of vector encodingALK or NPM-ALK. The total amount of DNA was maintained constant at 15 µg in each transfection by adding empty vector. Forty-eight hours after transfection the cells were harvested and immunoprecipitation was performed with an antibody reactive against the myc-tags of PLC1 and PLC2 (A and C) or with an antibody reactive against ALK and NPM-ALK (B and D). The samples were then subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1 and PLC2, and an antibody reactive against ALK for ALK and NPM-ALK. IP: immunoprecipitation, WB: Western blotting
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3.4.4 The activation of PLC isozymes is mediated by the kinase activity of ALK and NPM‐ALK.
To proof, that the activation of PLC isozymes by ALK and NPM-ALK is a direct effect of the kinase activity of ALK and its fusion protein NPM-ALK, kinase-dead mutants of ALK (ALKI1250T) [228] and NPM-ALK (NPM-ALKI310T) were established and tested for their I1250T ability to activate PLC1 and PLC2 in transfected COS-7 cells. ALK was originally detected in neuroblastoma and was shown to be a kinase-dead RTK [228]. The kinase dead NPM-ALK mutant, NPM-ALKI310T, was established by substituting the corresponding isoleucine in the ALK part of NPM-ALK. As shown before, PLC2 is activated by ALK, whereas the kinase-dead mutant of ALK failed to activate PLC2 (Figure 3-13A).
Furthermore, the activation of PLC1 by ALK as well as by NPM-ALK was abrogated by introducing point mutations into the kinases, leading to kinase-dead RTKs. Therefore, the activation of PLC isozymes by ALK and NPM-ALK is mediated by the kinase activity of the enzymes and is therefore likely to be due to the phosphorylation of PLC2 tyrosine residues, which are not identified yet.
Figure 3-13: Effect of ALK and NPM-ALK kinase-dead mutants on the activity of PLC isozymes. A, COS-7 cells were cotransfected as indicated at the abscissa with 500 ng of either empty vector (Co.), 300 ng 79 Results
of vector encoding PLC1 (PLC1), 500 ng of vector encoding PLC2 (PLC2), or 150 ng of vector encoding
PLC2 (PLC2) together with 250 ng of either empty vector (Control), vector encoding ALK (ALK), vector encoding ALKI1250T (I1250T), vector encoding NPM-ALK (NPM-ALK), or vector encoding NPM-ALKI310T (I310T). The total amount of DNA was maintained constant at 750 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2,5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1 and PLC2, an antibody reactive against PLC2 for PLC2, and an antibody reactive against ALK for ALK, ALKI1250T, NPM-ALK, and NPM-ALKI310T.
It is noticeable that the expression of the kinase-dead mutants is obviously lower than the expression of the wild-type enzymes, and that furthermore the expression of the lipases also decreased when coexpressed together with the kinase-dead mutants. This effect is apparently also observable with PLC2 and PLC2 that are not activated by NPM-ALK or ALK and NPM-ALK, respectively (Figure 3-13B). To further investigate if the binding of
NPM-ALK to PLC1 is also dependent on the kinase activity of the fusion protein immunoprecipitation studies were performed. As shown in the left panel of Figure 3-14 wild-type NPM-ALK binds to PLC1 whereas the binding of the kinase-dead mutant was markedly reduced. The possibilty that the reduced association of NPM-ALKI310T is due to a lower expression could be excluded by western blot analysis of the whole cell lysates used for immunoprecipitation, where NPM-ALK and the kinase-dead mutant showed a similar expression (Figure 3-14, right panel). The results presented here indicate that the phosphorylation-induced actvation as well as the association of PLC1 and NPM-ALK are dependent on an active kinase domain.
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I310T Figure 3-14: Association of NPM-ALK and NPM-ALK with PLC in intact cells. COS-7 cells were transfected with 15 µg per well of either empty vector (Control), 10 µg per well of vector encoding PLC1
(PLC1) and PLC2 (PLC2), or 5 µg per well of vector encoding ALK (ALK) or NPM-ALK (wt) or NPM- I310T ALK (I310T), or cotransfected with 10 µg per well of vector encoding PLC1 or PLC2 together with 5 µg per well of vector encoding NPM-ALK or NPM-ALKI310T. The total amount of DNA was maintained constant at 15 µg in each transfection by adding empty vector. Left panel, forty-eight hours after transfection the cells were harvested and immunoprecipitation was performed with an antibody reactive against the myc- tag of PLC1. The samples were then subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1, and an antibody reactive against ALK for NPM-ALK and NPM-ALKI310T. Right panel, 10 µl of the whole-cell lysates used for immunoprecipitation were subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for I310T PLC1, and an antibody reactive against ALK for NPM-ALK and NPM-ALK . IP: immunoprecipitation, WB: Western blotting
3.4.5 Tyrosine residues 664 in NPM‐ALK and 1604 in ALK are not essential for the complex formation with PLC isozymes in transfected COS‐7 cells.
The tyrosine phosphorylation site Y664 in NPM-ALK was found to be essential for the Y664F complex formation between the kinase and PLC2. It was shown that NPM-ALK no longer forms complexes with or activates PLC in transfected Ba/F3 cells [9]. The PLC binding site is located at the C-terminus of NPM-ALK and therefore is also present in the full-length ALK enzyme. It is hence likely that Y1604 in ALK also utilizes PLC isozymes 81 Results for signal transduction. The two mutants NPM-ALKY664F and ALKY1604F were constructed and tested for their ability to activate PLC1 and PLC2 (Figure 3-15). Surprisingly, and in contrast to the results presented by Bai et al. [9], NPM-ALKY664F, as well as ALKY1604F, activated PLC2 or both isozymes, PLC2 and PLC1, in intact COS-7 cells to an extend that was only slightly diminished compared to the wild-type enzymes (Figure 3-15A), while the expression levels of the kinases and the lipases were similar (Figure 3-15B).
Figure 3-15: Effect of ALKY1604F and NPM-ALKY664F on the activity of PLC isozymes. A, COS-7 cells were cotransfected as indicated at the abscissa with 500 ng of either empty vector (Co.), 300 ng of vector encoding PLC1 (PLC1), or 500 ng of vector encoding PLC2 (PLC2) together with 250 ng of either empty vector (Control), vector encoding ALK (ALK), vector encoding ALKY1604F (Y1604F), vector encoding NPM- ALK (NPM-ALK), or vector encoding NPM-ALKY664F (Y664F). The total amount of DNA was maintained constant at 750 ng by adding empty vector. Twenty four hours after transfection, the cells were incubated for 20 h in the presence of myo-[2-3H]inositol (2,5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and
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immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1 and PLC2 and an antibody reactive against ALK for ALK, ALKY1604F, NPM-ALK, and NPM-ALKY664F.
The obtained results where further strengthened by demonstrating the association of PLC1 Y664F and NPM-ALK . COS-7 cells were cotransfected with DNA encoding PLC1 and DNA Y664F encoding NPM-ALK or NPM-ALK , and PLC1 was selectively immunoprecipitated using an antibody reactive against the c-myc epitope. A clear coprecipitation was observed for NPM-ALK as well as for its Y664F variant (Figure 3-16).
Y664F Figure 3-16: Association of NPM-ALK with PLC1. COS-7 cells were transfected with 15 µg per well of either empty vector (Control), 10 µg per well of vector encoding PLC1 (PLC1), or 5 µg per well of vector encoding NPM-ALK (wt) or NPM-ALKY664F (Y664F), or cotransfected with 10 µg per well of vector Y664F encoding PLC1 together with 5 µg per well of vector encoding NPM-ALK (wt) or NPM-ALK (Y664F). The total amount of DNA was maintained constant at 15 µg in each transfection by adding empty vector. Forty-eight hours after transfection the cells were harvested and immunoprecipitation was performed with an antibody reactive against the myc-tag of PLC1. The samples were then subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope for PLC1, and an antibody reactive against ALK for NPM-ALK. IP: immunoprecipitation, WB: Western blotting
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3.5 Characterization of PLC2 deletion mutants associated with PLAID
Genomic deletions in the PLCG2 gene were associated with a novel disease pattern named
PLAID (phospholipase C-2-associated antibody deficiency and immune dysregulation) including amongst other symptoms cold-induced urticaria, immunodeficiency, and autoimmunity [190]. However, the impact of cold temperatures on the activity of the
PLC2 deletion mutants and the mechanistical implications leading to the described phenotype are unknown so far.
3.5.1 PLC219 and PLC220‐22 are specifically activated at subphysiological temperatures.
Variants of human PLC2 carrying deletions corresponding to those described by Ombrello et al. [190] and designated PLC219 and PLC220-22, lacking the amino acids encoded by exons 19 and 20-22 of the human PLCG2 gene, were expressed in transiently transfected COS-7 cells and maintained for 24 h at 37 °C. Radiolabeling of the cells with [3H]inositol was then performed for 20 h at temperatures ranging from 39 °C to 25 °C, followed by measurement of inositol phosphate formation. Figure 3-17A shows that expression of the two PLC2 deletion mutants at 37 °C caused slight increases in inositol phosphate formation in comparison to expression of the wild-type enzyme. Consistent with earlier results [190], these increases were approximately 2.8- and 3.6-fold for PLC219 and PLC220-22, respectively, relative to the increase over basal activity observed for wild-type PLC2. Much more strikingly, however, there was a marked stimulation of inositol phosphate formation when cells expressing either deletion mutant were incubated at subphysiologic temperatures. In both cases, there was a biphasic stimulatory response with declining temperatures, with a maximum at 31 °C and a gradual reduction upon further cooling to 25 °C. There was only a modest monophasic decrease in inositol phosphate formation in cells expressing wild-type PLC2 and in control cells when the temperature was reduced from 39 °C to 25 °C. Strikingly, at 31 °C, the increase in inositol phosphate formation in cells expressing PLC219 and PLC220-22 was enhanced approx. 480 ± 91- and 430 ± 84-fold (means ± SEM) relative to the increase over basal 84 Results
inositol phosphate formation observed in cells expressing wild-type PLC2. Figure 3-17B shows that the expression of wild-type PLC2 decreased with decreasing incubation temperature, while the expression of both PLC219 and PLC220-22 took slight increases at intermediate temperatures ranging from 35 °C to 31 °C and from 35 °C to
25 °C, respectively. While reduced expression of wild-type PLC2 at lower temperatures may explain, at least in part, the monophasic decrease in inositol phosphate formation by this enzyme, the limited magnitude of the changes observed in Figure 3-17B for PLC219 and PLC220-22 argues against a critical role of fluctuating enzyme expression in the marked changes of inositol phosphate formation evident in Figure 3-17A.
Figure 3-17: Specific activation of PLC219 and PLC220-22 at subphysiological temperatures. COS-7 cells were transfected with 500 ng each per well of either empty vector (), or vector encoding either wild-type PLC2 (●), PLC219 (▼), or PLC220-22 (▲). A, twenty-four hours after transfection, the cells were incubated for a further 20 h in individual incubation chambers at the temperatures indicated at the abscissa, in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. The levels of inositol phosphate formation at 37 °C are shown in expanded scale on the right vertical axis (open symbols). The values correspond to the means ± standard error of triplicate determinations. The statistical significance of the difference between the increases in inositol phosphate formation over basal activity (□) caused by expression of wild-type PLC2 (○), PLC219 (), and PLC220-22 () at 37 °C was determined using the unpaired t test with two-tailed P values: wild-type
PLC2 vs. PLC219, P = 0.0616; wild-type PLC2 vs. PLC220-22, P = 0.0231 (not shown). B, COS-7 cells were transfected as described above and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against PLC2.
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The determination of the 10 °C temperature coefficient (Q10) value, widely used to characterize the regulation of transient receptor potential (TRP) channels by temperature
[40], for wild-type PLC2, PLC219, and PLC220-22 is shown in Figure 3-18. While only a single linear component with a Q10 value of 4.6 (corresponding to a 4.6-fold decrease in activity per 10 °C) was evident for wild-type PLC2 between 39 °C and 25 °C, both PLC219 and PLC220-22 displayed two separate phases of opposite signs and markedly distinct Q10 values. Specifically, while Q10 was as high as 6745 for the marked increase in activity between 39 °C and 33 °C, it was lower by more than three orders of magnitude and not different, by sign and by magnitude, by global curve fitting analysis from the Q10 value describing the decrease in activity of the wild-type enzyme, 4.6, between 31 °C and 25 °C. Thus, it appears that the major functional consequence of the two deletions is their increased activity upon cooling from 37 °C to approximately 31 °C. The decrease in activity observed at temperatures below 31 °C appears to be a property inherently present in the remaining elements of wild-type PLC2.
Figure 3-18: Q10 analysis of the activities of wild-type PLC2, PLC219, and PLC220-22. The individual temperatures T were plotted against A , with the maximum activity of PLC 19 at 31 °C i log 2 10 Aref chosen as the reference activity Aref and reference temperature, Tref, respectively. The data of the linear components was analysed by non-linear least square curve fitting to a polynominal first order (straight line) equation. The slopes of the curves of PLC219 and PLC220-22 were not significantly different by clobal curve fitting using shared parameters from each other and, between 25 °C and 31 °C, from slope obtained for wild-type PLC2 (P = 0.9356 and P = 0.2121, respectively). 86 Results
The influence of a decrease in incubation temperature on the abundance of inositol phospholipids in transfected COS-7 cells is shown in Figure 3-19. Of note, PLC is capable of hydrolyzing PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 [146, 220, 221]. There was a monophasic loss of inositol phospholipids with decreasing temperature to approximately 50 % and 8 % at 31 °C and 25 °C, respectively, of the level observed at 37 °C. While the loss between 31 °C and 25 °C may explain some of the decline in inositol phosphate formation by PLC219 and PLC220-22 at these low temperatures (cf. Figure 3-17), the loss between 37 °C and 31 °C would be expected to counteract the increased inositol phosphate formation by the two deletion mutants in this range of temperatures, if the amount of radiolabeled inositol phospholipids available for hydrolysis ever becomes limiting in transfected cells. Thus, the degree of stimulation of the two enzymes between 37 °C and 31 °C may actually be higher than evident in Figure 3-17A.
Figure 3-19: Effect of subphysiological temperatures on cellular inositol phospholipid levels. COS-7 cells were transfected with 2 µg/well of empty vector. Twenty-four hours after transfection, the cells were incubated for a further 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa. The amount of [3H]inositol present in the culture medium after radiolabeling of the cells (; right vertical axis) and the cellular formation of inositol phospholipids (●; left vertical axis) was then determined as described under Materials and Methods.
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3.5.2 The activation of PLC219 and PLC220‐22 occurs posttranslationally and is reversible.
The next experiment was designed to examine whether stimulation of PLC219 and
PLC220-22 by cool temperatures occurs during or after mRNA translation. To this end, cells expressing either wild-type PLC2, PLC219, or PLC220-22 were treated with 100 µg/ml cycloheximide during [3H]inositol radiolabeling at either 37 °C or 31 °C. Figure 3-20B, shows that addition of cycloheximide caused a halt in the approximately two-fold increase in expression of both wild-type and deletion mutant PLC2 at both 37 °C and 31 °C, indicating that cycloheximide was in fact effective as a protein biosynthesis inhibitor in this experiment. As shown in Figure 3-20A, however, cycloheximide had only a minor effect on the increased formation of inositol phosphates by the two deletion enzymes at 31 °C versus 37 °C. These results clearly indicate that the stimulatory effect of subphysiologic temperatures on deletion mutant PLC2 activity may occur at the posttranslational stage.
Figure 3-20: Effect of cycloheximide on the activity of PLC219 and PLC220-22 at 31 °C. COS-7 cells were transfected as indicated at the abscissa with 500 ng of vector encoding PLC2 (Wt), PLC219
(19), or PLC220-22 (20-22). A, twenty-four hours after transfection, the cells were incubated for a further 20 h at 31 °C or 37 °C in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml), 10 mM LiCl, and as indicated in the absence or presence of 100 µg/ml cycloheximide (CHX), and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described 88 Results above.Twenty four hours after transfection cells from one well were lysed in 100 µl of SDS-PAGE sample buffer (Co., lane 1) or incubated for another 20 h in individual incubation chambers at 37 °C (lanes 2 and 3) or 31 °C (lanes 4 and 5), as indicated in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 100 µg/ml cycloheximide (CHX). Cells from one well were then lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of PLC2, PLC219 and PLC220-22.
To examine whether the stimulatory effects of the 19 and 20-22 deletions on PLC2 activity were reversible, wild-type PLC2, PLC219 and PLC220-22 were incubated for the last four hours of the transfection protocol, i.e. in the absence of radiolabeled inositol phospholipid substrate, at either 37 °C or 31 °C and then radiolabeled with [3H]inositol at one of the two temperatures. Figure 3-21, right panel, shows that the incubation protocol had no effect on the expression of the three enzymes. As shown in the corresponding left panel, preincubation of the three enzymes at 31 °C had no effect on their ability to promote inositol phosphate formation. Only when cells were incubated at 31 °C in the presence of [3H]inositol, enhanced inositol phosphate formation was evident by the two deletion mutants, in contrast to wild-type PLC2.
Figure 3-21: The activation of PLC219 and PLC220-22 by subphysiological temperatures is reversible. Left panel, COS-7 cells were transfected with 500 ng each per well of either vector encoding wild-type PLC2 (Wt), PLC219 (19), or PLC220-22 (20-22). Left panel, twenty hours after transfection the cells were pre-incubated for four hours in individual incubation chambers at 31 °C (31→31;
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31→37) or 37 °C (37→37). The cells were then incubated for another 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31 °C (31→31) or 37 °C (31→37;37→37) and the levels of inositol phosphates were then determined. Right panel, COS-7 cells were transfected and incubated as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of PLC2, PLC219, and PLC219-22.
Figure 3-22 shows a time-course of the changes of inositol phosphate formation by wild- type and mutant PLC2 upon changes in incubation temperature from 37 °C to 31 °C and vice versa. Both deletion mutants were activated with no apparent lag time with cooling to 31 °C. However, deactivation of both enzymes by warming to 37 °C required a lag time of approximately 10 min to come into effect. Taken together, the results suggest that activation of PLC219 and PLC220-22 by cool temperatures is a rapid and slowly, but fully reversible process.
Figure 3-22: Time course of the specific activation and inactivation of PLC219 and PLC220-22 by changes to and from a subphysiological temperature. Left panel, COS-7 cells were transfected with 500 ng each per well of either vector encoding wild-type PLC2 (Wt, ●), PLC219 (19, ▲), or PLC220-22 (20-22, ▼). Twenty-four hours after transfection, the cells were incubated for a further 20 h at 37 °C, in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. From the time point 0 min on (37→31 °C) the cells were incubated in individual incubation chambers at 31 °C, and at the time point 40 min (31→37 °C) the cells were placed back in the incubator at 37 °C for another 30 min. The levels of inositol phosphate formation of cells incubated at 37 °C for the whole time are shown as open symbols. Right panel, COS-7 cells were transfected as described above, and cells
90 Results from one well were lysed in 100 µl of SDS-PAGE sample buffer at the indicated time points. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of PLC2, PLC219, and PLC219-22.
3.5.3 The deletion of PLCG2 exons 19 and 20‐22 synergize to
promote PLC2 activation.
The fact that either of the two PLC2mutants, PLC219 and PLC220-22, were activated by cool temperatures to a similar degree raised the question as to the effect of a combined deletion of the residues encoded by exons 19 through 22. Figure 3-23 shows that the compound deletion mutant, designated PLC219-22, displayed a markedly
(approximately 24-fold) enhanced activity in comparison to PLC219 and PLC220-22 already upon incubation of the cells at 37 °C.The stimulatory effect of cool temperatures was assayed at two expression levels of the deletion mutants. In both cases, the stimulatory effect of cooling from 37 °C to 31 °C was markedly higher for PLC219-22 than for both
PLC219 and PLC220-22. At both temperatures, 37 °C and 31 °C, the deletions of
PLCG2 exons 19 and 20-22 clearly synergized to promote activation of PLC2.
Figure 3-23: Effect of deleting exon 19 to 22 of PLCG2 on the enzymatic activity at 37 °C and 31 °C. Left panel, COS-7 cells were transfected as indicated at the abscissa with 500 ng each per well of either
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vector encoding wild-type PLC2 (Wt), or 500 ng, 20 ng or 10 ng of vector encoding PLC219 (19),
PLC220-22 (20-22), or PLC219-22 (19-22). The total amount of DNA was maintained constant at 500 ng by adding empty vector. Twenty-four hours after transfection, the cells were incubated for a further 20 h at 31 °C or 37 °C in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. Right panel, COS-7 cells were transfected with 500 ng of vector encoding wild-type PLC2 (Wt), PLC219 (19), PLC220-22 (20-22), or PLC219-22 (19-22). Twenty-four hours after transfection, the cells were incubated for a further 20 h at 37 °C. Cells from one well were then lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of
PLC2, PLC219, PLC220-22 and PLC219-22.
3.5.4 The temperature induced activation mediated by exon
deletions is an isozyme specific effect for PLC2.
The structural organization of PLC2 between its two catalytic subdomains X and Y is very similar to that of its close relative PLC1 [289]. To address the question, whether deletion of residues in PLC1 corresponding to those encoded by exons 19 and 20-22 of PLCG2 has similar functional consequences, the relevant mutants of human PLC1 were produced. Since the human PLCG1 and PLCG2 genes exhibit distinct patterns of genomic organization, the deletions in PLC1 do not correspond to specific exons in PLCG1. Hence, the mutants are designated PLC1"19" and PLC1"20-22". Figure 3-24A shows that both PLC1"19" and PLC1"20-22", in contrast to their PLC2 counterparts, were already highly constitutively active at 37 °C (54- and 37-fold increases in inositol phosphate formation, respectively, in comparison to the increase caused by wild-type
PLC1 relative to control). Furthermore, only minor changes in their activity were observed upon reduction of the incubation temperature from 37 °C to 27 °C. Within the same range of temperatures, there were only minor changes in inositol phosphate formation by wild- type PLC1. Figure 3-24B shows that there were only subtle, if any changes in protein expression of the PLC enzymes with decreasing temperatures, except for PLC1"20-22" and PLC220-22, which showed increased expression. These changes notwithstanding, the results shown in Figure 3-24 clearly indicate that the temperature sensitivities of PLC1 and PLC2 carrying deletions corresponding to exons 19 and 20-22 of PLCG2 are distinct.
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Figure 3-24: Effect of the deletion of PLC1 residues corresponding to exon 19 or exon 20-22 of PLCG2 on the activity of PLC1 at subphysiological temperatures. A, COS-7 cells were transfected with 500 ng each per well of vector encoding either wild-type PLC1 (1Wt, ●), PLC1 (1”19”, ▼),
PLC1 (1”20-22”, ▲), PLC219(219, ), or PLC220-22(220-22, ). Twenty-four hours after transfection, the cells were incubated for a further 20 h in individual incubation chambers at the temperatures indicated at the abscissa, in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of
PLC1, PLC1668-707, PLC1708-828, PLC219, and PLC219-22.
3.5.5 Stimulation of basal PLC2 activity by deletions within the SH domain tandem does not correlate with activation by cool temperatures.
The region encoded by exons 20 to 22 of PLCG2 contains an octapeptide (YRKMRLRY) at the end of SH2C that is absolutely conserved in PLC1 and has previously been shown to mediate inhibition of PLC2 in trans and in cis [59, 88, 91]. To determine whether deletion of the octamer, designated PCI (for phospholipase C inhibitor), is sufficient to mediate sensitivity of PLC2 to cool temperatures, the deletion mutant PLC2PCI was expressed in COS-7 cells and functionally compared to PLC220-22 and wild-type PLC2. At the 93 Results same time, three other mutants carrying deletions known to promote constitutive activity of
PLC2 at 37 °C, PLC2SA, PLC2SH, and PLC2SH2C, were characterized. Figure
3-25 shows that PLC2PCI shared most features of its temperature sensitivity with
PLC220-22 and, by extension, with PLC219 (not shown): only a very slight (approximately 2.5-fold) enhancement of its activity at 37 °C, a marked activation upon lowering the incubation temperature from 37 °C to 31 °C, and a decrease in activity at temperatures below 31 °C. In contrast, PLC2SA, PLC2SH, and PLC2SH2C, although exhibiting constitutive activity at 37 °C as reported earlier for both PLC1 and
PLC2 [59, 77], showed an only modest, further increase in activity with cooling, which was monophasic for PLC2SH2C and only vaguely biphasic for PLC2SA and
PLC2SH.
Figure 3-25: Effect of subphysiological temperatures on the activities of PLC2PCI, PLC2SA,
PLC2SH and PLC2SH2C. A, COS-7 cells were transfected with 50 ng each per well of vector encoding either wild-type PLC2 (●), PLC220-22 (▲), or PLC2PCI () (left panel), or with 10 ng each per well of vector encoding either wild-type PLC2SA (○), PLC2SH (□), or PLC2SH2C (◊) (right panel). Twenty- 94 Results four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of
PLC2 and its deletion mutants.
A more comprehensive comparison of the effects of the various SH tandem constituents, alone or in combination, on PLC2 constitutive activity and sensitivity to cool temperatures is shown in Figure 3-26. As described before for PLC1 and PLC2 [59, 77], deletion of
SH2N did not markedly affect basal activity at 37 °C, while deletion of SH2C, caused an approximately 2.6-fold increase. Unlike observed previously, however, deletion of SH3 also caused an increase in basal activity, which was even slightly higher (approximately
4.1-fold) than that monitored for deletion of SH2C, but lower than that for deletion of all three SH domains (approximately 7.5-fold). The increases for the variants lacking two SH domains, PLC2SH2NSH2C, PLC2SH2CSH3, and PLC2SH2NSH3, were approximately 3.6-, 1.9-, and 2.4-fold, respectively. While deletion of SH2N did not change the temperature sensitivity of PLC2 relative to the wild-type enzyme and deletion of all three SH domains resulted in a PLC2 mutant largely insensitive to cool temperature, all other deletion mutants showed a clear sensitivity. Maximal sensitivity (approximately
6.1-fold) was observed for PLC2SH2CSH3, followed, in that order, by SH2NSH3,
SH2NSH2C, SH2C,and SH3.
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Figure 3-26: Effect of subphysiological temperatures on the activities of PLC2 deletion mutants. COS- 7 cells were transfected with 500 ng each per well of empty vector (Co.) and vector encoding wild-type
PLC2 (Wt), or with 10 ng each per well of vector encoding PLC2SH2N (SH2N), PLC2SH2C (SH2C),
PLC2SH3 (SH3), PLC2SH2NSH2C (SH2NSH2C), PLC2SH2CSH3 (SH2CSH3), PLC2SH2NSH3
(SH2NSH3), or PLC2SH (SH). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31 °C or 37 °C, and the levels of inositol phosphates were then determined.
3.5.6 The hypermorphic mutations Ali5 and Ali14 in combination
induce temperature‐mediated activation of PLC2.
The PLC2 point mutants identified in the two mouse disease models Ali5 and Ali14, D993G and Y495C, respectively, have previously been shown to cause constitutive activation of PLC2 at 37 °C [1, 56, 60, 301]. In one of the models, Ali5, autoinflammatory dermatitis commenced in the superficial layers of the paws and ears, i.e. in cool body regions [301]. It has been noted that this distribution resembles those of cutaneous granulomatous lesions in patients with PLAID [190]. We therefore examined and Ali5, compared the effect of cool temperatures on the activity of the mutants PLC2 Ali14 Ali5/Ali14 PLC2 , and the compound mutant PLC2 in intact cells. Figure 3-27A, left panel, shows that all three mutants exhibit moderately enhanced activity at 37 °C Ali5/Ali14 Ali5 Ali14 (PLC2 > PLC2 > PLC2 ) as decribed before [60]. Importantly, however, the three mutants displayed markedly different sensitivities to cool temperatures. Thus, while 96 Results
Ali5/Ali14 the response of PLC2 to decreasing temperatures closely resembled the pattern Ali14 observed for PLC220-22 (Figure 3-27A, center panel), PLC2 showed only a minor, monophasic increase in activity, which was opposite to the decrease observed for wild-type Ali5 PLC2 (Figure 3-27A, right panel). PLC2 displayed an intermediate phenotype, nonetheless showing a 3.9-fold activation upon cooling from 37 °C to 31 °C in comparison to wild-type PLC2.
Ali5 Ali14 Figure 3-27: Effect of subphysiological temperatures on the activities of PLC2 , PLC2 , and Ali5/Ali14 PLC2 . A, left panel, COS-7 cells were transfected with 500 ng each per well of either empty vector D993G Y495C (Co.), or vector encoding either wild-type PLC2 (PLC2), PLC2 (Ali5), PLC2 (Ali14), or Y495C/D993G PLC2 (Ali5/14). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31°C or 37°C, and the levels of inositol phosphates were then determined. Center and right panel, COS-7 cells were transfected with 500 ng each per well of vector encoding either wild-type PLC2 (PLC2, ○), D993G Y495C Y495C/D993G PLC220-22 (20-22, ▲), PLC2 (Ali5, ), PLC2 (Ali14, ), or PLC2 (Ali5/14, ●). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa, and the levels of inositol phosphates were then determined. B, left, center, and right panel, COS-7 cells were transfected as described above, and cells from one well were lysed in 100 µl of SDS-PAGE
97 Results sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an Ali5 Ali14 Ali5/Ali14 antibody reactive against the c-myc epitope of PLC2, PLC2 , PLC2 , PLC2 , and PLC220-22.
3.5.7 The temperature‐induced activation of PLC219 and
PLC220‐22 is distinct from a loss of autoinhibition.
Figure 3-28 shows a summary of the effects of the PLC2 mutations analyzed so far, in which the constitutive activities of the mutants at 37 °C were plotted against their activation by cool temperature (31 °C). Analyzing the results by cluster robustness analysis [139], it was found that the functional phenotypes fell into three clusters: one characterized by high constitutive activity at 37 °C (SA, SH, SH2C, SH3, and Ali5), one by high activation by cold (19, 20-22, PCI, and 19-22), and a third, intermediate one, consisting of mutants that could not be clearly categorized into one of the other two. These results suggest that the activation pattern of the two PLAID mutants, PLC219 and
PLC220-22, is distinct form a mere loss of autoinhibition, and indicate, by extension, that it is based on a specific molecular mechanism(s).
Figure 3-28: Summary and clustering of the effects of the PLC2 mutations. The ratios of the activities of the mutants and the activity of the wild-type enzyme at 37 °C corresponding to constitutive activity, were 98 Results
plotted against the ratios of the mutant activities at 31 °C and 37 °C, representing PLC2 activation by cool temperatures. Since some activities determined in cells expressing wild-type or variant PLC2 were similar to background inositol phosphate formation in the absence of exogeneous PLC2, the former values were used to calculate the ratios throughout. The values shown on both axes thus represent minimum estimates. The results of ensemble clustering (majority vote) for the leave-one-out clusterings are indicated by three different colours.
3.5.8 A hypermorphic missense mutation in PLC2 associated with APLAID is only limited activated by cool temperatures.
A hypermorphic missense mutation in PLCG2 was associated with a dominantly inherited autoinflammatory disease with immunodeficiency, termed APLAID (autoinflammation and PLC2-associated antibody deficiency and immune dysregulation). In contrast to PLAID, APLAID patients do not suffer from cold-induced urticaria. The detected missense mutation is located in the SH2C domain, resulting in a replacement of serine 707 by a S707Y tyrosine residue [311]. Figure 3-29A shows that the expression of PLC2 at 37 °C leads to an about 16.2-fold increase in inositol phosphate formation compared to cells expressing wild-type PLC2, which is in accordance with previously published data [311]. S707Y Despite the fact that APLAID patients do not suffer from cold urticaria, PLC2 displayed an intermediate sensitivity to cold temperatures as shown in Figure 3-29A. However, the fold activation upon cooling from 37 °C to 31 °C is about 20.6 magnitudes lower than the one observed for PLC220-22 in this experiment. The expression levels of the enzymes were similar and did not change significantly with decreasing temperatures (Figure 3-29B).
99 Results
S707Y Figure 3-29: Effect of subphysiological temperatures on the activity of PLC2 . A, COS-7 cells were transfected with 500 ng each per well of vector encoding either wild-type PLC2 (Wt, ○), PLC220-22 (20-22, ▲), or PLCS707Y (S707Y, ). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected and incubated at the temperatures described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
3.5.9 Phosphorylation of Y733 plays a crucial role in the
temperature‐mediated activation of PLC2.
The effect of decreasing temperatures on the phosphorylation-induced activation of PLC2 was investigated by using the phosphomimetic mutants that were already characterized in section 3.3. As shown in Figure 3-30A the two phosphomimetic mutants, Y733E/Y753E/Y759E/Y1197E Y733E/Y753E/Y759E/Y1197E/Y1217E PLC2 and PLC2 that showed the highest basal activity at 37 °C are, in contrast to all other phosphomimetic mutants, further activated at 31 °C (about 3.4-fold and 3.9-fold compared to their activity at 37 °C, respectively). The expression levels of the mutants at 31 °C did not significantly differ from those at 37 °C (Figure 3-30B).
100 Results
Figure 3-30: Specific activity of PLC2 phosphomimetic mutants at 37 °C and 31 °C. A, COS-7 cells were transfected as indicated at the abscissa with 500 ng of empty vector (Co.), vector encoding PLC2 (Wt) Y733E Y753E Y759E Y753E/Y759E or PLC2 phosphomimetic mutants PLC2 (733), PLC2 (753), PLC2 (759), PLC2 Y733E/Y753E/Y759E Y753E/Y759E/Y1197E Y753E/Y759E/Y1217E (753/759), PLC2 (733/753/759), PLC2 (753/759/1197), PLC2 Y753E/Y759E/Y1197E/Y1217E Y733E/Y753E/Y759E/Y1197E (753/759/1217), PLC2 (753/759/1197/1217), PLC2 Y733E/Y753E/Y759E/Y1197E/Y1217E (733/753/759/1197), PLC2 (733/753/759/1197/1217). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2- 3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31°C or 37°C, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected and incubated at the temperatures described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
101 Results
The critical role of phosphorylation of Y733 for the activation of PLC2 at subphysiological temperatures was further characterized as shown in Figure 3-31A. The quadruple phosphomimetic mutant (Y753E/Y759E/Y1197E/Y1217E) in which phosphorylation of all four well known phosphorylation sites was mimicked and the PLC2 mutant in which phosphorylation of Y733 alone was mimicked showed no or only a minor monophasic response to decreasing temperatures. In contrast, the combination of both mutants, resulting in the quintuple phosphomimetic mutant displayed a clear sensitivity to cool temperatures that also showed the characteristic biphasic stimulatory response with decreasing temperatures that was observed before for PLC220-22. Nevertheless, the fold activation upon cooling from 37 °C to 31 °C is about 29 magnitudes lower than the one observed for PLC220-22 in this experiment. The expression levels of PLC220-22 and the quintuple phosphomimetic mutant did not change significantly with decreasing temperatures (Figure 3-31B).
Figure 3-31: Effect of subphysiological temperatures on the activities of PLC2 phosphomimetic mutants. A, COS-7 cells were transfected with 500 ng each per well of vector encoding either wild-type Y733E Y753E/Y759E/Y1197E/Y1217E PLC2 (2Wt, □), PLC220-22 (20-22, ), PLC2 (Y733E, ), PLC2 (4YtoE, Y733E/Y753E/Y759E/Y1197E/Y1217E ), or PLC2 (5YtoE, ● ). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa, and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected and incubated at the temperatures described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
102 Results
3.5.10 The activation of PLC2 by cool temperatures and RacGTPases is competitive.
Beside the activation by tyrosine phosphorylation, PLC2 can also be activated by RacGTPases. The experiment shown in Figure 3-32 was designed to determine the effect of decreasing temperatures on the ability of constitutively active Rac2G12V to activate G12V PLC219 in comparison to wild-type PLC2. At 37 °C, Rac2 caused similar enhancements of the activity of both enzymes (Figure 3-32A, left panel). Lowering the incubation temperature, however, took a very different effect on Rac2G12V-mediated activation of the two enzymes. While only minor changes were observed for wild-type G12V PLC2, there was a progressive loss of the stimulatory effect of Rac2 on PLC219, ranging from about 27.2-fold at 37 °C, to about 1.3-fold at 31 °C, and no effect at 27 °C. This loss is unlikely to be due to exhaustion of the inositol phospholipids substrate at lower temperatures, since PLC219 is well capable of producing even higher levels of inositol phosphates when expressed at higher density (Figure 3-32A, right panel). It thus follows that activation of the PLC219 deletion mutant studied herein by cool temperatures is competitive with its activation by Rac2G12V.
103 Results
G12V Figure 3-32: Effect of subphysiological temperatures on the Rac2 -mediated activation of PLC2 and PLC219. A, Left panel, COS-7 cells were cotransfected with 500 ng each per well of vector encoding either wild-type PLC2 (, □) or 100 ng of vector encoding PLC219 (●, ○) together with 25 ng per well of either empty vector (open symbols) or vector encoding Rac2G12V (closed symbols). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2- 3H]inositol (2.5 µCi/ml) and 10 mM LiCl at the temperatures indicated at the abscissa, and the levels of inositol phosphates were then determined. Right panel, COS-7 cells were transfected with either 500 ng per well of vector encoding wild-type PLC2 (□) or increasing amounts (10 ng, 20 ng, 50 ng, 100 ng, 200 ng, 500 ng) of vector encoding PLC219 (○).The total amount of DNA was maintained constant at 500 ng by adding empty vector. Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31°C. Inositol phosphate formation was then determined as described under Materials and Methods. B, COS-7 cells were transfected and incubated at the temperatures described above, and cells from one well were lysed in 100 µl of SDS- PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope.
The experiment shown in Figure 3-33 examines the influence of the PLC219 expression level on the stimulatory effect of Rac2G12V. Two markedly different patterns were 104 Results
G12V observed. At 37 °C, stimulation of PLC219 by Rac2 was lost upon lowering the enzyme´s cellular expression level. In contrast, at 31 oC, enzyme stimulation was markedly enhanced with decreasing expression (Figure 3-33, left panel). There was no difference in G12V the sensitivity of PLC219 to Rac2 under these two conditions (Figure 3-33, right G12V panel).Given that Rac2 mediates translocation of PLC2 from the cytosol to the membrane fraction, i.e. the site of the enzyme substrate [168, 203], these findings suggest that enhanced membrane translocation by Rac2G12V is only effective at low cellular expression of PLC219 at 31 °C. According to this model, at higher expression levels,
PLC219 would already be present in the membrane compartment at concentrations sufficient to account for high inositol phosphate formation and, hence, less sensitive to further stimulation by activated Rac.
G12V Figure 3-33: Cool temperature shifts the requirement of PLC2 for Rac2 -mediated activation to G12V lower cellular PLC2 expression levels without altering the enzymes sensitivity to Rac2 . Left panel, COS-7 cells were cotransfected with increasing amounts (10 ng, 20 ng, 50 ng, or 100 ng) of vector encoding G12V PLC219 together with 25 ng per well of either empty vector (, □) or vector encoding Rac2 (●,○).The total amount of DNA was maintained constant at 500 ng by adding empty vector. Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2,5 µCi/ml) and 10 mM LiCl at 31 °C (closed symbols) or 37 °C (open symbols). Inositol phosphate formation was then determined as described under Materials and Methods. Right panel, COS-7 cells were transfected with either 100 ng () or 10 ng () per well of vector encoding PLC219 or 20 ng G12V per well of vector encoding Rac2 (□, ○) or cotransfected with 100 ng () or 10 ng (●) of vector encoding
PLC219 together with increasing amounts (0.5 ng, 1 ng, 2 ng, 5 ng, 10 ng, or 20 ng) of vector encoding Rac2G12V.Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation
105 Results chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31 °C (●,○,) or 37 °C
(,□,). Inositol phosphate formation was then determined as described under Materials and Methods.
3.5.11 The cold‐induced activation of PLAID PLC2 mutants is mediated by the split PH domain.
The activation of PLC2 by Rac is mediated by the internal, split PH domain of the enzyme [28, 275]. The interdependence of the stimulatory effects of Rac2G12V and cooling prompted us to determine the role of this domain in PLC2 deletion mutant activation by cooling. Replacement in spPH of PLC2 of a tryptophan residue conserved in all PH domains [196] by alanine has previously been shown to result in a loss of PLC2 stimulation by Rac, but not by tyrosine phosphorylation [59]. Figure 3-34 shows that the W899A replacement completely abrogates the response of the enzyme to cool temperature activation. Furthermore, replacement of one or the other half of the split PH domain of
PLC2 by the corresponding portions of PLC1, previously shown to block activation of the mutant enzymes by Rac, but to take no effect on their catalytic activity [275], also eliminated enzyme activation by cool temperatures. In contrast, replacement of both PH domains halves, which does not convey Rac sensitivity to PLC2 [275], fully rescued the response of PLC2 to cooling. These results indicate that a functional split PH domain, not necessarily the one genuinely contained in PLC2, is required for the sensitivity of PLC2 to cool temperatures.
Two residues of the N-terminal half of the PLC1 spPH domain, Y509 and F510, have previously been shown to be important for the ability of spPHN to cooperate with SH2C to maintain PLC1 in an inactive conformation [48, 60] and to reside, by NMR mapping, at the interface of spPH and SH2C [27]. To examine the role of the interaction in mediating the cool temperature response of PLC2, the two residues were substituted by alanine residues in PLC219 carrying the entire spPH of PLC1 (PLC2-11-19), thus generating the mutant PLC2-11-19-Y509A/F510A. Consistent with earlier findings [48, 60], there was a slight increase in basal activity of the latter mutant in comparison to PLC2-11-19.
Remarkably, however, and in striking contrast to PLC2-11-19, there was no response of the mutant to a decrease in incubation temperature from 37 °C to 31 °C. These results
106 Results
suggest that stimulation of the PLAID PLC2 deletion mutants by cool temperatures is dependent on the integrity of the site on spPHN that normally interacts with SH2C. Hence, it is likely that a functional interaction between this site and a structural element of the SH domain tandem retained in the deletion mutants mediates their cool temperature sensitivity.
Figure 3-34: Role of the spPH domain in the specific activation of PLC219 at subphysiological temperatures. A, COS-7 cells were transfected with 500 ng each per well of vector encoding either wild- W899A type PLC2 (Wt), PLC219 (19), PLC219 (W899A19), PLC2-21219 (12-19), PLC2-22119 Y509A/F510A (21-19), PLC2-21119 (11-19), PLC2-211 (Wt-11), or PLC2-21119 (11-19-Y509A/F510A). Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31 °C or 37 °C, and and the levels of inositol phosphates were then determined. B, COS-7 cells were transfected and incubated as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-
PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of PLC2 and PLC2 mutants.
107 Results
The observation that PLC2SH, a mutant lacking the entire SH tandem, displays enhanced basal activity, but is largely insensitive to activation by cold is remarkable, because this mutant lacks all constituents thought to mediate autoinhibition of the enzyme (Figure 3-35A). On the one hand, this finding is very well consistent with and confirms the notion that activation by cold and loss of autoinhibition by SH tandem constituents are independent processes. On the other hand, it is rather puzzling because cold activation appears to be both dependent on and independent of specific constituents of the SH tandem (cf. Figure 3-25, Figure 3-26, and Figure 3-35A). However, given the importance of the split PH domain for cold activation of PLC2 emerging from the experiments shown in Figures 3-31, 3-32, and 3-33, the possibility remained that activation by cold temperatures is sterically restricted in the mutant PLC2SH by the (artificial) covalent linkage between the two halves of the split PH domain. To examine this possibility, we took advantage of previous findings suggesting that PLC isozymes can be expressed as two independent polypeptide fragments containing either the catalytic subdomain X or Y [94, 309] (section
3.2). Thus, PLC2SH was expressed either as a single polypeptide and as two separate chains, encompassing either the sequence from the amino terminus up to and including spPHN or the sequence beginning with and including spPHC and extending to the carboxyl- terminus (cf. Figure 3-6). The cells were then analyzed and compared for inositol phosphate formation at 37 °C and 31 °C. Figure 3-35B shows that expression of the
N-terminal or the C-terminal half of PLC2 did not lead to enhanced inositol phosphate formation. In marked contrast, when both halves were coexpressed, basal inositol phosphate formation at 37 °C was markedly enhanced. More importantly, however, decreasing the incubation temperature to 31 °C caused a substantial, approximately 6.8-fold enhancement of PLC activity, in striking contrast to cells expressing the SH deletion mutant of PLC2, where no change in activity was observed. These results strongly suggest that the structural element(s) mediating cold activation of PLAID PLC2 mutants reside outside the SH tandem and that its responsiveness to cool temperatures is blocked by covalent linkage between the two constituents of the PLC2 split PH domain.
108 Results
Figure 3-35: Covalent linkage of the split PH domain halves blocks PLC2 sensitivity to cool temperatures. A, Schematic representation of the deletions within the SH domain tandem. A linear sketch of the region between residues 515 and 839 of human PLC2 is shown. The positions of the two repeats detected within the two SH2 domains (KDGTFLVR/RDGAFLIR and GRVQHCRI/GKVKHCRI; SH2N/SH2C, identical residues underlined) and of the various deletions used in this study are indicated. B, COS-7 cells were transfected with 500 ng each per well of either empty vector (Co.) or vector encoding wild-type PLC2
(PLC2), 10 ng of vector encoding PLC2SH (SH), 250 ng of vector encoding either PLC2-X (X) or
PLC2-Y (Y), or 250 ng each of both of the latter two vectors (X+Y).Twenty-four hours after transfection, the cells were incubated for 20 h in individual incubation chambers in the presence of myo-[2-3H]inositol (2.5 µCi/ml) and 10 mM LiCl at 31 °C or 37 °C, and and the levels of inositol phosphates were then determined. C, COS-7 cells were transfected and incubated as described above, and cells from one well were lysed in 100 µl of SDS-PAGE sample buffer. An aliquot was subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-myc epitope of PLC2 and PLC2 mutants.
109 Discussion
4 Discussion
PLCs are signaling molecules that catalyze the hydrolysis of phosphatidylinositol 4,5- bisphosphate to produce the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate and the protein kinase C-activating second messenger diacylglycerol [171, 186, 251]. The mammalian PLCs are divided into six subfamilies, designated , , , , , and [99, 129, 181, 225, 254, 255, 313]. Within the PLC subfamily, the two PLC isoforms share a number of features that are distinct from those of the other PLC subfamilies. The most striking difference is the insertion of additional domains between the catalytic subdomains X and Y. This specific array (SA) contains a second, split pleckstrin homology (spPH) domain, consisting of two halves separated by two Src homology 2 (SH2) domains and one
Src homology 3 (SH3) domain. Activation of PLC2 upon B cell antigen receptor (BCR) stimulation has been implicated to be a critical step in the BCR-mediated Ca2+ signaling
[81]. Therefore, it is important to understand the mechanisms of how the activity of PLC2 is stimulated and inhibited.
The need for a stringent regulation of PLC activity in unstimulated cells becomes apparent on closer examination of the balance of energy of a cell. A constitutively active PLC isozyme would imply a tremendous waste of energy, needed for the resynthesis of the PLC substrate PIP2 out of DAG and inositol (5 equivalents of ATP) [212]. Furthermore, the 2+ depletion of PIP2 and the persisting Ca signalling may lead to regulatory feedback mechanisms that cause insensitivity of the cells to extracellular stimuli. The physiological consequences of constitutive activation in the case of PLC2 were reported to be severe defects in immune regulation leading to autoimmunity and immune dysregulation in mouse models as well as in humans [1, 190, 301, 311]. Therefore, a better knowledge of the mechanisms of autoinhibitory regulation of the PLC2 isozyme is of exceptional importance.
110 Discussion
4.1 Autoinhibitory and phosphorylation‐induced
regulation of PLC2 activity
The involvement of PLC X-Y linkers in the intramolecular regulation were already revealed in the 1990s by several different experimental approaches, all of which have one common ground: the deletion of either parts of or the whole X-Y linkers or the disruption of the structure of these linkers. The deletion of PLC1 aa 517-883 encompassing the SH2SH2SH3 tandem and, unknown to the authors at that time, parts of the spPH domains
[93], as well as the coexpression of amino and carboxy terminal halves of PLC1 [94] and
PLC2 [309] to assemble enzymes lacking the SH domains and parts of the spPH domains in case of PLC1 or the X-Y linker in case of PLC2, created constitutively active PLC isozymes. Similarly, protease cleavage of PLC1 [39, 55], PLC2 [226], and PLC1 [62] within the X-Y linker region, disrupting the structure of this region, also resulted in an increase in basal PLC activity. Furthermore, PLC1 and PLC1 were inhibited in trans by peptides corresponding to sequences within the X-Y linkers [91]. All described approaches finally led to one common result, namely the loss of autoinhibitory regulation of the appropriate PLC isozymes. The `hinged´ lid model was proposed as a general model for the autoinhibitory regulation of PLC isozymes [77]. This model is based on biochemical analysis [83] as well as on the crystal structure of PLC2 in complex with Rac1 [117] and describes the occlusion of the active site of PLC isozymes by their X-Y linkers. Upon activation, PLC isozymes are recruited to and orientated at plasma membranes at which the negatively charged X-Y linker is repelled from the negatively charged membrane, revealing the active site and allowing substrate entry and processing. Since the X-Y linker of PLC isozymes is, in contrast to all other PLC isozymes, composed of multiple domains and does not exhibit large accumulations of negatively charged side groups, it is likely that these PLCs are subject to different autoinhibitory mechanisms.
The results of this work show that the deletion of either the whole specific array (SA) or just the SH2SH2SH3 (SH) domain tandem resulted in a marked increase in basal activity of the enzymes (both about 40-fold compared to wild-type), suggesting that PLC2 is negatively regulated by its specific array (Figure 3-1). Since PLC2SA and PLC2SH did not show any obvious differences in PLC activity in transfected COS-7 cells, the most simple reasoning for this result would be that the spPH domains, present in PLC2SH but 111 Discussion
missing in PLC2SA are not involved in autoinhibitory regulation of the enzyme and that the deletion of the SH domain tandem is sufficient to overcome autoinhibition. However, the effect of the direct deletion of the spPH domains on the basal activity of PLC2 is discussed contrary in literature. The Katan group [59] reported that upon deletion of both halves of the spPH domain the basal activity of PLC2 increased whereas the Sondek group [77] stated that there is no change in the activity of the enzyme compared to wild-type
PLC2. The contradictory effects possibly arise from the different deletion strategies that were utilized by the two groups. While the Katan group directly deleted aa 471-513 and aa 841-913, the Sondek group replaced aa 464-515 and aa 852-921 by a G-S-G-S linker. With regard to the direct deletion of the spPH domains, there is no clear evidence for their involvement in PLC2 autoinhibitory regulation. However, results from our group could show that chimeric PLC1 mutants in whom the spPH domains were replaced by those of
PLC2 display an enhanced phospholipase activity in intact cells as well as in vitro. This was in particular the case when the N-terminal spPH (spPHN) domain was swapped, indicating a direct involvement of the spPH domains in the intramolecular regulation of
PLC1. Interestingly, the counterpart chimeric PLC2 mutants were not susceptible to enhanced basal activity [275]. Possibly, PLC2 underlies a more stringent intramolecular regulation than PLC1, or the spPH domain is not involved at all in this particular regulatory mechanism. Further evidence for the contribution of the spPHN domain in the regulation of PLC1 provided a study in which the SH2C domain was found to be involved in autoinhibitory regulation of PLC1 [48]. It was suggested by the authors that the spPHN domain (in particular residues Y509 and F510) regulates the accessibility of the SH2C domain, and thereby plays an ancillary role in the regulation of PLC1 activity. However, it appears that the identified residues in the spPH domain are not sufficient to fully overcome autoinhibition, but possibly drive the lipase to a higher susceptibility for the activation by upstream activators like Rho GTPases, tyrosine or non-tyrosine kinases [48]. This hypothesis was further supported by a point mutation in PLC2 (Ali14, Y495C) found in a murine model and corresponding to Y509 in PLC1. Although the Ali14-type mutation did not affect basal activity of PLC2, the lipase exhibited enhanced stimulation by Rac and an increased response to EGF stimulation in transfected cells [60]. Finally, NMR spectra revealed that the Ali14 mutation appears to be located at the interface of the spPH and
SH2C domains [27].
112 Discussion
In addition to the depicted role of the spPH domains in autoinhibitory regulation, both domains are of crucial importance for the Rac2-mediated activation of PLC2. Biochemical analysis, NMR spectra, as well as the crystal structure of the isolated spPH domain bound to Rac2 clearly demonstrated that Rac2 binds to PLC2 as well as to the isolated spPH domain [28, 275]. Taking these results into account it is to be expected that the PLC2SA mutant lacking the spPH domains is no longer activated by Rac2. This hypothesis was confirmed experimentally. Furthermore, the PLC2SH mutant, still harbouring spPH domains, was additionally stimulated by Rac2G12V (Figure 3-2), indicating (i) that the two halves of the spPH domain fold into a functional PH domain and (ii) that the SH domain tandem in not required for the activation of PLC2 by Rac2. Moreover, the results argue that the autoinhibitory regulation and the activation by Rho GTPases rely on distinct mechanisms that partially implicate common domains, in particular the spPH domain.
The PLC SH domain tandem is composed of two SH2 domains and one SH3 domain, at which each domain possesses particular functions. SH3 domains are known to interact with proteins containing proline rich sequences [306], whereas the SH2N domain is critical for binding to receptor tyrosine kinases or adaptor molecules like BLNK, that mediate translocation of the lipase to the plasma membrane [35, 49, 106, 107, 119, 206]. The function of the SH2C domain appears to be more directed to the intramolecular regulation of PLC isozymes. It was demonstrated for PLC1 that the deletion or the functional interruption of the SH2C domain leads to constitutive activation of the lipase [48, 77].
Likewise, the deletion of the SH2C domain of PLC2 also resulted in an increase in basal activity. In contrast, point mutations that specifically affect the function of the SH2 domains were not sufficient to enhance the basal activity of the enzyme. Also, the deletion of SH2N or SH3 had no impact on PLC activity [59]. Based on these results, one would expect that the insertion of the SH2C domain into PLC2SH should be adequate to reduce the elevated basal activity of the deletion mutant. Nevertheless, neither the insertion of the
SH2C domain nor the insertion of the SH2N or SH3 domains gave rise to a fully inhibition of the activity of PLC2SH (Figure 3-3). The observed inhibition for each of the SH domains ranged between 33 % and 47 %, indicating that each of the domains contribute equally to the autoinhibitory regulation of PLC2. Likewise, the insertion of the SH2C domain together with the SH2N domain, and the insertion of the SH2N domain together with the SH3 domain were not sufficient to reduce the basal activity of PLC2SH to the level of the wild-type enzyme. Intriguingly, the SH2C domain in combination with the SH3 113 Discussion domain exhibited the strongest inhibitory effect on the deletion mutant and inhibited the basal activity of PLC2SH by almost 88 % (Figure 3-3). A more detailed and comparative analysis of the activities of PLC2SH+SH2C, PLC2SH+SH3 and
PLC2SH+SH2C-SH3 revealed that both mutants in whom either the SH2C or the SH3 domain was inserted showed an about 6.8-fold higher activity than the mutant in which both domains in combination were inserted (Figure 3-4). The linker between the SH2C and the SH3 domain contains two important regulatory tyrosine phosphorylation sites (Y753 and Y759). Phosphorylation of PLC1 at the corresponding tyrosine residues mediates high intramolecular interactions with the SH2C domain resulting in conformational changes that release the enzyme from its autoinhibitory constraint [77]. However, insertion of the SH3 domain together with the SH2C-SH3 linker exhibited no further inhibitory effect on the activity of PLC2SH compared to the insertion of the SH3 domain alone (Figure 3-3).
Taken together, the results imply that the SH2C domain together with the SH3 domain and the SH2C-SH3 linker are the major elements involved in the autoinhibitory regulation of
PLC2. However, on the basis of the performed experiments, the role of the SH2C-SH3 domain linker is still undefined. There is the possibility that the SH2C domain together with the SH2C-SH3 linker in combination with phosphorylation of Y753 and Y759 contribute to autoinhbitory regulation, like it was described for PLC1 [77]. However, the direct deletion of the PLC2 SH2C domain, like it is described in literature [59], is not sufficient to address this hypothesis. To test the possibility that the observed inhibition of
PLC2SH activity by introducing the SH2C and SH3 domain is dependent on both domains or probably only on the SH2C domain together with the linker region, it would be adequate as a future experiment to insert the SH2C domain together with the linker region, containing Y753 and Y759, into PLC2SH.
As previously demonstrated for PLC2SH, also the appropriate insertion mutants are further activated by Rac2G12V (Figure 3-5). Impressively, only very small amounts of vector (1ng/well) encoding for the insertion mutants as well as for PLC2SH were needed for transfection to obtain an observable stimulation by Rac2G12V. In contrast, no stimulation of wild-type PLC2 was detectable under these conditions. These results point to an enhanced susceptibility of the desinhibited PLC2 deletion mutants to Rac2-mediated activation. The possibility that the binding of Rac2 leads to conformational changes in the three dimensional structure of PLC2, releasing the lipase from its autoinhibitory
114 Discussion constraints and thereby achieving a stimulated PLC activity was excluded by using FRET (fluorescence resonance energy transfer) analysis. At least in this kind of experiments,
Rac2 binding did not provoke major conformational rearrangements in the PLC2 structure
[59]. The same results were observed for PLC2, where the comparison of the crystal structure of PLC2 alone and in complex with Rac1 revealed that there are no structural changes upon binding of Rac1 [83, 117]. The proximity to membranes was shown to be essential for the Rac2-mediated activation of PLC2 [59]. However, the role of membrane proximity in the release of autoinhibition of PLC2, as it was described for PLC2 [83], has to be further clarified.
The functional reassembly of PLC1 [94] or PLC2 [309] out of independently expressed amino and carboxy terminal halves was initially used to study the formation of the catalytic site or the functional contribution of different domains to enzyme function, respectively. The physically association between the two halves of the catalytic domain was furthermore demonstrated by limited proteolysis of PLC1 within the SH domain tandem resulting in enhanced PLC activity [62]. A common outcome of these studies was (i) that both enzymes can reassemble from separately expressed halves and form a catalytically active enzyme and (ii) that in both cases the specific activation of the enzyme was elevated compared to the holoenzyme if the X-Y linker was missing. The same results were obtained in this work for independently expressed halves of PLC2, in which the
N-terminal part of the enzyme, reaching from the N-terminus up to the end of spPHN
(PLC2-X, aa 1-514), and the C-terminal part, starting at the beginning of spPHC and ending at the C-terminus of the enzyme (PLC2-Y, aa 842-1265), reassembled to a catalytically active enzyme that showed an elevated basal activity (about 20-fold) compared to the holo-PLC2 enzyme (Figure 3-6). This showed once again the role of the
SH domain tandem as an intrinsic negative regulator of PLC2 activity and furthermore demonstrates the formation of an intact catalytic TIM-barrel by the two halves, X and Y, of the catalytic domain. The complex between PLC2-X and PLC2-Y was additionally activated by Rac2G12V (Figure 3-6) suggesting the formation of an intact spPH domain, which is necessary for the Rac2-mediated activation of PLC2 [275]. This hypothesis is further supported by the three dimensional structure of the PLC1 spPH domain, exhibiting that the structure of the spPH domain is not changed by the insertion of the SH domain tandem [288]. The formation of intact domains out of two halves might be driven by at
115 Discussion least two distinct reasons: (i) both halves contain residues that are directly involved in the catalytic activity in case of the catalytic domains, or in the Rac2-mediated activation in case of the spPH domain, or (ii) only one of the halves contains the described residues and the other halve imparts the proper folding of the domain. In case of the PLC1 spPH domain it was shown that the isolated halves of the spPH domains remained unfolded and that correct folding was only achieved upon interaction with the complementary halve
[288]. Based on NMR data and the crystal structure of the 2 spPH domain bound to GTP-loaded Rac2G12V, the interaction site of Rac2 was mapped to the C-terminal halve of the spPH domain, in which residues K862, V893 and F897 were found to be important for the activation of PLC2 by Rac2 [28, 275]. Taken together, it is most likely that the C-terminal halve of the spPH domain mediates the interaction with Rac2 and the N-terminal halve is necessary to achieve the correct folding of the spPH domain.
Impressively, PLC2-X and PLC2-Y also formed complexes with full-length PLC2 enzymes in transfected COS-7 cells. This was demonstrated by coexpression of H327A/H372A PLC2 , in which two important residues in the catalytic domain X were replaced by alanin resulting in a catalytically inactive enzyme [235], together with PLC2-X, harbouring a functional X domain. The complementary experiment regarding the spPH F897Q domain was performed using PLC2 , which is insensitive to stimulation by Rac2 due to the mutation of a residue in the C-terminal spPH domain important for the interaction with Rac2 [275]. The results shown in Figure 3-7 arrestingly demonstrate that the X domain of PLC2-X can functionally restore the nonfunctional X domain of H327A/H372A PLC2 resulting in a catalytically active enzyme. The identical findings were F897Q observed for PLC2 in which the spPHC domain of PLC2-Y redressed the ability of the enzyme to be responsive to the activation by Rac2G12V. Another interesting observation emerging from this experiment is that no enhanced basal activity was observed upon coexpression of full-length PLC2 mutants and the truncated PLC2 halves. This is probably due to the presence of the SH domain tandem, which was shown to exert an intrinsic inhibitory effect on PLC2 (section 3.1.1). These results suggest that the SH domain tandem has not to be covalently linked between the two halves of the spPH domain to exert its inhibitory effect. Recombinant SH2SH2SH3 domain regions of PLC1 and
PLC2 inhibited phosphoinositide hydrolysis induced by PLC1, PLC1, PLC2, and PLC1 in vitro [91], supporting the above described hypothesis.
116 Discussion
Phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes. This reversible posttranslational modification is furthermore crucial for the assembly of multiprotein complexes. Antigen-mediated activation of the BCR entails the phosphorylation and activation of PLC2 at specific tyrosine residues located in the SH domain tandem, more precisely in the linker between the SH2C and SH3 domain (Y753 and Y759), and at the C-terminal end of the lipase (Y1197 and Y1217) [135, 283]. The replacement of Y753 and Y759 by phenylalanine residues revealed that both tyrosine residues are important for PLC2 signalling function in B cells [217]. However, the regulatory role of phosphorylation of Y1197 and Y1217 remains questionable and also the mechanistically impact of phosphorylation of different tyrosine residues on the activity of
PLC2 remains an open question. In order to study the direct impact of tyrosine phosphorylation on the activity of PLC2, we replaced all four known tyrosine phosphorylation sites alone or in combination, by glutamic acids, generating PLC2 phosphomimetic mutants. An acidic substitution, like glutamic acid or aspartic acid, can mimic the effects of tyrosine, serine or threonine phosphorylation by its negative charge. This is a widely used approach to study the effects of phosphorylation on the activity of proteins [47, 125, 200, 296]. The replacement of Y753 and Y759 alone or in combination by glutamic acid had no influence on the activity of PLC2. This could indicate that phosphorylation of Y753 and Y759 is not sufficient to activate PLC2 and that the phosphorylation sites on the C-terminus of the enzyme are additionally required to achieve activation of the lipase. In deed, mimicking phosphorylation of Y753 and Y759 together with Y1197 or Y1217 resulted in a 12.2-fold and 4.7-fold activation of PLC2 compared to the wild-type enzyme, respectively. The quadruple phosphomimetic mutant displayed an about 13-fold enhanced activity compared to wild-type PLC2 (Figure 3-8). At least three of the four common phosphorylation sites had to be phosphorylated to exert an activating effect on the lipase. This is consistent with the observation that chicken DT40 B cells expressing PLC2 mutants in which single tyrosine residues were substituted by phenylalanine exhibited a diminished Ca2+ mobilization whereas the expression of the quadruple YF mutant almost completely abolished Ca2+ signalling [283]. The results obtained indicate that mimicking tyrosine phosphorylation by substitution of the appropriate tyrosine residues with glutamic acid is a valuable tool to study the effect of tyrosine phosphorylation on the activity of at least Phospholipases C. It is worth mentioning that the chemical structure of phosphorylated tyrosine differs strongly from that of glutamate. Both of them are negatively charged, but glutamate is singly charged 117 Discussion whereas phosphorylated tyrosine is, nominally, doubly charged at physiological pH [44]. Furthermore, glutamate is much shorter than tyrosine and it totally lacks the aromatic ring.
However, in the case of PLC2 it seems that the negative charge of glutamate alone is sufficient to mimic the phosphorylation of tyrosine residues, since the impact of these substitutions led to changes in the activity of PLC2 that are in line with similar results obtained from other experimental approaches, as described above. To fully exclude the possibility that the absence of the aromatic ring of tyrosine itself has an influence on the activity of the enzyme it would be adequate to substitute the appropriate tyrosine residues for alanine.
A phosphoproteomic approach targeted on the survey of tyrosine kinase activity in lung cancer identified a novel phosphorylation site in PLC2, Y733, which is located at the
C-terminus of the SH2C domain and thus lies immediately upstream of the two established phosphorylation sites Y753 and Y759 [216]. Most interestingly, the SH2C domain per se and in particular an octapeptide, the PCI peptide (726YRKMRLRY733), including Y733, were shown to be involved in the autoinhibitory regulation of PLC2 [59, 87, 91].
Furthermore, phosphorylation induced activation of PLC1 was shown to be coupled to the release of autoinhibition of the enzyme [77]. Based on this knowledge it is most likely that phosphorylation of tyrosine residue 733 is a major contributive component in a PLC2 activating mechanism that potentially leads to tumorigenesis. As previously observed for Y753 and Y759, mimicking phosphorylation of Y733 alone had no impact on the activity of PLC2. However, mimicking phosphorylation of Y733, Y753 and Y759 in combination elevated PLC activity about 11-fold which is comparable to the measured activities of the Y753E/Y759E/Y1197E triple and quadruple phosphomimetic mutants PLC2 and Y753E/Y759E/Y1197E/Y1217E PLC2 (Figure 3-8). Despite the obvious difference in the location of Y1197 and Y733 in respect to Y753 and Y759, they seem to exert the same effect on the activity of PLC2. There are at least two possible explanations for this surveillance, (i) the three dimensional structure of PLC2 also brings Y1197 and Y1217 in direct spatial proximity to Y753 and Y759, or (ii) distinct mechanisms lead to the activation of PLC2 depending on the pattern of phosphorylated tyrosines. Intriguingly, mimicking phosphorylation of Y733 in the context of the two most actively phosphomimetic mutants Y753E/Y759E/Y1197E Y753E/Y759E/Y1197E/Y1217E PLC2 and PLC2 , led to a marked activation of PLC activity, 81-fold and 85.9- fold compared to PLC2, respectively (Figure 3-8). Thus,
118 Discussion
phosphorylation of Y733 seems to execute an exceeding effect on the activation of PLC2, which may play an important role in the pathogenesis of lung cancer.
All established phosphomimetic mutants are additionally activated by constitutively active Rac2G12V (Figure 3-9), suggesting that tyrosine phosphorylation-dependent activation and the Rac2-mediated activation of PLC2 are independent processes that both, separately as well as in combination, fulfil the requirements to activate PLC2. In line with the described results, mutations in the binding interface of Rac2 and PLC2 did not affect the phosphorylation induced activation of PLC2 downstream of the EGF receptor [28].
Similarly, abolishing the activation of PLC2 by Rac had no effect on the stimulation of the lipase by G subunits [117]. Strikingly, all phosphomimetic mutants that displayed an elevated activity compared to the wild-type enzyme also showed an increased basal activity in cells expressing wild-type Rac2 (Figure 3-9). By culturing the transfected COS-7 cells in cell culture medium containing serum, a small proportion of endogenously expressed Rac1 as well as over-expressed Rac2 exists in its activated GTP bound form. However, this small population of activated Rac2 is not sufficient to activate wild-type
PLC2 but seems to be adequate to induce a further activation of the constitutively active phosphomimetic mutants. This leads to the assumption that those mutants are more susceptible to Rac2-mediated activation. It seems that the intrinsic inhibition of PLC2 by autoinhibitory elements is a general barrier that has to be overcome to fully activate the lipase. This appears to be also the case for the activation of PLC2 by upstream regulators like Rho GTPases or receptor and non-receptor tyrosine kinases. If the intrinsic barrier, or in other words, the autoinhibitory regulation is weakened through certain events like phosphorylation of regulatory tyrosine residues or deletion of SH domains, it is easier for a second regulator, e.g. Rac2, to exert its activating effect on PLC2.
The apparently striking effect of the phosphorylation of tyrosine residue 733 on the activity of PLC2 and the potentially pathophysiological effects of this constitutive activation of the lipase regarding tumorigenesis incited the search for the kinase phosphorylating PLC2 on Y733. Using the PPSP algorithm, several kinases were suggested to be able to phosphorylate this specific tyrosine residue in PLC2 [299]. However, ALK seemed to be the most promising hit for two distinct reasons: (i) NPM-ALK, an ALK fusion protein, was shown to directly bind to SH2 domains of PLC, in which Y733 is located, resulting in
119 Discussion phosphorylation and subsequent activation of the lipase [9] and (ii) furthermore, a novel ALK fusion protein, EML4-ALK, was found in the same phosphoproteomic approach that also discovered Y733 as a novel phosphorylation site in PLC2 [216]. ALK displays the classical structural features of a receptor tyrosine kinase and belongs to the insulin receptor superfamily [16, 108]. NPM-ALK is an oncogenic constitutively active tyrosine kinase associated with ALCL and is created via the recurrent chromosomal translocation t(2;5)(p23;q35), which fuses the C-terminal half of ALK to the N-terminal half of nucleophosmin (NPM) [12, 63, 124, 164]. A further constitutively active ALK fusion protein, EML4-ALK, was shown to be associated with NSCLC [216, 239]. Full length- ALK, as well as constitutively active ALK mutants, ALKF1175L and ALKR1275Q, found to be mutated in sporadic and familial cases of neuroblastoma [7, 37, 145], activated PLC1 and
PLC2 in transfected COS-7 cells (Figure 3-11). The distinct stimulating impact of wild- type ALK, which is not constitutively active, on PLC activity is most likely due to the overexpression of the kinase. Constitutive phosphorylation and activation of ALK was also observed in human NBL (naval biosciences laboratory) cell lines that displayed a high expression of ALK, leading to mutation-independent activation of the kinase [198].
The proof of an interaction between ALK and the PLC isozymes was assessed by using a coimmunoprecipitation approach (Figure 3-12). PLC1 as well as PLC2 coprecipitated with ALK, indicating that the observed activation of both PLC isozymes is based on an association of the lipases with the kinase. More direct approaches to characterize the interaction of two proteins would be surface plasmon resonance (SPR) spectroscopy or fluorescence resonance energy transfer (FRET) analysis. However, there is a need of recombinant expressed and purified proteins for both of the two methods. So far, the production of recombinant soluble ALK protein failed using baculovirus infected Sf9 insect cells (data not shown).
As shown in Figure 3-10, ALK also activated the quintuple phosphomimetic mutant, in which all 5 known tyrosine phosphorylation sites were replaced by glutamic acid. This observation refuted the initial hypothesis that ALK is a potential kinase phosphorylating
PLC2 on tyrosine residue 733. The same results were obtained for all other phosphomimetic mutants as well (data not shown). Therefore, Y733, Y753, Y759, Y1197, and Y1217 seem to be no or not the only phosphorylation sites in PLC2 that are targeted by ALK. The identification of the ALK-specific phosphorylation sites could be conducted 120 Discussion
by a mass spectromic approach in the future. PLC2 was only marginal stimulated by ALK and by the constitutively active ALK mutants (Figure 3-10 and Figure 3-11). Thus ALK seems to be no interaction partner of PLC2. Furthermore, the expression of PLC2 is restricted to cells of the haematopoietic system [30, 120, 192, 237], whereas expression of full-length ALK is absent in cell lines of the haematopoietic system [52]. This would also mean, that the observed association of ALK and PLC2, which is also only expressed in haematopoietic cells [253], has probably no physiological impact.
Most interestingly, the activation of PLC isozymes by NPM-ALK appears to be isozyme specific. PLC1 is clearly activated by NPM-ALK whereas PLC2 did not show any change in PLC activity by coexpression with NPM-ALK (Figure 3-10). NPM-ALK is associated with ALCL which is a type of non-Hodgkin lymphoma involving aberrant T cells. The predominantly expressed PLC isoform in these cells is PLC1 [253], emphasizing the isozyme selective activation by NPM-ALK. Furthermore, PLC1 was identified to associate with NPM-ALK in Karpas299 cells by coimmunoprecipitation and subsequently tandem mass spectrometry (MS/MS) [46]. In this work, the association of NPM-ALK and
PLC1 was demonstrated by coimmunoprecipitation experiments, whereas no or only a very weak interaction was detectable for PLC2 (Figure 3-12). The results further confirm the observed isozyme specific activation of PLC isozymes by NPM-ALK in transfected COS-7 cells.
EML4-ALK did not exert any effect on the activity of PLC1, PLC2 or PLC2 in transfected COS-7 cells (Figure 3-10). This may indicate that EML4-ALK does not regulate the PLC pathway. Pharmacologic inhibition of EML4-ALK by small molecule tyrosine kinase inhibitors led to the downregulation of the Ras/MEK/ERK and PI3K/Akt pathways. In contrast, no or only a minor downregulation of STAT3 phosphorylation was observed in the same experimental approach [137]. So far, there is no indication in literature about a link between the PLC pathway and EML4-ALK. However, the results obtained here could possibly imply that EML4-ALK exhibits its mitogenic activity through other downstream signalling pathways than the PLC pathway.
The ability of NPM-ALK to activate PLC1 was found to be due to the phosphorylation of the lipase. Tyrosine phosphorylation of PLC was exclusively detectable in BaF3
121 Discussion lymphocyte cells that stably overexpressed NPM-ALK. Furthermore, this phosphorylation of PLC led to enhanced inositol phosphate levels, corroborating the activation of PLC
[9]. In order to ensure that the observed activation of PLC1 and/or PLC2 by ALK and NPM-ALK in transfected COS-7 cells is also dependent on the phosphorylation of the lipases, kinase-dead mutants of both kinases were tested for their ability to activate PLC isozymes. In line with the results described in literature, kinase-dead mutants of ALK as well as of NPM-ALK failed to activate PLC1 and/or PLC2 (Figure 3-13). These results imply that the presumably phosphorylation-induced activation of PLC isozymes by ALK and/or NPM-ALK and at least the binding of NPM-ALK to PLC1 (Figure 3-14) is strictly dependent on the intrinsic kinase activity of ALK. It was also demonstrated for other NPM-ALK interacting molecules like pC130 CAS (p130 Crk-associate substrate), Grb2, JAK3, Shp2, or Gab2, that the interaction is dependent on the catalytic activity of NPM- ALK, as there was no association with NPM-ALK kinase dead mutants [4, 215, 269, 282, 304]
Phosphopeptide competition experiments identified tyrosine residue 664 in NPM-ALK as the binding site for PLC isozymes. Furthermore, Y664 was shown to be an autophosphorylation site in vivo that mediates the complex formation between NPM-ALK and PLC. Expression of NPM-ALK in Rat-1 fibroblasts and Ba/F3 lymphocyte cells was shown to lead to transformation of these cells, whereas NPM-ALKY664F lost the ability to transform Rat-1 fibroblasts as well as Ba/F3 cells. Thus, the activation of PLC seems to be essential for the transduction of a mitogenic signal by NPM-ALK [9]. In contrast to the described observations, the results of this work show that NPM-ALKY664F was able to activate PLC1 in transfected cells to a level that is comparable to wild-type NPM-ALK (Figure 3-15). Furthermore, a clear association of both enzymes in transfected COS-7 cells was demonstrated by coimmunoprecipitation (Figure 3-16). The same results were observed for full-length ALK in which the corresponding tyrosine was replaced by Y1604F phenylalanine. ALK behaved as wild-type ALK in regard to the activation of PLC1 and PLC2 (Figure 3-15). In line with the described results, Bischof et al. reported that a truncated NPM-ALK mutant lacking the C-terminal 154 amino acids, including Y664, still exhibited its transforming ability on Fr3T3 fibroblasts [17]. It might be possible that the observed effects are dependent on the used cell-types.
122 Discussion
4.2 Molecular mechanisms underlying the human hereditary disease PLAID
The results presented in this work show that deletion of residues encoded by exons 19 and
20-22 from PLC2 causes both a mild constitutive activation of inositol phosphate formation at 37 °C and a marked, several-hundred-fold enhancement of this activity in response to very small decreases in temperature, e.g. to 31 °C, upon expression of the mutant enzymes in intact cells. These functional responses are qualitatively and quantitatively similar for both types of mutants. While the former changes are consistent with those reported earlier for transfected cells, the latter are much higher than increases in 2+ intracellular calcium [Ca ]i observed when peripheral blood B cells of PLAID patients are exposed to cold. In the latter case, differences to normal B cells were only observed at temperatures below 29 °C, not exceeding a maximum of an about two-fold increase at 21 °C [190]. The results shown here suggest that the primary defect of the mutant enzymes, i.e. sensitivity to stimulation by cool temperatures, emerges at temperatures only a few degrees below the normal body temperature. These findings are in agreement with clinical findings on patients with a deletion of exons 20-22, where only very subtle cooling, such as the one caused by a tear rolling down the cheek of an affected family member at room temperature caused symptoms within one minute [73]. Of note, even at an ambient, comfortable temperature of 27 °C, the temperature in many cutaneous and subcutaneous regions is already somewhat lower than the esophageal core temperature of 37 °C [284], such that only minimal further temperature decreases by evaporative heat loss may suffice to cause maximal PLC2 activation. The considerable decrease of the stimulatory effect observed at temperatures below 31 °C may explain why PLAID patients are typically negative in the cold stimulation time test (CSTT), involving cooling with ice, and upon cold-water immersion [73, 190]. The time course of PLC2 deletion mutant activation and its reversible nature closely matches the clinical observation on PLAID patients of an immediate onset of inflammatory symptoms upon evaporative skin cooling and a somewhat slower resolution of the symptoms developing over the course of
30 minutes of rewarming at room temperature [73]. Mouse PLC2 carrying a gain-of- Ali5 function mutation, D993G, in the catalytic region, PLC2 , and causing spontaneous autoinflammation and autoimmunity shows a similar, albeit less dramatic response to cold temperatures [301]. This suggests that some of the lesions noticed in the affected animals,
123 Discussion such as inflammation in superficial skin layers, may in fact be caused by further activation Ali5 S707Y of PLC2 by cool temperatures. Intriguingly, PLC2 , found in APLAID patients, that do not suffer from cold urticaria [311], is further activated by cool temperatures, even though to a much lower extend than it was observed for PLC219 and PLC220-22. APLAID is a dominantly inherited autoinflammatory disease with immunodeficiency which is characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency [311]. In contrast to PLAID, unstimulated B cells from APLAID patients show an enhanced intracellular Ca2+ release, which may lead to B cell anergy, as discussed below. Hence, an effect of this could be a desensitization of B cells or even mast cells to a further response to S707Y e.g. cool temperatures. Due to the only moderate response of PLC2 to cool temperatures it is also possible that APLAID patients only suffer from a very mild form of cold urticaria which was possibly not assessed in the phenotypical analysis of the patients so far. Similarly, also phosphomimetic mutants of PLC2 showed a further activation by Ali5 cool temperatures, with an intensity that is comparable to those observed for PLC2 and S707Y PLC2 . These results suggest that a hyperphosphorylation of PLC2 can result in a sensitivity of the enzyme to cool temperatures.
Three mechanisms should be considered to explain the sensitivity of the PLC2 mutants to cold temperatures observed in this study [270]. First, other cold-sensitive molecules endogenously present in COS-7 cells might indirectly cause activation of the mutants, e.g. via a soluble mediator or via altered protein-protein-interaction patterns. Second, the mutants may be specifically enabled to sense temperature-mediated changes in the physical properties of the plasma membrane phospholipid bilayer containing the enzymes´substrate(s). The third possibility is that a cool temperature sensor is intrinsically present in the PLC2 mutants. We do not think, however, that the first possibility is a likely one. Thus, among the known cold-temperature-sensitive molecules, TRPA1, TRPM8, and TRPC5, the former two responded to cold temperatures within lower temperature ranges [41] and endogenous expression of the latter two was not evident in COS-7 cells [127, 161, 214, 264]. While we cannot at present formally exclude the second mechanism, we note that the phase transition temperature of DPPC (Dipalmitoylphosphatidylcholine) (Tm) in artificial phospholipid membrane vesicles is 41 °C [105] and thus outside the range of temperatures mediating activation of the PLC2 mutants. However, the behavior of native
124 Discussion plasma membranes may be different. Experiments in cell-free systems using soluble PLC substrate (e.g. [96]) will allow addressing this question in the future.
If the structural element mediating the response to cool temperatures resides on the enzyme itself, the simplest interpretation of the results would be that removal of the portions encoded by exons 19 or 20-22 causes a loss of an autoinhibition, since the two regions overlap with regions identified before [27, 59, 77, 79] or in this study (see section 3.1) to be autoinhibitory at 37 °C. Comparison of the functional consequences of deletion and point mutations within the SH tandem on constitutive activity at 37 °C vs. stimulation by cool temperatures, e.g. 31 °C, shows that the mutants can be grouped into three distinct categories and that the two functional consequences are not necessarily correlated with each other (Figure 3-28). While this would not formally exclude a loss of autoinhibition, acquisition of an autostimulatory mechanism sensitive to decreasing temperatures appears as an alternative explanation. At first glance, a structural element residing in the SH domain tandem of PLC2 would appear a likely candidate for triggering such a stimulation. However, the deletion experiments shown in Figure 3-26 revealed that none of the constituents of the entire SH tandem is specifically required for enzyme activation by cool temperatures and that even a deletion mutant lacking both of the repeats shared between the two SH2 domains (PLC2SH2NSH2C) (Figure 3-26, Figure 3-35A) is activated by incubation at 31 °C.
Thus, the possibility remains that temperature-sensitivity resides in the split PH domain and that this sensitivity is specifically kept in check by either the intact, native SH tandem, by its SH2C-SH3 portion, or by a covalent linkage between the two split PH domain halves. The fact that the cool-temperature-sensitivity is lost when non-homonymous split
PH domain halves are present in PLC2, when the tertiary structure of the PH domain is disrupted by the W899A mutation [60], or when two residues of its N-terminal half, Y509 and F510, are replaced by alanine residues, is consistent with this view. The latter residues have recently been mapped, by NMR titration analysis, to the interface of spPH and SH2C and suggested to be involved in SH2C-mediated autoinhibition [27]. The fact that removal of the two residues is also effective in the absence of almost the entire SH2C domain in
PLC219 is difficult to reconcile with an important role of a spPH-SH2C interaction in cool-temperature regulation of mutant PLC2. However, chemical shift perturbations observed in NMR experiments probing protein-protein-interactions may imply structural
125 Discussion reorientation of residues upon binding rather than direct involvement in the interaction surface [187]. This and the weak interaction between the two domains observed in [77] is consistent with additional functions of these two spPH domain residues. Interestingly, Ali14 Y509 of PLC1 corresponds to Y495 of PLC2, which is mutated to C495 in PLC2 and Ali14/Ali5 the double mutant PLC2 . The considerable stimulatory effect of the Ali14 mutation Ali5 on the stimulation of PLC2 by cool temperature (cf. Figure 3-27, left panel) suggests there may loss- and gain-of function mutations in this locus with regard to cool- temperature sensitivity of PLC2.
The thermodynamic changes of PLC219 and PLC220-22 induced by cool temperatures exceed those reported for other temperature-regulated proteins, such as thermoTRPs, where Q10 values between approximately 10 and > 100 have been reported [41]. However, higher Q10 values are not without precedence in the literature. For example, the step of heat damage to the development of the posterior crossvein of Drosophila that is most sensitive to increasing temperature showed a Q1 of 1.8, corresponding to a Q10 of about 360 [86, 172]. Clapham and Miller recently developed a thermodynamic framework for understanding temperature sensing by transient receptor potential (thermoTRP) channels [41]. It is based on the requirement, under the laws of thermodynamics, of unusually large
conformational standard-state enthalpies, H 0 , in the opening of temperature-sensitive TRP channels. The authors suggest that thermoTRP channel gating is accompanied by large changes in molar heat capacity, CP , and postulate that hot- and cold-sensing TRPs
operate by identical conformational changes. For the cold-activated TRP channels, H 0 is
-1 -1 between -40 kcal mol and -112 kcal mol . According to H 0 20ln Q10 , the enthalpies
-1 are even higher, approximately -176 kcal mol , for PLC219 and PLC220-22. The -1 -1 molar heat capacity of tempTRPs, CP , is in the order of 3-5 kcal mol K . Since high heat capacity increases accompany the transfer of nonpolar compounds from small molecules into water, these values have been taken by Clapham and Miller to suggest that on the order of 10 to 20 non-polar side chains are unburied from the thrust of each subunit of the tetrameric thermoTRPs upon activation. Figure 4-1 shows that CP is even higher, -1 -1 approximately 35 kcal mol K , for PLC219 and PLC220. If the thermodynamic framework developed for the thermoTRPs apply to the deletion mutants of PLC2, this would suggest exposure of an even higher number of hydrophobic residues to the aqueous solvent upon cool-temperature-mediated enzyme activation. 126 Discussion
Figure 4-1: Temperature dependence of conformational equilibrium constant. The effect of incubation temperature on the conformational equilibrium constant K is shown for PLC219 (▲) and
PLC220-22 (▼). The data was fit to equation (4) (section 2.6) by non-linear least squares curve fitting to estimate CP and T0. The two parameters were not significantly different from each other by clobal curve fitting using shared parameters for the curves of PLC219 and PLC220-22 (P = 0.1746). The region of the curve that has not been accessed experimentally is drawn by a dot-dashed line. The relationships between -1 -1 -1 -1 temperature and K proposed by [41] for thermoTRPs for CP = 3 kcal mol K and CP = 5 kcal mol K , using K0 = 0.01 and T0 = 25 °C, are shown in comparison. The dashed line marks the half-activarion level of the equilibrium. The positions of T0 are marked by dotted lines.
The marked sensitivity of the two PLAID PLC2 mutants to cool temperatures may explain the gain-of-function symptoms observed in all PLAID patients such as cold urticaria [73,
190]. They are likely to be caused by cold-temperature-mediated activation of PLC2 in cutaneous mast cells and monocytes/macrophages, where the enzyme plays important roles in FcR- and FcR-mediated inflammatory skin reactions [287]. Symptomatic allergic and autoimmune disease, occurring in 56 % and 26 % of PLAID patients and potentially precipitated by enhanced basal (37 °C) or cool-temperature-mediated activation of PLC2 in mast and other immune cells, may also fall into this category. The latter may include B cells and dendritic cells. Loss-of-function symptoms, such as antibody deficiency, recurrent sinopulmonary infection, and symptoms resembling certain forms of common variable immunodeficiency [222], detected in 75 %, 44 %, and 11 %, respectively, of PLAID patients, are more difficult to explain. In this context, it is interesting to note that 127 Discussion constant antigen receptor occupancy and signaling, including an increased basal 2+ 2+ basal concentration of intracellular Ca ([Ca ]i ) is required for the maintenance of B cell anergy, which is characterized by the persistence in the periphery of B cells unresponsive to immunogen [13, 75, 82, 314]. The latter change is thought to downmodulate the expression of surface IgM, but not IgD BCRs [314], most likely by activating ERK and 2+ NFAT pathways [18, 219], resulting a dampened [Ca ]i response to surface IgM ligation. 2+ A defect leading to impaired BCR-induced increases in [Ca ]i has been described for type Ia patients with common variable immunodeficiency (Freiburg classification) and shown to be associated with a reduction in IgD-IgM- CD27+ class-switched memory B cells, hypogammaglobulinemia, and autoimmune dysregulation [65], all of which are observed in PLAID patients. Defective Ca2+ signaling leading to reduced memory B cell subsets has also been observed in pediatric patients with CVID disorders, with B cells displaying a normal surface phenotype, but disrupted BCR-CD20 dissociation [265]. Importantly, in normal individuals there is a continuum of anergy or unresponsiveness to anti-IgM stimulation in the mature B-cell compartment [314]. In normal B cells, anergy is rapidly reversed after dissociation of self antigen, e.g. by using hapten competition, and these cells 2+ basal regain antigen responsiveness [75]. In PLAID B cells, [Ca ]i would be expected to be elevated, either chronically due to constitutively enhanced PLC2 mutant activity at 37 °C or intermittently during passage through cool body regions of the patient, causing reversible reductions of B cell responsiveness. Some of the trans-inhibitory mechanisms observed in anergic B cells, inhibiting the signaling of G-protein-coupled chemokine receptors [22], may be operative even in other immune cells, such as neutrophils, potentially contributing to the loss-of-immune-response phenotype observed in PLAID patients.
An interesting question raised by the dramatic functional consequence of the PLCG2 deletions observed in PLAID patients is whether the genomic regions encompassing exons 19 through 22 are subject to alternative processing of pre-messenger RNA [128, 278], leading to exclusion of residues encoded by exons 19 and/or 20-22 from the mature PLC2 protein. This appears as an important issue, since almost 90 % of human genes undergo alternative splicing with a minor isoform frequency of 15 % or more, with variations, intraindividually, between tissues and between individuals [278]. Pertinent to this issue, variations in position -15 from the exon 19 splice acceptor site have been detected in about one third of the examined samples and suggested to be potentially involved in altering the
128 Discussion efficiency of splicing at this site [276]. Very recently, alternative RNA splicing has been shown to be pervasive across immune system lineages [57]. Intriguingly, one of the two + changes observed for PLC2-encoding mRNA in mouse CD19 B cells is skipping of exons 20-22. The marked similarity between the genomic organization of the mouse Plcg2 and the human PLCG2 genes raises the possibility that this alteration may also exist in humans to convey cool-temperature-sensitivity to PLC2 in certain cell types and/or particular individuals. Four single nucleotide polymorphisms (SNPs) in the PLCG2 gene associated with myocardial infarction (rs4072683), stroke (rs4889428), left ventricular hypertrophy (rs1143689), and arterial diseases (rs13331678) can be found in the NCBI (National Center for Biotechnology Information) Gene data base. The SNPs were identified in the course of the Cardiovascular Health Study (CHS) [66] and the NHLBI Family Heart Study (FamHS) [84]. The SNPs associated with myocardial infarction, stroke and arterial diseases are located in the introns between exon 1 and 2, 9 and 10, and 20 and 21 of the PLCG2 gene, respectively. The impacts of these SNPs on splicing or other regulatory mechanisms which affect the transcription of the gene are unknown so far. The fourth SNP, associated with hypertrophy of the left ventricle, is located in exon 15 and represents a synonymous variant of the coding amino acid.
Cold environmental temperatures are associated with a number of human disease states, including, e.g., exercise-induced asthma and acute cardiovascular events [166, 263]. While the pathophysiology of both states is certainly complex and still incompletely understood, both are associated with cell types functionally dependent on PLC2-mediated signaling. Thus, substantial evidence suggests that exercise-induced asthma is effected by the release of mediators from mast cells and other inflammatory cells of the airways [33, 197, 244]. During quiet breathing of room air, the temperatures in the upper trachea and subsegmental bronchi were approximately 32.0 °C and 35.5 °C, respectively [167]. With increasing ventilation, these temperatures fell to approximately 29.2 °C and 32.9 °C, respectively, with normal room temperature and to 20.6 °C and 31.6 °C with cold air, respectively. Thus, under the latter conditions, even in subsegmental bronchi, temperatures are well within the range of those promoting maximal activation of the PLC2 deletion mutants studied here. Although TRPM8 has been implicated in mediating the response of asthmatic subjects to cold temperatures by induces mucin hypersecretion from bronchial epithelial cells, a much lower temperature of 18 °C has been used in these studies as a cold stimulus for extended time periods [151]. Since the 1920s, numerous studies have suggested that
129 Discussion acute coronary events are more frequent during the cold winter season [292], especially in conjunction with physical exercise, such as snow shoveling [112, 121, 184]. Although an increase in myocardial oxygen demand, due to a cold-induced increase in peripheral vascular resistance, may be causative in these situations, coronary vasoconstriction or the absence of normal coronary vasodilation during exercise could also be involved [121]. Kounis syndrome is the concurrence of acute coronary syndromes with conditions associated with mast cell activation [138]. While the syndrome is usually connected to hypersensitivity reactions, activation of mast cells by cold temperatures, either at pulmonary or extrapulmonary sites such as the skin and subcutaneous tissues may also play a role. In this context, it is important to note that the hyperactive PLC2 variant harboring the Ali5 mutation has been shown to cause platelet hyperreactivity and a prothrombotic phenotype in mice [56]. Furthermore, PLC2 has long been known to be the predominant PLC subtype, together with PLC1, in proliferating smooth muscle cells, suggesting that the enzyme may play a particular role in vascular lesions [90].
130 Summary
5 Summary
The generation of the two second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by inositol-phospholipid-specific phospholipases C (PLCs) from its substrate phosphatidylinositol 4,5-bisphosphate (PIP2) constitutes one of the main signaling responses to extracellular stimuli. The produced second messengers IP3 and DAG finally promote the release of Ca2+ from intracellular stores and the activation of protein kinase C, respectively. There are six subfamilies of mammalian PLCs, designated , , , , , and . Among these subfamilies, the PLC isozymes take a particular position because of their structural organization, which reveals a unique linker inserted between the two halves of the catalytic subdomains X and Y. This specific array (SA) contains a second, split pleckstrin homology (spPH) domain, consisting of two halves separated by two Src homology 2 (SH2) domains and one Src homology 3 (SH3) domain. The activity of the PLC isozymes is regulated by several inter- and intramolecular mechanisms, constituting a complex regulatory network. Deregulated PLC activity is associated with several severe immunological phenotypes in mice and in human, and with several types of cancer, which demonstrates the absolute necessity to understand the regulation of these important signalling molecules.
While the activation of PLC2 by Rac GTPases is fairly well understood and characterized on the molecular level, the autoinhibitory regulation and the phosphorylation-induced activation of the lipase still remain undefined. In this work, it is shown that PLC2 is autoinhibited by its specific array, and that the main elements involved in this autoinhibitory regulation are the C-terminal SH2 (SH2C) domain together with the SH3 domain. This was investigated by deleting the SH2SH2SH3 domain tandem of PLC2 resulting in a constitutively active enzyme, PLC2SH, in transfected COS-7 cells. The insertion of SH2C domain together with the SH3 domain inhibited the activity of
PLC2SH efficiently, whereas the insertion of the N-terminal SH2 (SH2N) domain and other combinations of SH domains only exhibited a minor inhibitory effect on the constitutively active deletion mutant.
131 Summary
Most interestingly, two regulatory phosphorylation sites, Y753 and Y759, are located in the identified autoinhibitory region, more precisely in the linker between the SH2C and the
SH3 domain. To investigate the impact of phosphorylation on the activity of PLC2, phosphomimetic mutants were established by replacing the appropriate tyrosine residues by glutamic acid. In the present thesis it was demonstrated that mimicking phosphorylation of PLC2 is an appropriate tool to study the effect of phosphorylation on the activity of the lipase. Furthermore, it was demonstrated that all four regulatory phosphorylation sites known in PLC2 are essential for the activation of the enzyme. Most interestingly, mimicking phosphorylation on Y733, which is a novel PLC2 phosphorylation site identified in non-small cell lung cancer (NSCLC) had the most striking effect on the activity of the lipase pointing to a role of PLC2 in the tumorigenesis of NSCLC and prompting the question to the kinase phosphorylating PLC2 on Y733. On the basis of an in silico analysis anaplastic lymphoma kinase (ALK) was predicted to phosphorylate Y733. However, the results of this study show that Y733 is no or not the only site targeted by ALK. Nonetheless, it was clearly demonstrated that ALK activates and associates with
PLC1 as well as with PLC2 in transfected COS-7 cells and that this activation is due to a phosphorylation of the lipases on a so far unknown tyrosine residue. Furthermore, it was shown that NPM-ALK, an ALK fusion protein, activates and binds to PLC1 but not to
PLC2 and that the activation as well as the association of both molecules depends on the kinase activity of NPM-ALK.
Quite recently, deletions of exon 19 and exon 20-22 in the PLCG2 gene were shown to cause a novel disease pattern, designated PLAID (phospholipase C-2-associated antibody deficiency and immune dysregulation) and characterized by immunodeficiency, autoimmunity and cold urticaria. In this work it was demonstrated that the two PLC2 deletion mutants, PLC219 and PLC220-22, which were shown to be associated with PLAID, are specifically activated by cool temperatures. This was investigated by incubation of transfected COS-7 cells at different temperatures and the subsequently measurement of inositol phosphate formation. The activation of the PLAID mutants by cool temperatures has been shown to be a reversible process that competes with the Rac2- mediated activation of the lipase. Furthermore, the mechanism underlying the described sensitivity of the mutants to cool temperatures has been shown to be dependent on the integrity of the spPH domain which is distinct from the mechanism by which autoinhibition of PLC2 is regulated. 132 References
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164 Acknowledgements
Acknowledgements
Though only my name appears on the cover of this dissertation, a great many people have contributed to its production. I owe my gratitude to all those people who have made this dissertation possible.
Foremost, I would like to express my sincerest gratitude to my supervisor and promoter Prof. Dr. Peter Gierschik, for his constant guidance, support, motivation and untiring help during the course of my PhD.
I also appreciate my advisory committee, Prof. Dr. Ralf Marienfeld and Prof. Dr. Reinhard Wetzker for all their guidance and valuable comments on my research, especially through my intermediate examinations.
Thank you also to PD Dr. Hans Kestler and Johann M. Kraus for doing the cluster analysis.
I owe particular thanks to Dr. Claudia Walliser who always supported me in any possible way. With her in-depth expertise in the scientific field she always provided me efficient assistance and support in experimental as well as in theoretical aspects. Thank you Claudia!
In my daily work I have been blessed with a friendly and cheerful group of colleagues. Thanks for the pleasant working atmosphere go to Norbert Zanker, Patricia Schilling, Zinna Rasonabe, Angela Mansard, Torben Langer, Jennifer Haas, Maren Lillich, Dr. Barbara Möpps, Dr. Carolin König, Dr. Petra Vatter, Carolin Weiss, Susanne Gierschik, Vincenzina Palmieri and all the other members of the institute.
Special thanks go to Norbert, who always provided me with valuable information and input for discussions beyond the scientific field ;)
A special thank also goes to my brother Armin who developed and built up the individual incubation chambers. Without his technical support an outstanding and important part of this thesis would not have been possible.
At the end, I want to say that this dissertation would not be possible with the great support of my parents, my sister Carina and my brother Armin.
One of the most important persons in my life, Christoph, supported and encouraged me all the time. Thank you!!!
165 Curriculum Vitae
Curriculum Vitae
Personal Name Anja Jasmin Bühler Date of Birth June 10th 1984 Place of Birth Ulm Nationality German
Education since August 2009 Ph. D. thesis under the supervision of Prof. Dr. Peter Gierschik
October 2007 - Master of Science (M.Sc.) program in biochemistry at Ulm May 2009 University; grade: 1.4; Master Thesis at the Institute of Pharmacology and Toxicology under the supervision of Prof. Dr. Peter Gierschik: “Molecular mechanism of autoinhibitory regulation
of phosphoinositide-specific phospholipase C-γ2” (grade 1.0).
October 2004- Bachelor of Science (B.Sc.) program in biochemistry at Ulm October 2007 University; grade: 2.2; Bachelor Thesis at the Institute of Pharmacology and Toxicology under the supervision of Prof. Dr.
Peter Gierschik: “Analysis of the regulatory PHN-SH2-SH2-SH3-
PHC-Domain of phosphoinositide-specific phospholipase C-γ2” (grade 1.0).
2001-2004 Abitur Ernährungswissenschaftliches Gymnasium Valckenburgschule Ulm
1995-2001 Mittlere Reife Ulrich von Ensingen Realschule Ulm
Languages English Good in writing, reading and speaking Spanish Basic knowledge
166 Publications
Publications
Everett, K.L., Buehler, A., Bunney, T.D., Margineanu, A., Baxendale, R.W., Vatter, P., Retlich, M., Walliser, C., Manning, H.B., Neil, M.A.A., Dunsby, C., French, P.M.W., Gierschik, P., and Katan, M. (2011) Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase C-2. Molecular and Cellular Biology. 31; 6: 1240-1251
Gierschik, P., Buehler, A., Walliser, C. (2012) Activated PLCγ breaking loose. Structure. 5, 20: 1989-1990
Buehler, A., Walliser, C., Haas, J., Kraus, J.M., Filingeri, D., Havenith G., Kestler, H.A., Milner, J.D., and Gierschik, P. (2013) Cool-temperature-mediated activation of human hereditary PLAID variant phospholipase C-2. submitted for publication
167 Appendix
Appendix
Circuit diagram of individual incubation chambers
168 Statutory Declaration
Statutory Declaration
I hereby declare that I wrote the present dissertation with the topic:
Molecular mechanisms regulating phospholipase C-2 activity independently and used no other aids than those cited. In each individual case, I have clearly identified the source of the passages that are taken word for word or paraphrased from other works.
I also hereby declare that I have carried out my scientific work according to the principles of good scientific practice in accordance with the current „Satzung der Universität Ulm zur Sicherung guter wissenschaftlicher Praxis“ [Rules of the University of Ulm for Assuring Good Scientific Practice].
Ulm, 12.12.13
______Anja Bühler
169