Identification of the Rac Gtpase Activating Protein Rho GAP 22 As a New Akt Substrate Defines a Novel Mechanism for Insulin Regulation of Cell Motility

Identification of the Rac GTPase Activating Protein Rho GAP 22 as a New Akt Substrate Defines a Novel Mechanism for Insulin Regulation of Cell Motility

Alexander Francis Rowland

Submitted for the degree of Doctor of Philosophy

Garvan Institute of Medical Research

Faculty of Medicine

University of New South Wales

ABSTRACT

Insulin exerts many of its metabolic actions via the canonical PI3K/Akt pathway leading to phosphorylation and 14-3-3 binding of key metabolic targets. Recognising this, the goal of my thesis was to identify novel actions of insulin. To accomplish this I have conducted a screen to identify insulin-responsive 14-3-3 binding phosphoproteins and in so doing have identified a GTPase Activating Protein (GAP) for Rac1 called Rho GAP 22. Insulin increased 14-3-3 binding to Rho GAP 22 by 4 fold and this effect was PI3Kinase and Akt dependent. Using semi-quantitative mass spectrometry we identified two putative 14-3-3 binding sites (Ser16 and Ser397) within Rho GAP 22 and insulin increased phosphorylation at both of these sites. Mutagenesis studies revealed a complex interplay in phosphorylation at these two sites. Mutating Ser16 to Ala almost completely abolished 14-3-3 binding to Rho GAP 22 in vivo but not in vitro. Phosphorylation of Ser16 both in vitro and in vivo was Akt-dependent and inhibition of Akt prevented 14-3-3 binding to Ser16 in vitro. A truncated Rho GAP 22 protein lacking the N-terminal PH domain and Ser16 interacted with 14-3-3 in vivo and this interaction was Akt dependent. The Ser397 site in Rho GAP 22 is located close to the GAP domain and so it is likely that phosphorylation at this site regulates GAP activity. Hence, this may serve as a novel mechanism by which Akt regulates Rac1 GTP Loading and activity. I found that over-expression of a mutant Rho GAP 22, which is unable to bind 14-3-3, reduced cell motility in NIH 3T3 cells. This defect was not observed in cells overexpressing mutant Rho GAP 22 which is unable to bind 14-3-3 and is catalytically inactive. Thus, I propose that insulin and possibly other growth factors may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of Rho GAP 22 leading to modulation of Rac1 activity.

ii TABLE OF CONTENTS

ABSTRACT ...... ii TABLE OF CONTENTS ...... iii ACKNOWLEDGEMENTS ...... vi CONFERENCE PRESENTATIONS ...... vii LIST OF FIGURES ...... viii LIST OF TABLES ...... ix LIST OF ABBREVIATIONS ...... x CHAPTER 1 General Introduction ...... 1 Insulin and Glucose Homeostasis...... 1 The Insulin Signalling Pathway ...... 1 The Protein Kinase Akt ...... 4 The Regulation of Akt...... 8 Activation of Akt ...... 9 Negative Regulators of Akt ...... 14 Akt is a Major Regulator of Cellular Metabolism ...... 15 Glucose Uptake ...... 16 Glucose Disposal ...... 17 Protein Synthesis ...... 18 Other Functions of Akt ...... 19 Akt in Cell Growth and Survival...... 19 Akt and the Regulation of Cell Proliferation ...... 19 Akt and the Regulation of Cardiac Function ...... 20 Akt and Cell Migration ...... 21 The Akt phosphorylation motif ...... 22 Akt Signals Through 14-3-3 ...... 23 14-3-3 Biology ...... 23 History of 14-3-3 ...... 24 The 14-3-3 Protein Family ...... 25 Structure of 14-3-3 ...... 26 Regulation of 14-3-3 ...... 30 Functions of 14-3-3 ...... 31 The “14-3-3 Interactome” ...... 34 Using 14-3-3 as a Tool...... 35 Aims...... 36 CHAPTER 2 Materials and Methods...... 37 Materials ...... 37 Plasmids and Constructs ...... 38 Cell Lines ...... 38 Methods ...... 39 Cell Culture ...... 39 Production of Retrovirus Using Plat-E cells ...... 39 Retroviral Infection of NIH3T3 Fibroblasts and Generation of Stable Cell Lines ...... 39 Transient Transfection of CHO IR/IRS-1 Cells Using Lipofectamine LTX ...... 40 SILAC Labelling of L6 Cells ...... 40 Production of GST Fusion Proteins ...... 41 Production of 6xHis Fusion Proteins ...... 42 Coupling GST or 6xHis Fusion Proteins to CNBR Sepharose ...... 42 Production of Rho GAP 22 Antibody ...... 42 Production of the pSer22 and pSer397 Rho GAP 22 Antibodies ...... 43 iii 14-3-3 Pulldown from L6 Myotubes or Mouse Quadriceps or MEF cells ...... 43 Immunoprecipitation (non FLAG) ...... 44 Immunoprecipitation Using FLAG Antibody ...... 45 Western Blotting and SDS-PAGE ...... 45 Peptide Competition Assays ...... 46 In-gel Tryptic Digest for Peptide Identification by LC MS/MS...... 46 Peptide Identification by LC MS/MS ...... 47 In-vitro Phosphorylation using Recombinant Akt...... 48 Cell Motility Assays ...... 48 CHAPTER 3 Identifying Novel Akt Substrates Using 14-3-3 Affinity Chromatography and SILAC...... 50 Introduction...... 50 Isotope Labelling and Quantitative Mass Spectrometry...... 51 Combining SILAC and 14-3-3 ...... 55 Results ...... 57 Akt Substrates Identified in this Study...... 57 Bioinformatic Analysis of Novel Akt Substrates ...... 60 Novel Proteins Identified in this Study Bind 14-3-3 in vivo ...... 61 Discussion ...... 64 CHAPTER 4 Rho GAP 22 is an Insulin-Responsive 14-3-3 Binding Protein ...... 67 Introduction...... 67 Rho GAP 22 in the Literature ...... 67 The Rho GAP Family of Proteins ...... 69 Domain Organisation of Rho GAP 22 ...... 72 Results ...... 74 Rho GAP 22 Binds 14-3-3 in an Insulin-Responsive Manner ...... 74 Production of a Rho GAP 22 Antibody ...... 76 Endogenous Rho GAP 22 Binds 14-3-3 in an Insulin-Responsive Manner ...... 78 Rho GAP 22 Binds 14-3-3 in Isolated Mouse Quadriceps ...... 80 Full-length Rho GAP 22 Binds 14-3-3 Directly ...... 80 Discussion...... 84 CHAPTER 5 Mapping the 14-3-3 Binding Site and its Kinase ...... 87 Introduction...... 87 Results ...... 89 Phosphopeptide Identification using Semi-Quantitative Mass Spectrometry ...... 89 14-3-3 Binds Rho GAP 22 at Ser16 and Ser395 ...... 92 Production of Phospho-Rho GAP 22 Antibodies ...... 94 Insulin Treatment Causes Phosphorylation at Ser16 and Ser395 ...... 98 Phosphorylation at Ser16 and Ser395 is Reduced by Inhibition of Akt ...... 100 Inhibition of Akt Reduces 14-3-3 Binding to Endogenous Rho GAP 22 ...... 102 Akt is Capable of Phosphorylating Rho GAP 22 in vitro ...... 104 Akt is Necessary for Insulin-Stimulated 14-3-3 Binding to Rho GAP 22 ...... 108 Discussion ...... 110 Phosphorylation of Rho GAP 22 by Akt ...... 110 The Gatekeeper Hypothesis ...... 113 Chapter 6 A Functional Role for the Rho GAP 22/ 14-3-3 Interaction ...... 116 Introduction...... 116 Functions of 14-3-3 ...... 116 Rho GTPase Biology...... 117 iv Rho GTPases and the Regulation of the Cytoskeleton in Cell Migration ...... 121 Rho GTPases and Cell Adhesion ...... 125 Results ...... 127 Generation of Polyclonal Cell Lines ...... 127 Cell Migration Studies ...... 130 Discussion ...... 132 Chapter 7 General Discussion ...... 134 Akt and 14-3-3 ...... 134 Novel Roles for Insulin Identified in this Study ...... 135 The Novel Akt/14-3-3 Substrate Rho GAP 22...... 137 The role of the PH domain of Rho GAP 22 ...... 139 Interaction of Rho GAP 22 with Other Proteins ...... 139 The Specificity of Rho GAP 22 for Rac...... 140 Rho GAP 22 and Other Functions of Rac...... 140 The Role of Rho GAP 22 in Insulin Signalling ...... 141 Concluding Remarks...... 141 REFERENCES ...... 143

v ACKNOWLEDGEMENTS

There are a number of people who I would to thank for their help and support throughout the life of my PhD. First and foremost, I owe a great deal to my supervisor, Prof. David James. His unwavering support and boundless passion for science, not to mention his insatiable hunger for data, provide the ideal environment for a budding scientist. He has provided unlimited help and assistance throughout the tenure of my PhD, and was always able to lend a willing ear.

A number of people have provided invaluable technical assistance in this course of this project. Dr Mark Larance was instrumental in setting up the SILAC experiments, and in helping me to get started on this project, mainly by answering far too many questions. Dr Will Hughes taught me valuable microscopy skills, was instrumental in establishing the cell motility assays and was always willing and able to answer even the most naïve questions. Dr James Burchfield suffered mightily as he taught me the ins and outs of the IMARIS software system. Dr Katarina Mele helped with the analysis of the cell motility data. Drs Jacqueline Stöckli, Georg Ramm and Kyle Hoehn were always happy to help with any and all technical questions I had.

I would also like to thank the members of the James lab, past and present, for their friendship and assistance during my PhD. It has been a pleasure to work with you all, and I consider all of you friends, as well as colleagues. I have never worked with a better group of people. A special thankyou goes out to Dr Jamie Lopez, who showed me the ropes when I was fresh out of undergrad.

I would also like to thank the other members of the Garvan community who have been my friends as well as colleagues. We’ve had a lot of fun in the past few years, and I suspect it’s the only thing that’s kept me sane. Thank you.

vi CONFERENCE PRESENTATIONS

Queenstown Molecular Biology Meeting 2nd-4th September 2008, Queenstown, NZ Poster Presentation: Characterisation of the novel 14-3-3 binding protein ARHGAP22

69th American Diabetes Association Meeting 5th-9th June 2009, New Orleans, Louisiana, USA Oral Presentation: Identification of a novel insulin regulated pathway involving phosphorylation of and 14-3-3 binding to a Rac1 GAP, ARHGAP22

10th Hunter Cellular Biology Meeting 16th-19th March 2010, Pokolbin, NSW Poster Presentation: Identification of a novel insulin regulated pathway involving phosphorylation of and 14-3-3 binding to a Rac1 GAP, Rho GAP 22

vii LIST OF FIGURES

Figure 1-1: Akt plays a central role in the insulin signalling pathway...... 3 Figure 1-2: A model for the structural basis of Akt regulation...... 13 Figure 1-3: The Structure of 14-3-3...... 29 Figure 1-4: Functional Outcomes of 14-3-3 Binding...... 33 Figure 3-1: A sample SILAC spectra showing the isotope-induced mass difference of the same peptide from three different cell populations...... 54 Figure 3-2: Summary of Experimental Procedure for the SILAC screen for novel 14-3-3 binding proteins...... 56 Figure 3-3: Confirmation that proteins identified by SILAC are true insulin - responsive 14-3-3 binding proteins...... 63 Figure 4-1: Phylogenetic structure of the Rho GAP family of proteins...... 71 Figure 4-2: Bioinformatic analysis of Rho GAP 22 ...... 73 Figure 4-3: Rho GAP 22 binds 14-3-3 in response to insulin stimulus ...... 75 Figure 4-4: Characterisation of the Rho GAP 22-specific antibody and tissue distribution of Rho GAP 22...... 77 Figure 4-5: Endogenous Rho GAP 22 binds 14-3-3 in response to insulin treatment. ... 79 Figure 4-6: Rho GAP 22 binds 14-3-3 in isolated mouse quadriceps...... 82 Figure 4-7: Full-length Rho GAP 22 binds 14-3-3...... 83 Figure 5-1: Insulin stimulation increases phosphorylation at Ser16, Ser359 and Ser395. ... 91 Figure 5-2: Insulin stimulation increases 14-3-3 binding to Ser16 and Ser395...... 93 Figure 5-3: Both phospho-specific Ser22 and Ser397 antibodies are specific for their cognate sites in Rho GAP 22...... 96 Figure 5-4: The phospho-specific Rho GAP 22 antibodies bind only their cognate phosphopeptide...... 97 Figure 5-5: PDGF stimulation in NIH3T3 fibroblasts causes phosphorylation at Ser16 and Ser395...... 99 Figure 5-6: Insulin-stimulated phosphorylation of Ser16 and Ser395 is inhibited by Wortmannin and Akti...... 101 Figure 5-7: Insulin-stimulated 14-3-3 binding to endogenous Rho GAP 22 is inhibited by Wortmannin and Akti...... 103 Figure 5-8: Recombinant Akt2 can phosphorylate Ser16 of Rho GAP 22 in vitro...... 105 Figure 5-9: Wortmannin and Akti inhibit insulin-stimulated 14-3-3 binding to myc- p68RacGAP...... 107 Figure 5-10: Akt is required for insulin-stimulated 14-3-3 binding to Rho GAP 22. ... 109 Figure 5-11: The regulation of dual 14-3-3 binding to Rho GAP 22 may be under conformational control...... 112 Figure 6-1: Regulation of small GTPases such as Rac1...... 120 Figure 6-2: Over-expression of various Rho GAP 22 mutants in NIH3T3 cells...... 129 Figure 6-3: Inhibition of 14-3-3 binding to Rho GAP 22 causes a defect in cell motility...... 131

viii LIST OF TABLES

Table 1-1: Known Akt Phosphorylation sites in the Human Proteome...... 7 Table 3-1: Insulin-responsive proteins identified by SILAC and 14-3-3 affinity chromatography...... 59 Table 3-2: Bioinformatic analysis of novel 14-3-3 binding proteins identified in this study...... 61 Table 5-1: Sequences of predicted 14-3-3 binding sites in Rho GAP 22 compared to known 14-3-3 binding motifs...... 87

ix LIST OF ABBREVIATIONS

ADP adenosine 5-diphosphate ATP adenosine 5-triphosphate 4E-BP1 4E-binding protein 1 AANAT arylalkylamine N-acetyltransferase Abl Abelson murine leukaemia viral oncogene homolog 1 AGC protein kinase A/cGMP-dependent protein kinase/protein kinase C Arp2/3 actin related protein 2/3 AS160 Akt substrate of 160 kDa BAD Bcl-2-associated death promoter BCA bicinchoninic acid Bcr breakpoint cluster region protein CamKII calcium/calmodulin-dependent protein kinase II cAMP cyclic AMP Cbl casitas B-lineage lymphoma cDNA complementary deoxyribonucleic acid CHO Chinese hamster ovary CNBR Cyanogen Bromide C-terminus carboxyl terminus DEAE Diethylaminoethyl cellulose DMEM Dulbecco's modified Eagle's medium DNA deoxyribonucleic acid EDTA ethylenediaminetetra-acetic acid eNOS endothelial nitric oxide synthase ERK extracellular signal–related kinase FACS Fluorescence-activated cell sorting FCS fetal calf serum FOXO Forkhead box O FRET Förster resonance energy transfer g unit of gravity GAP GTPase activating protein GDP guanine diphosphate x GEF guanylnucleotide exchange factor GFP green fluorescent protein GLUT glucose transporter GS glycogen synthase GSK glycogen synthase kinase GST glutathione-S-transferase GSV GLUT4 storage vesicles GTP guanine triphosphate h hour HEK human embryonic kidney His histidine HM hydrophobic motif IGF-1 Insulin-like growth factor 1 IP immunoprecipitation IPTG isopropyl-b-D-thiogalactosidase IR insulin receptor IRS insulin receptor substrate JNK c-Jun N-terminal kinase kDa kilodalton m milli M molar MAPK mitogen-activated protein kinase MEF mouse embryonic fibroblast min minute mRNA messenger RNA mTOR mammalian target of rapamycin NFAT nuclear factor of activated T cells N-terminus amino terminus N-WASP neural Wiskott-Aldrich syndrome protein PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PDGF platelet derived growth factor

xi PDK1 3-phosphoinositide dependent protein kinase-1 PH pleckstrin homology PHLLP PH domain leucine-rich repeat protein phosphatase PI(3,4)P2 phosphatidylinositol 3,4 bisphosphate PI(3,4,5)P3 phosphatidylinositol 3,4,5 triphosphate PI3K phosphatidylinositide 3-kinase PIF PKC-related kinase-2 -interacting fragment PIKfyve phosphoinositide kinase for five position containing a fyve finger PKA protein kinase A PKB protein kinase B PKC protein kinase C PlatE Platinum-E PM plasma membrane PP2A protein phosphatase 2A PRAS40 proline-rich Akt substrate of 40 kDa PSG penicillin/streptomycin/L-glutamine PTEN phosphatase and tensin homolog deleted on chromosome ten PVDF polyvinylidene difluoride RAC related to A and C kinases Rheb Ras homolog enriched in brain RNA ribonucleic acid s second SDS sodium dodecyl sulphate SHIP2 SH2-containing Inositol phosphatase SILAC Stable Isotope Labelling of Amino acids in Cell culture SMG7 suppressor with morpho-genetic effects on genitalia protein SNAP soluble NSF attachment protein SNARE soluble NSF attachment protein receptor TBS tris-buffered saline TCEP Triscarboxyethylphosphine TPR tetratricopeptide repeat TSC tuberous sclerosis complex v/v volume per volume

xii VAMP vesicle associated membrane protein w/v weight per volume micro

xiii CHAPTER 1

General Introduction

Insulin and Glucose Homeostasis.

The primary fuel source for mammalian cells is glucose. Mammals are subject to wide swings in glucose uptake, dependent on the interval between meals and the foodstuffs ingested. Despite this, blood glucose concentrations are maintained within a narrow range at all times. Excursions in blood glucose levels outside of this physiologic range have dire consequences, ranging from insidious damage to the microvasculature that eventually results in kidney disease and blindness, to coma and death if there is insufficient blood glucose to maintain normal brain functionality. Mammals have evolved an exquisite mechanism for maintaining stable blood glucose concentration. In the fasting state, blood glucose is maintained either via hepatic glycogenolysis, where liver glycogen is broken down to glucose, or via gluconeogenesis, where 3C intermediates such as lactate are converted back to glucose in the liver. After a meal, the peptide hormone insulin is secreted from cells, located in the islets of Langerhans in the pancreas. Insulin inhibits glucose output from the liver and it promotes the uptake of glucose from the blood stream into skeletal muscle and adipose tissue (Shepherd and Kahn 1999).

The Insulin Signalling Pathway

The main mechanism by which insulin stimulates the uptake of glucose from the blood stream is via the insulin-regulated protein, GLUT4. GLUT4 is a member of the glucose transporter family of proteins, the 13 members of which are 12-transmembrane spanning proteins that facilitate the transport of glucose from the extracellular space to the cytosol in an energy-independent manner (Joost, Bell et al. 2002). Disruption of the GLUT4 gene in knockout mice either in a whole body context (Rossetti, Stenbit et al. 1997; Stenbit, Tsao et al. 1997), in skeletal muscle (Zisman, Peroni et al. 2000), or in adipose tissue (Abel, Peroni et al. 2001) results in insulin resistance and glucose 1 intolerance. Conversely, overexpression of GLUT4 in skeletal muscle or adipose tissue decreases blood glucose levels and confers increased insulin sensitivity (Liu, Gibbs et al. 1993; Ikemoto, Thompson et al. 1995). These studies underscore the central role GLUT4 plays in whole-body glucose metabolism. In the basal state, GLUT4 is excluded from the cell surface and is thought to reside in a specialised storage compartment known as GLUT4 storage vesicles (GSVs) as well as in the endosomal system and the trans-Golgi network (Bryant, Govers et al. 2002). Insulin stimulation results in a redistribution of GLUT4 from the intracellular storage compartment to the PM (Slot, Geuze et al. 1991). However, the signalling pathway linking insulin binding to its receptor with GLUT4 exocytosis has not been completely elucidated. In insulin-responsive tissues such as skeletal muscle and adipose tissue, insulin signalling is initiated following binding of insulin to the -subunits of the insulin receptor on the cell surface. This results in the autophosphorylation of the -subunits and activation of the receptor’s intrinsic tyrosine kinase activity (Gammeltoft and Van Obberghen 1986). This tyrosine kinase activity phosphorylates insulin-receptor substrate (IRS) proteins and c-Cbl. Phosphorylated IRS proteins recruit phosphatidylinositol 3-kinase (PI3K) through the p85 regulatory subunit leading to the activation of the catalytic subunit p110. PI3K catalyses the formation of phosphatidylinositol (3,4,5)P3 from phosphatidylinositol (4,5)P2 on the cytosolic leaflet of the plasma membrane. Phosphatidylinositol (3,4,5)P3 acts as a docking site for a kinase known as Akt {Kanzaki, 2006 #233}{Franke, 1995 #245}. The recruitment of Akt to the plasma membrane and its subsequent activation is a major node in the insulin signalling pathway. Active Akt sets in motion the diverse functions of the insulin signalling pathway (Fig.1-1).

Figure 1-1: Akt plays a central role in the insulin signalling pathway. The insulin signalling cascade results in the activation of the protein kinase Akt, which transduces many of insulin’s physiological effects. Phosphorylation of many targets of the insulin signalling pathway by Akt results in binding of 14-3-3, suggesting that 14-3-3 translates Akt phosphorylation into a functional outcome.

3 The Protein Kinase Akt

The protein kinase Akt, also known as Protein Kinase B (PKB) is a member of the AGC (cAMP-dependent, cGMP-dependent and protein kinase C) family of serine/threonine dependent protein kinases. Kinases in this family display a large amount of sequence homology within their kinase domains, although they generally show little homology outside of this domain. This lack of sequence homology is thought to account for their varied regulation. Three groups independently identified Akt in 1991. The first group cloned c-Akt, the cellular homolog of the transforming oncogene v-Akt in the mouse leukaemia virus AKT8 (Staal 1987; Bellacosa, Testa et al. 1991). Two other groups cloned Akt based on homology of its kinase domain to protein kinase A and protein kinase C (Coffer and Woodgett 1991; Jones, Jakubowicz et al. 1991). The first protein identified as an Akt substrate was GSK-3 (Cross, Alessi et al. 1995). Since then, well over 100 substrates have been reported. Indeed, a search of the online database PhosphoSitePlus (Hornbeck, Chabra et al. 2004) reveals 111 Akt substrates in humans, with 142 unique phosphorylation sites (Table 1). Akt is now recognised as a central signalling node in the control of intracellular signalling and metabolism.

4 UNIPROT Phosphorylated Protein Name Accession Residue P63104 14-3-3 S58 Q9UKV3 Acinus S1180 P07550 ADRB2 S346 P31749 Akt1 T72, S246 P10275 AR S213, S791 P53365 Arfaptin 2 S260 O60343 AS160 T642 Q59GL6 ASK1 S163 P54253 Ataxin-1 S775 Q92934 Bad S99 O95999 Bcl-10 S218, S231 O43521 Bim S87 P15056 B-Raf S365, S429 P38398 BRCA1 T509, S694 Q07352 BRF1 S92, S203 Q9Y2V2 CaRHSP1 S52 P55211 Casp9 S196 P78371 CCT2 S259 P24941 CDK2 T39 Q15027 CENTB1 S554 Q99490-2 CENTG1 isoform 2 S629 O15519 CFLAR S273 O14757 Chk1 S280 P41279 Cot S400 P35222 CTNNB1 S552 Q92879 CUGBP1 S28 Q96F86 EDC3 S161 P21453 EDG-1 T236 P29474 eNOS S614, S1176 P03372 ER-alpha S167 Q15910 EZH2 S21 P15311 Ezrin T567 O15360 FANCA S1149 Q8NCD3 FLEG1 S486 Q14315 FLNC S2233 Q9Y261 FOXA2 T156 P55316 FOXG1 T279 Q12778 FOXO1A T24, S256, S319 O43524 FOXO3A T32, S253 P98177 FOXO4 T32, S197, S262 Q9UQC2 Gab2 S159 P15976 GATA-1 S310

5 UNIPROT Phosphorylated Protein Name Accession Residue P23769 GATA2 S401 Q3V6T2 Girdin S1417 P49840 GSK3A S21 P49841 GSK3B S9 P33778 H2B S36 P09601 HMOX1 S188 P09651 hnRNP A1 S199 P04792 HSP27 S82 O43464 HtrA2 S212 P42858 Huntingtin S421 O15111 IKK-alpha T23 P51617 IRAK1 T100 P35568 IRS-1 S629 Q92945 KHSRP S193 Q15BH2 Kv11.1 isoform 5 T897 Q05195 Mad1 S145 Q00987 MDM2 S166, S186 O15151 MDM4 S367 Q9UBP6 METTL1 S27 Q16584 MLK3 S674 Q13043 MST1 T120 P42345 mTOR T2446, S2448 Q9UN36 NDRG2 S332, T348 Q7Z5N3 NEDD4L S342, S428 Q12906 NFAT90 S647 S648, S703, P19634 NHE1 S796 O60285 NuaK1 S600 P22736 Nur77 S351 P38936 p21Cip1 T144, S145 S10, T157, P46527 p27Kip1 T198 Q09472 p300 S1834 P14598 p47phox S304, S328 Q53EL6 PDCD4 S67, S457 Q15121 PEA-15 S116 O60825 PFKFB2 S466, S483 Q16875 PFKFB3 S461 Q9Y3M2 PGEA1 S20 Q9Y2I7 PIP5K S307 P19174 PLCG1 S1248 P62136 PPP1CA T320 Q96B36 PRAS40 T246

6 UNIPROT Phosphorylated Protein Name Accession Residue Q96S44 PRPK S250 P18031 PTP1B S50 P63000 RAC1 S71 Q9H6Z4 RANBP3 S126 P10276 RARA S96 Q9H4X1-2 RGC32 isoform 2 S45 Q9Y3C5 RNF11 T135 Q04912 Ron S1394 P45985 SEK1 S80 Q7Z6J0 SH3RF1 S304 P12755 SKI T458 P78362 SRPK2 T492 Q8IYJ3 SYTL1 S241 P17542 TAL-1 T90 Q86TI0 TBC1D1 S237, T596 O14746 TERT S227, S824 Q86V81 THOC4 S33, T218 Q92547 TOPBP1 S1159 P54274 TRF1 T273 P53804 TTC3 S378 S939, S981, P49815 Tuberin T1462 S351, S745, P55072 VCP S747 P30291 Wee1 S642 Q9H4A3 WNK1 T60 P98170 XIAP S87 P46937 YAP1 S127 P67809 YB-1 S102 Q15942 Zyxin S142

Table 1-1: Known Akt Phosphorylation sites in the Human Proteome. The online database PhosphoSitePlus was used to list all known Akt substrates, and the residues that are phosphorylated, in humans. PhosphoSitePlus collates curated information from both high and low-throughput sources (www.phosphosite.org).

7 There are three isoforms of Akt in mammals; each shares the same domain organisation and are encoded by separate genes sharing greater than 85% homology (Kandel and Hay 1999). Akt1 is ubiquitously expressed, while expression of Akt2 is highest insulin- responsive tissues such as heart, liver, kidney and skeletal muscle (Altomare, Guo et al. 1995). The expression of Akt3 is limited to the brain and testes (Brodbeck, Cron et al. 1999). The varied expression profile of the different isoforms raised the possibility that each isoform plays a specific role. Several studies in mice knocked out for a single Akt gene have supported this hypothesis. Deletion of the Akt1 gene results in growth retardation, defects in adipogenesis and increased neonatal mortality, but has no effect on glucose metabolism (Chen, Xu et al. 2001; Cho, Thorvaldsen et al. 2001). Conversely, deletion of Akt2 results in a specific defect in glucose metabolism{Cho, 2001 #420}{Bae, 2003 #9}. Deletion of Akt3 results in retardation of normal brain development but has no effect on glucose metabolism (Easton, Cho et al. 2005; Tschopp, Yang et al. 2005). Although these studies suggest distinct roles for the various Akt isoforms, evidence has emerged that there is a degree of redundancy in the system. The most obvious argument in favour of this is that single Akt gene deletions are viable. It is interesting however, that simultaneous deletion of two isoforms is lethal. Akt1/2 double knockouts die shortly after birth and have various developmental defects (Peng, Xu et al. 2003). MEF cells derived from these mice are viable and significant apoptosis is only induced after 80% knockdown of the remaining Akt3 by siRNA, indicating that only small amounts of Akt are required for cell survival (Liu, Shi et al. 2006). Double knockout of Akt1/3 confers embryonic lethality, but interestingly this is avoided by expression of Akt1 from a single locus, once again indicating that small amounts of Akt are sufficient (Yang, Tschopp et al. 2005). The lethal consequence of knocking out two Akt isoforms indicates that there is substantial overlap in function between these isoforms, which is not shared with the remaining isoform. The various isoforms of Akt are thus best described as having distinct roles in cellular metabolism, but there is a degree of redundancy built into the system.

The Regulation of Akt.

The regulation of Akt’s activity is closely related to its structure. Full activation of the kinase activity requires multiple post-translational modifications that induce

8 conformational changes in Akt and activate its kinase activity. Akt possesses an N- terminal PH domain, which is important in mediating its localisation to the plasma membrane via its interaction with phospholipids, followed by a short linker region, which connects the PH domain to the catalytic domain. C-terminal to the kinase domain is a hydrophobic motif (HM) that is required for activation of the kinase activity of Akt {Alessi, 1996 #421}(Yang, Cron et al. 2002). Activation of Akt requires phosphorylation at Ser473 and Thr308; these residues are conserved in all three isoforms (Alessi, James et al. 1997).

Activation of Akt

Activation of the kinase activity of Akt requires three major steps. Firstly, it must be recruited to the plasma membrane via its PH domain, a process that is dependent on the presence of the phospholipids generated by the activity of PI3K. The PH domain of Akt binds both PI(3,4)P2 and PI(3,4,5)P3, with some studies suggesting a preference for PI(3,4,5)P3 (Franke, Yang et al. 1995; Stokoe, Stephens et al. 1997). Mutations in the PH domain that reduce phospholipid binding inhibit Akt activation (Bellacosa, Chan et al. 1998), whereas mutants that increase phospholipid binding results in constitutive Akt activation (Franke, Yang et al. 1995). The relocalisation of Akt to the plasma membrane from the cytosol is crucial for its activity; Akt mutants that are constitutively targeted to the plasma membrane are constitutively active (Kohn, Summers et al. 1996). Binding of PI(3,4,5)P to the PH domain induces a conformational change in Akt, which is thought to be a necessary step prior to its activation by PDK-1 (Thomas, Deak et al. 2002; Milburn, Deak et al. 2003; Calleja, Alcor et al. 2007). The second and third activation steps both require phosphorylation of Akt, a mechanism of protein regulation common to almost all kinases and first described in 1959 when muscle phosphorylase b kinase was first identified as a phosphoprotein (Krebs, Graves et al. 1959). Akt 1 was discovered to be phosphorylated in vivo at four sites (Ser124, Thr308, Thr450 and Ser473) (Alessi, Andjelkovic et al. 1996). The phosphorylation of the Ser124 and Thr450 residues is thought to be an important priming step, which allows subsequent activation; these residues are phosphorylated independently of growth factor stimulation (Bellacosa, Chan et al. 1998). Activation of the kinase activity of Akt

9 requires phosphorylation of the Ser473 and Thr308 residues, which is dependent on growth factor stimulation (Alessi, Andjelkovic et al. 1996). Mutation of these sites to alanine (T308A/S473A) inhibits the activation of Akt and thus its activity. In the reciprocal experiment, the phospho-mimetic mutation T308D/S473D causes constitutive Akt activity (Bellacosa, Chan et al. 1998). This method of activation by phosphorylation is common to all AGC family protein kinases. There are several conserved phosphorylation sites in AGC kinases that regulate their function. The first site is located in an activation loop in the centre of the kinase domain, analogous to Thr308 in Akt. Phosphorylation of this activation loop is thought to occur via one of two main processes. The first, autophosphorylation, is best demonstrated by the cAMP-dependent kinase, also known as PKA. PKA is a holoenzyme and in its inactive state it exists as a complex of two catalytic subunits bound to two regulatory subunits. Binding of cAMP to the regulatory subunits causes a conformational change, dissociation of the tetrameric complex and phosphorylation of target proteins by the catalytic subunits. When the catalytic subunit is expressed recombinantly in bacteria, it is capable of autophosphorylation (Yonemoto, McGlone et al. 1997). Phosphorylation at this site was later shown to be important in allowing the activation loop in the kinase domain to re-align and allow substrate binding (Knighton, Zheng et al. 1991). In contrast to PKA, other AGC family kinases such as Akt rely on another kinase to phosphorylate this residue in the activation loop. In Akt, this site is Thr308 and the kinase that phosphorylates it was independently discovered by two groups to be the 3-Phosphoinositide-dependent protein kinase-1, also known as PDK-1 (Alessi, Deak et al. 1997; Stokoe, Stephens et al. 1997). PDK-1 appears to be constitutively active, and is found both at the plasma membrane and in the cytosol, implying that it is not a major node in the regulation of Akt activity (Alessi, Andjelkovic et al. 1996; Currie, Walker et al. 1999). Consistent with its role in regulating Akt; animal studies utilising a tissue-specific knock in of a mutant PDK-1 that was unable to bind PI(3,4,5)P3 demonstrated a reduction in Akt activity and also insulin resistance {Bayascas, 2008 #431}. Phosphorylation of Thr308 by PDK-1 causes a conformational change in the activation loop, causing it to “flip out” of the active site and allowing binding of both ATP and the substrate protein (Franke, Hornik et al.). The second regulatory phosphorylation site, Ser473 is present in the C-terminal HM of Akt. The search for the kinase that phosphorylates Ser473, the so-called “PDK-2” has

10 been long and controversial. Various candidate molecules have been proposed, including PKC, the integrin-linked kinase, ATM, DNA-PK and auto-phosphorylation by Akt itself (Toker and Newton 2000; Dong and Liu 2005). However, the mammalian target of rapamycin (mTOR) in complex with mLST8, mSin and rictor (the mTORC2 complex) has recently emerged as a strong candidate for the Ser473 kinase. Sarbassov et al showed that the mTORC2 complex is necessary for Ser473 phosphorylation and directly phosphorylates Akt at Ser473 (Sarbassov, Guertin et al. 2005). In addition, mTORC2 was shown to be the sole kinase responsible for Ser473 phosphorylation in the insulin-responsive 3T3-L1 adipocyte cell line (Hresko and Mueckler 2005). Tissue specific knockout of rictor in skeletal muscle and adipose tissue resulted in decreased insulin-stimulated glucose uptake and GLUT4 translocation {Kumar, 2008 #428}{Kumar, 2010 #429}. Phosphorylation of Ser473 has been proposed to play a dual role in the activation of Akt. This residue lies within the HM motif at the C-terminus of Akt. This motif has been proposed to act as a docking site for PDK-1 in other AGC family kinases (Frodin, Antal et al. 2002), but also acts as an allosteric regulator of Akt activity. The Akt/PDK-1 interaction is mediated by this motif and this is essential for phosphorylation at Thr308 (Frodin, Antal et al. 2002). Moreover, by associating with the PKC-related kinase-2 -interacting fragment (PIF) pocket it stabilises the N lobe of the enzyme and increases its kinase activity (Balendran, Casamayor et al. 1999). Phosphorylation of Ser473 causes the HM motif to engage with the N lobe of the kinase domain, causing a conformational change resulting in the active conformation of the kinase, a process dependent on interaction between phosphorylated Thr308 and the C helix of the kinase domain (Yang, Cron et al. 2002). Finally, it should be noted that Akt lacks a third regulatory phosphorylation site that is found in other AGC family kinases. In PKA, the phosphorylated residue at this motif (Ser338) anchors the C-terminus at the top of the upper lobe of the kinase domain, the phosphorylated residue forming a tight turn. Mutation of this residue destabilises the kinase domain in PKA (Yonemoto, McGlone et al. 1997), but mutation of the corresponding residue in Akt (Thr450) has no effect on kinase activity (Bellacosa, Chan et al. 1998; Toker and Newton 2000). Based on these structural data, an attractive model for the activation of Akt emerges. PI3K-generated phospholipids recruit both Akt and PDK1 to the plasma membrane, where the Ser473 residue of the HM motif of Akt is phosphorylated. Phosphorylation of

11 Ser473 increases the net negative charge of the HM motif, facilitating its interaction with PDK1 by means of the PIF pocket. This interaction stabilizes and activates PDK1 in order to phosphorylate Thr308 (Franke, Hornik et al. 2003). Release of PDK-1 is catalysed by the preference of phosphorylated Ser473 to interact with its own PIF pocket (Fig. 1-2). However, it has also been shown that phosphorylation of Thr308 can occur before phosphorylation of Ser473 (Toker and Newton 2000). The requirement for Ser473 phosphorylation prior to that of Thr308 has also been challenged by experiments using an animal knockout of Sin1, a component of the mTORC2 complex. Despite loss of Ser473 phosphorylation, phosphorylation at Thr308 was maintained both in knock-out mice and derived MEFs (Jacinto, Facchinetti et al. 2006). Additionally, tissue-specific knockout of rictor does not result in impaired phosphorylation of all Akt substrates, phosphorylation of GSK3 at Ser9 remains unaffected {Kumar, 2008 #428}{Kumar, 2010 #429}. The requirement of PDK-1 binding to the HM motif as a requirement for activation of Akt has also been challenged by experiments using genetic disruption of the PIF pocket {Bayascas, 2006 #432}{Collins, 2003 #262}. However, this effect appears to be unique to Akt and HM motif binding is required in other AGC family kinases (Collins, Deak et al. 2005).

Figure 1-2: A model for the structural basis of Akt regulation. PI3K activity at the PM generates PI(3,4,5)P3 which serves as a docking site (1) for the PH domains of Akt (purple) and its activator PDK-1 (yellow). Binding of PI(3,4,5)P3 induces a conformational change in Akt and allows PDK-1 to phosphorylate Thr308 (2a) located on the activation loop of the kinase domain (KD). The affinity of Akt for PDK-1 is enhanced by phosphorylation of Ser473 by the mTORC-2 complex (2b), the putative PDK2. Ser473 lies in the hydrophobic motif (HM) of Akt and phosphorylation at Ser473 allows the HM motif to bind the PIF pocket of PDK-1. Akt dissociates from PDK-1 by means of the HM motif (3), which preferentially binds the PIF pocket of Akt. Akt activity is down-regulated by the activity of lipid phosphatases such as SHIP2 and PTEN which dephosphorylate PI(3,4,5)P3, and by direct dephosphorylation by protein phosphatases such as PP2A (4). See text for further discussion.

13 Most models of Akt activation are inferred from similar studies using other AGC family kinases. However, as discussed above, there are several differences in the mode of activation between Akt and other AGC family kinases. What is not clear is whether Akt interacts with PDK-1 solely at the plasma membrane, or whether it binds PDK-1 in the cytosol and translocates as a complex. A recent study using Förster resonance energy transfer (FRET) suggested that Akt binds PDK-1 in the cytosol, and this complex translocates to the plasma membrane after growth factor stimulation (Calleja, Alcor et al. 2007). However, an interaction between the PH domain and the kinase domain is suggested to prevent phosphorylation of Thr308 by PDK-1 and thus activation. This repression is relieved by binding of PI(3,4,5)P by the PH domain, inducing a conformational change in Akt and allowing activation via PDK-1 (Calleja, Alcor et al. 2007).

The activation of Akt’s kinase activity therefore requires three steps; translocation to the plasma membrane and binding of PI(3,4,5)P and subsequent phosphorylation of Thr308 by PDK-1 and Ser473 probably by the mTORC2 complex. However, the order of these phosphorylation events is not yet clear, and the spatial regulation of the interaction with PDK-1 remains contentious.

Negative Regulators of Akt

The main method by which Akt signalling is terminated is generally thought to be via the action of lipid phosphatases at the PM. By converting PI(3,4,5)P3 to PI(3,4)P2, these enzymes eliminate binding sites for Akt at the PM, preventing its activation by mTORC2 and PDK-1. Two major phosphatases are thought to be involved. The first, the SH2-containing Inositol phosphatase (SHIP2) dephosphorylates PI(3,4,5)P3 on the five position of its Inositol ring to produce PI(3,4)P2 (Cantley 2002). The other is the phosphatase and tensin homolog deleted on chromosome ten (PTEN) which converts PI(3,4,5)P3 to PI(4,5)P2 by dephosphorylating PI(3,4,5)P3 at the D3 position (Cantley 2002). Consistent with its role in regulating Akt, PTEN is a known tumour suppressor; loss of heterozygosity at the PTEN locus is the most common mutation in the development of malignant gliomas {Bigner, 1988 #422}. The role of PTEN in regulating insulin signalling in vitro was uncovered by experiments that showed that

14 overexpression of PTEN in adipocytes resulted in a reduction in insulin-stimulated phosphorylation of Akt and S6 kinase (Nakashima, Sharma et al. 2000). Knockout studies have demonstrated that PTEN is essential for normal embryogenesis {Cristofano, 1998 #423}{Stambolic, 1998 #424}. The role of PTEN in dephosphorylating PI(3,4,5)P3 was uncovered using animal models. Immortalised murine fibroblasts from PTEN -/- animals had hyper-phosphorylated Akt and elevated levels of PI(3,4,5)P3 when compared to fibroblasts from PTEN +/- animals {Stambolic, 1998 #424}. Akt activity can also be down-regulated by the actions of protein phosphatases. Protein phosphatase 2A can directly dephosphorylate Akt at Thr308 and Ser473 both in vitro and in vivo (Meier, Thelen et al. 1998). The PH domain leucine-rich repeat protein phosphatase (PHLLP) can also dephosphorylate Akt at Ser473, but not Thr308 (Gao, Furnari et al. 2005). Interestingly, the two identified isoforms of PHLLP selectively dephosphorylate specific isoforms of Akt. This could allow differential control of isoform-specific Akt signalling (Brognard, Sierecki et al. 2007).

Akt is a Major Regulator of Cellular Metabolism

Akt controls multiple intracellular signalling pathways, and thus regulates many aspects of cellular biology. Akt has been implicated in cellular processes as diverse as cell growth and survival, glucose metabolism, proliferation, and cell migration. What is clear is that a single Akt substrate may be involved in the regulation of multiple cellular processes; it is not simply a case of one substrate for each function. For example, the transcription factor FOXO is negatively regulated by Akt-mediated phosphorylation, blocking transcription of target genes that promote apoptosis e.g. the pro-apoptotic Fas ligand (Brunet, Bonni et al. 1999). However, in the liver, the activity of FOXO in conjunction with PGC-1 promotes glucose output via the transcription of gluconeogenic genes (Puigserver, Rhee et al. 2003). Once again, this activity is down- regulated by Akt-mediated phosphorylation and nuclear export of FOXO.

15 Glucose Uptake

The major mechanism by which Akt regulates glucose metabolism is by controlling the transport of the facilitative glucose transporter GLUT4 from an intracellular storage compartment to the plasma membrane in response to an insulin stimulus. The consequence of GLUT4 translocation to the PM is a net 10-40 fold increase of the glucose influx into the cell (Whiteman, Cho et al. 2002). This role of Akt is limited to insulin-responsive tissues that express the GLUT4 protein, namely skeletal muscle, brown and white adipose tissue and cardiac muscle (James, Brown et al. 1988). A breakthrough in determining the insulin signalling pathway distal to Akt activation came in 2002, with the discovery of the Akt substrate of 160 kDa (AS160, also known as TBC1D4). The protein was first identified in insulin-stimulated 3T3-L1 adipocytes using an antibody specific for the Akt phosphorylation motif (Kane, Sano et al. 2002). This protein was found to be a Rab GTPase activating protein, although the identity of its target Rab is controversial and remains an area of fiercely active research. AS160 associates with GSVs under basal conditions, and is released into the cytosol after insulin stimulation (Larance, Ramm et al. 2005). This association is essential in inhibiting the translocation of GLUT4 to the PM under basal conditions. While insulin dependent phosphorylation of AS160 is essential for GLUT4 trafficking to the PM, the dissociation of AS160 from GLUT4 containing membranes may not be necessary. (Stockli, Davey et al. 2008). Insulin stimulation causes AS160 phosphorylation at 5 sites (Ser318, Ser570, Ser588, Thr642 and Thr751) and mutation of four of these sites to alanine (S318A, S588A, T642A, T751A) (AS160-4P) results in reduced GLUT4 translocation to the PM in response to insulin treatment (Sano, Kane et al. 2003). Phosphorylation of Thr642 via Akt phosphorylation induces 14-3-3 binding at this site. Interestingly, by inducing constitutive 14-3-3 binding at this site it is possible to overcome the block in GLUT4 translocation induced by the AS160-4P mutant (Ramm, Larance et al. 2006). Although the exact role of AS160 in mediating GLUT4 translocation is not yet clear, the current hypothesis is that the function of AS160 is to retain GLUT4 within the cell by inactivating its cognate Rab protein. Phosphorylation of AS160 by Akt in response to an insulin stimulus recruits 14-3-3, which binds to AS160 and inhibits its GAP activity, allowing activation of the Rab protein. The closest homolog of AS160, TBC1D1, also plays a role in GLUT4 translocation (Roach, Chavez et al. 2007). TBC1D1 is also a GAP, and its domain organisation is 16 identical to AS160. In a similar fashion to AS160, insulin stimulation causes phosphorylation of TBC1D1 at a site which is predicted to be recognised by Akt (Thr596). Ablation of this site by the T596A mutation causes a block in GLUT4 translocation in adipocytes, an outcome that was dependent on the GAP activity of TBC1D1 (Roach, Chavez et al. 2007). Other Akt substrates involved in GLUT4 trafficking have also been identified, although their roles are less well defined. Ablation of the Akt phosphorylation site by mutation to alanine in the PI(3)P 5-kinase PIKfyve (PhosphoInositide Kinase for five position containing a fyve finger) enhances insulin-stimulated translocation of GLUT4 to the PM (Berwick, Dell et al. 2004). Akt substrates have also been identified in the putative final step of GLUT4 translocation, fusion of GLUT4-containing transport vesicles to the PM. This membrane fusion event is mediated by the action of the SNARE proteins Syntaxin 4, SNAP-23 and VAMP 2 (Rea, Martin et al. 1998). The interaction between VAMP 2 and Syntaxin 4 is regulated by a number of factors, one of which is the protein Synip, an Akt substrate. Phosphorylation of Synip in response to insulin stimulus dissociates it from Syntaxin 4 and allows VAMP 2 binding and GLUT4 vesicle fusion with the PM (Yamada, Okada et al. 2005).

Glucose Disposal

The vast majority of glucose taken up from the blood stream by insulin-dependent transport is stored as glycogen in skeletal muscle (Shulman, Rothman et al. 1990). Insulin regulates the incorporation of glucose into glycogen by stimulating the activity of glycogen synthase (GS), the enzyme that catalyses the final step in glycogen synthesis. Akt is involved in the regulation of GS activity by controlling the activity of glycogen synthase kinase (GSK), which phosphorylates GS in the basal state and inactivates it. A unique requirement of GSK3’s kinase activity is that many of its substrates require a “priming” phosphorylation, located C-terminal to the GSK3 phosphorylation site {Fiol, 1987 #426}. After insulin stimulation, active Akt phosphorylates GSK at several sites including Ser9, inactivating the kinase activity of GSK (Cross, Alessi et al. 1995). The phosphorylated N-terminus then acts as a pseudo- substrate, occupying the same binding site as substrates that have undergone a “priming” phosphorylation {Frame, 2001 #425}. Rendering GSK insensitive to Akt

17 phosphorylation by the S9A mutation results in suppression of insulin-stimulated GS activity in adipocytes (Summers, Kao et al. 1999). Interestingly, phosphorylation of Ser9 of GSK causes 14-3-3 binding in the brain (Agarwal-Mawal, Qureshi et al. 2003). However, the relative contribution of Akt in controlling glycogen synthesis is not yet clear. The regulation of GS activity is complex and is also controlled by several Akt independent mechanisms.

Protein Synthesis

The promotion of cell growth by Akt occurs mostly through its control of cell metabolism via the mammalian target of rapamycin, mTOR. In complex with Raptor, LST8 and PRAS40, the mTORC1 complex, mTOR acts as a central integrator of several signalling pathways and is itself a serine/threonine kinase. Active mTORC1 promotes protein synthesis by regulating the activity of the ribosomal S6 kinase and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) stimulating the elongation and initiation steps of translation, respectively (Schmelzle and Hall 2000). Akt is capable of directly phosphorylating mTOR on Ser2448 and phosphorylation of this residue generally correlates with mTOR activation (Scott, Brunn et al. 1998). However, mutation of this residue to alanine does not inhibit S6 kinase activation, leaving its significance unclear (Sekuli, Hudson et al. 2000). Instead, Akt is thought to regulate the activity of mTOR indirectly, controlling the activity of the small GTPase Rheb via the GTPase activating protein tuberous sclerosis complex 2 (TSC2). TSC2 is an Akt substrate, and when non-phosphorylated it heterodimerises with TSC1 and has GAP activity towards Rheb (for a full discussion of the biology of small GTPases and GAPs, please refer to chapter 4) (Inoki, Li et al. 2002; Tee, Fingar et al. 2002). Akt phosphorylates TSC2 at Ser939 and Thr1462, which may sequester TSC2 away from TSC1, inhibiting the GAP activity of the complex and relieving the repression of Rheb (Li, Inoki et al. 2003). However, the mechanism by which Rheb activates mTORC1 is still unclear (Bhaskar and Hay 2007). Akt also regulates the activity of the mTORC1 complex by phosphorylating the proline-rich Akt substrate of 40 kDa (PRAS40) at Thr246 (Kovacina, Park et al. 2003). PRAS40 binds to mTORC1 and negatively regulates its activity (Sancak, Thoreen et al. 2007). The phosphorylation of PRAS40 by

18 Akt induces 14-3-3 binding, dissociating PRAS40 from mTORC1, allowing activation (Vander Haar, Lee et al. 2007).

Other Functions of Akt

Akt in Cell Growth and Survival.

The role of Akt in cell survival is closely linked to the control of apoptosis. Akt generally acts to promote cell survival by inhibiting the function of pro-apoptotic processes and proteins. Akt was recognised as playing a critical role in promoting cell survival before the relevant substrates were discovered (Kulik, Klippel et al. 1997). A classical mechanism by which Akt promotes cell survival is by controlling the activity of various members of the Bcl-2 family of proteins, whether by transcriptional regulation or direct phosphorylation. As already mentioned, FOXO transcribes several pro-apoptotic genes, and its activity is inhibited by phosphorylation by Akt. Besides the Fas ligand, FOXO transcribes Bim, a proapoptotic member of the Bcl-2 family of mitochondrial outer membrane proteins. Transcription of Bim by FOXO is believed to contribute to apoptosis following cytokine withdrawal via mitochondrial disruption (Dijkers, Birkenkamp et al. 2002). Direct phosphorylation of another member of the Bcl-2 family, the Bcl-2 homology domain 3 only promoter (BAD) at Ser136 by Akt induces the 14-3-3 mediated dissociation of BAD from its target proteins, promoting survival (Datta, Dudek et al. 1997; Datta, Katsov et al. 2000). Bim and BAD both lie on the mitochondrial pathway of apoptosis, culminating in the activity of caspase proteins which are initiated by the activation of caspase 9 upon cytochrome c release from the mitochondria. There is some evidence that Akt can down-regulate the activity of caspase 9 by phosphorylating its pro-caspase form, albeit at a non-canonical Akt phosphorylation site (Cardone, Roy et al. 1998).

Akt and the Regulation of Cell Proliferation

Akt was first identified as an oncogene, so it is perhaps not surprising that it plays a role in the regulation of cellular proliferation, a process that is intimately linked to cancer progression. Akt probably has an indirect effect on the cell cycle via its control of

19 protein synthesis by mTOR, leading to increased synthesis of proteins involved in cell cycle entry. Akt can also indirectly influence cell cycle progression via GSK. GSK phosphorylates several proteins important in cell cycle control, such as transcription factors c-jun and c-myc as well as G1 cyclins D and E. Phosphorylation of these proteins by GSK, targets them for proteasomal degradation (Diehl, Cheng et al. 1998; Welcker, Singer et al. 2003; Yeh, Cunningham et al. 2004; Wei, Jin et al. 2005). The inhibition of GSK activity by Akt could therefore be expected to protect these proteins from degradation. However, Akt also plays a more direct role in regulating the cell cycle through several substrates that are directly involved in cell cycle control. The cyclin-dependent kinase inhibitor 1B (also known as p27Kip1) controls G1 entry by binding to, and preventing the activation of, cyclin E-CDK2 or cyclin D-CDK4 complexes. Akt can phosphorylate p27Kip1 at Thr157 (Liang, Zubovitz et al. 2002; Shin, Yakes et al. 2002; Viglietto, Motti et al. 2002), inducing 14-3-3 binding and sequestering p27Kip1 in the cytosol (Sekimoto, Fukumoto et al. 2004), allowing G1 entry and cell cycle progression. Akt is also capable of influencing p27Kip1 indirectly, controlling its expression via FOXO (Medema, Kops et al. 2000). Akt has also been reported to phosphorylate the cyclin-dependent kinase inhibitor p21Cip1/WAF1 on Thr145. Similarly to its regulation of p27, this phosphorylation event leads to p21Cip1/WAF1 cytosolic localization (Zhou, Liao et al. 2001) although this has not been reported to be 14-3-3 dependent. Akt has also been reported to phosphorylate the DNA damage checkpoint kinase Chk1 at Ser280 (King, Skeen et al. 2004). This promotes progression through mitosis by sequestering Chk1 in the cytosol where its cannot interact with DNA damage sensing kinases such as ATM and ATR (Puc, Keniry et al. 2005). Interestingly, Chk1 has also been reported to bind 14-3-3, which affects its localisation. However, this 14-3-3 binding does not appear to be via a canonical phosphoserine/threonine motif, rather by a novel hydrophobic binding mode (Dunaway, Liu et al. 2005).

Akt and the Regulation of Cardiac Function

Cardiac muscle is a dynamic tissue that can remodel itself in response to changes in its workload. The resulting increase in tissue mass is due to the increase in volume of individual cardiomyocytes, rather than proliferation {Hill, 2008 #409}. This

20 hypertrophy can be both a normal physiological response to exercise, and a maladaptive response to a pathological or genetic insult. Several lines of evidence have implicated the PI3K/ Akt pathway in the regulation of cardiac hypertrophy. Cardiac-specific overexpression of constitutively active PI3K in transgenic mice resulted in a form of adaptive hypertrophy {Shioi, 2000 #410}. Akt1 null mice, in addition to a global reduction in organ size and growth retardation {Chen, 2001 #270} are defective in exercise induced cardiac hypertrophy {DeBosch, 2006 #408}. Conversely, cardiac- specific overexpression of constitutively active Akt in transgenic mice resulted in hypertrophy {Shioi, 2002 #411} but also resulted in contractile dysfunction in some models {Taniyama, 2005 #412}. To investigate the role of Akt in the progression of adaptive hypertrophy to dysfunction, Shiojima et al employed a transgenic system that allowed for inducible cardiac-specific expression of Akt {Shiojima, 2005 #413}. This study demonstrated that acute overexpression of Akt resulted in adaptive hypertrophy, but chronic overexpression lead to pathological hypertrophy possibly as the result of a disconnect between hypertrophy and coronary angiogenesis {Shiojima, 2005 #413}. Taken together, these data indicate that Akt is a positive regulator of physiological cardiac hypertrophy and a negative regulator of pathological hypertrophy. Akt regulates the adaptive hypertrophic response by signalling through both GSK3 and the mTOR pathway. GSK3 negatively regulates several hypertrophic transcription factors such as NFAT, c-Myc and -catenin {Proud, 2004 #414}, phosphorylation of GSK3 by active Akt relieves this repression as described above. Evidence linking mTOR to hypertrophy arose via use of the mTOR inhibitor rapamycin. Rapamycin treatment reduces cardiac hypertrophy caused by pressure overload, a pathological hypertrophic model {Shioi, 2003 #415}{McMullen, 2004 #416}. As mTOR is a master regulator of protein synthesis, it is perhaps not surprising that it is involved in hypertrophy; an increase in cell size requires an increase in protein synthesis. However, this does not seem to be mediated via the S6 kinases, as cardiac specific deletion of both S6K1 and S6K2 has no effect on the development of hypertrophy {McMullen, 2004 #417}.

Akt and Cell Migration

There have been several tantalising hints that Akt is involved in the regulation of cell migration. At the leading edge of the migrating cell, the activity of PI3K generates

21 PI(3,4,5)P. The presence of this second messenger at the leading edge is necessary in directed migration (Kolsch, Charest et al. 2008), and may trigger lamellipodia formation towards an attractant during chemotaxis (Arrieumerlou and Meyer 2005). Although PI(3,4,5)P binds Akt, in this case the preferred target is believed to be the small GTPase Rac, which localises to the leading edge during directed migration (Nobes, Hawkins et al. 1995). However, the Rac effector protein Pak is capable of targeting Akt to the plasma membrane where it can be activated by PDK-1 (Higuchi, Onishi et al. 2008). This pathway is important for cell migration in endothelial cells (Primo, di Blasio et al. 2007). Specific activation of Akt1 has also been found to decrease mammary epithelial cell migration (Yoeli-Lerner, Yiu et al. 2005). It has been proposed that this is due to Akt1-mediated degradation of nuclear factor of activated T cells (NFAT) transcription factors. In the absence of Akt1 activation, NFAT is proposed to initiate transcription of genes that promote migration and invasion (Yoeli-Lerner, Yiu et al. 2005). A second study also found that knockdown of Akt1, but not Akt2, leads to an increase in the migration of mammary epithelial cells. Loss of Akt1 leads to an increase in extracellular signal–related kinase (ERK) activation, leading to increased migration (Irie, Pearline et al. 2005). These effects may well be cell-type specific. The exact opposite effects have been observed in the control of fibroblast migration (Zhou, Tucker et al. 2006).

The Akt phosphorylation motif

The minimal recognition motif for Akt phosphorylation is RXRXXp(S/T) where X is any amino acid {Obata, 2000 #20}. This recognition motif was first identified by screening degenerate peptide libraries based on the Akt phosphorylation site on GSK for their ability to be phosphorylated in vitro by Akt {Alessi, 1996 #427}. That initial study described the minimal recognition motif as RXRYZpS where Y and Z are small amino acids other than glycine and is a bulky hydrophobic residue such as phenylalanine or leucine. The most important feature of the recognition site is the presence of arginine at the -3 and -5 position, mutation of either of these residues drastically reduces phosphorylation {Alessi, 1996 #427}. The requirement for arginine at the -5 and -3 position was confirmed by crystallisation studies, these residues interact with residues in the substrate binding cleft of Akt {Yang, 2002 #250}.

22 Subsequent studies using oriented degenerate peptide libraries predicted the optimal recognition sequence to be RLRXRTYpSFG and by screening a phage expression library determined the minimum Akt recognition sequence to be RXRXXp(S/T) {Obata, 2000 #20}. This recognition sequence is similar to that of other basophilic kinases such as PKC, PKC, and PKC which also phosphorylate substrates that have arginine at the -3 and -5 positions and phenylalanine at the +1 position. The different substrate specificities for Akt and PKCs is thus mediated by residues 3’ to the phosphorylated residue; PKCs prefer basic or aromatic residues at the +2 position whereas Akt’s optimal motif contains a glycine in this position {Obata, 2000 #20}.

Akt Signals Through 14-3-3

The phosphorylation of many Akt substrates creates a binding motif for another class of proteins, the 14-3-3 family. This is not surprising, as Akt and 14-3-3 share the same recognition motif (Obata, Yaffe et al. 2000). In addition, 14-3-3 is responsible for translating many Akt phosphorylation events into a function. In most of the functions of Akt that are discussed above, 14-3-3 plays a role. For example, many of the antiapoptotic effects of Akt are mediated by its regulation of the transcription factor FOXO. Akt dependent phosphorylation of FOXO results in the binding of 14-3-3 to FOXO, leading to the translocation of FOXO into the cytosol and thus inhibition of its transcriptional activity (Zhao, Gan et al. 2004). The involvement of 14-3-3 in so many functions of Akt leads to the hypothesis that 14-3-3 binding is a general mechanism by which Akt controls many aspects of cellular behaviour. Although a great deal is known about Akt, it is likely that more remains to be discovered. I propose that new Akt substrates can be discovered by using 14-3-3 as a tool.

14-3-3 Biology

The 14-3-3 protein family is a group of highly conserved acidic proteins of approximately 30 kDa that are expressed in a wide range of tissues. Exclusively eukaryotic, they have been implicated in a range of cellular processes due to their function as adaptor molecules and mediators of intracellular signalling.

23 History of 14-3-3

The protein family now known as 14-3-3 was first identified in a systematic study of the bovine brain proteome (Moore and Perez 1967). This is the origin of their unusual name; it refers to the fraction they eluted in DEAE-cellulose chromatography and the migration position in subsequent starch–gel electrophoresis. Thus, it was found in the 14th fraction that eluted off the DEAE cellulose column, and was found in fraction 3.3 of the electrophoresis. That 14-3-3 was originally discovered in the brain is no surprise because it constitutes almost 1% of the total soluble protein in that tissue (Boston, Jackson et al. 1982). However, it is now recognised that 14-3-3 is expressed in a wide variety of tissues (Celis, Gesser et al. 1990). Although the 14-3-3 family of proteins was first identified in 1967, it was not until 1987 that functional data appeared. 14-3-3 proteins were first implicated in neurotransmitter synthesis via their activation of tyrosine and tryptophan monooxygenases, which are the rate limiting enzymes in catecholamine and dopamine biosynthesis (Ichimura, Isobe et al. 1987). Evidence that 14-3-3 was involved in regulating signal transduction came with the discovery that 14-3-3 was an inhibitor of protein kinase C in the brain (Toker, Ellis et al. 1990). However, it was not until 14-3-3 was shown to interact with several oncogenic proteins that interest in the family was piqued. 14-3-3 was reported to be phosphorylated by Bcr-Abl, the oncogenic fusion protein formed from the Breakpoint Cluster Region protein (Bcr) and Abelson murine leukaemia viral oncogene homolog 1 (Abl). However, 14-3-3 is not a substrate for Abl (Reuther, Fu et al. 1994). 14-3-3 was also shown to be associated with the polyomavirus middle T antigen (Pallas, Fu et al. 1994). A major leap in the understanding of 14-3-3 biology came with the recognition that 14-3-3’s binding to Raf-1 was dependent on binding to a phosphoserine residue (Muslin, Tanner et al. 1996). The 14-3-3 binding motifs were originally described as RXXpSXP and RXXXpSXP (Yaffe, Rittinger et al. 1997), however other motifs have since been described. For a full discussion of the different modes of 14-3-3 binding, please see Chapter 5. To date, more than 200 proteins have been reported to interact with 14-3-3 (Aitken 2006).

24 The 14-3-3 Protein Family

14-3-3 is believed to have evolved very early in the eukaryotic lineage and most eukaryotes express at least one isoform. The number of 14-3-3 isoforms per species ranges from one in D. discoideum, two in C. elegans and S. cerevisiae to 12 in A. thalia (Rosenquist, Sehnke et al. 2000). There are seven known isoforms in mammals, designated , , , , , , and , each isoform named for its elution profile in a reversed- phase high-performance liquid chromatography purification of bovine brain (Ichimura, Isobe et al. 1988). Originally, and isoforms were also described, however these were later found to be the phosphorylated forms of the and isoforms, respectively (Aitken, Howell et al. 1995). There is a surprisingly large amount of sequence identity between isoforms across species. This may be as high as 96-100% when comparing the same isoform in mammalian species, dropping to only 46% when comparing different isoforms from the same species (Rosenquist, Sehnke et al. 2000). An enduring question in the study of 14-3-3 is why are so many isoforms necessary? Three possible reasons have been advanced (Rosenquist, Sehnke et al. 2000). First, different isoforms are expressed at varying levels at different stages of development. For example, during heart development in Rattus the expression of 14-3-3 and Raf-1 are co-ordinately regulated (Luk, Ngai et al. 1998). This hypothesis implies that different isoforms have specific roles and interactions. Secondly, there may be isoform- specific subcellular localisation. This has been demonstrated in Arabidopsis, where several 14-3-3 isoforms were found in the chloroplast (Sehnke, Henry et al. 2000), although this was later suggested to be the result of isoform-specific protein binding (Paul, Sehnke et al. 2005). The third hypothesis which is essentially a combination of the first two suggests that there are isoform-specific interactions between 14-3-3 and its target proteins. Indeed, it was reported that the A20 protein preferentially associates with 14-3-3 in vivo (Vincenz and Dixit 1996) and it has been demonstrated that homodimers of different isoforms bind with varying affinity to a peptide encoding the non-canonical 14-3-3 binding motif YpTV from Arabidopsis H+ ATPase AHA2 (Rosenquist, Sehnke et al. 2000). However, other studies examining binding to higher affinity ligands such as the canonical mode II 14-3-3 binding motif were unable to demonstrate isoform specificity (Yaffe, Rittinger et al. 1997; Rittinger, Budman et al. 1999). It is therefore possible that isoform-specific binding only occurs for low affinity interactions. It was initially proposed that isoform-specific dimerisation would allow for 25 control of substrate specificity, however it was later shown that most isoforms can form mixed hetero-dimers in vitro and that at least the and isoforms can heterodimerise in vivo (Jones, Ley et al. 1995). This adds further complication to isoform-specific substrate recognition, especially as there may be limits on which heterodimers actually occur in vivo (Chaudhri, Scarabel et al. 2003). For almost all known 14-3-3 interactors, a detailed study of isoform binding specificity has not been performed. It is becoming clear that the 14-3-3 is different to most other isoforms, both in structure and function. 14-3-3 is primarily expressed in epithelial cells (Leffers,

Madsen et al. 1993), where it has been implicated in G2/M checkpoint control and its expression is induced by p53 in response to DNA damage (Hermeking, Lengauer et al. 1997). It is the most evolutionarily diverse isoform, and in contrast to other isoforms exists preferentially as a homodimer (Wilker, Grant et al. 2005). This preference for homodimerisation in can be explained by structural differences from other isoforms (Wilker, Grant et al. 2005). The isoform has numerous amino acid differences in the region that mediates dimer formation when compared to other 14-3-3 isoforms. These substitutions favour homodimer formation by forming additional stabilising interactions in the case of homodimerisation and at the same time, make the formation of heterodimers energetically unfavourable. The substrate specificity of can be also be attributed to protein-protein interactions outside of the phosphopeptide binding cleft, which is identical to other isoforms (Wilker, Grant et al. 2005).

Structure of 14-3-3

14-3-3 is a highly acidic protein (pI ~ 4.6) with a molecular weight of approximately 30 kDa. The sequence and structure of 14-3-3 appears to be quite unlike anything else in the proteome. The only significant structural homology appears to be with the suppressor with morpho-genetic effects on genitalia protein (SMG7) which regulates nonsense mediated mRNA decay. Although SMG7 has less than 10% sequence identity to 14-3-3, the tertiary structure of one of its tetratricopeptide repeat (TPR) domains is almost identical to a 14-3-3 monomer (Fukuhara, Ebert et al. 2005). Interestingly, SMG7 is also a phosphopeptide binding protein, possibly suggesting that a number of 14-3-3-like proteins exist but have not been described as such due to low sequence homology.

26 The crystal structure of both the and isoforms of 14-3-3 were solved in 1995. Both structures were highly homologous with each forming a horseshoe shaped dimer and each monomer consisting of 9 anti-parallel helices. The interactions between these helices result in a very rigid structure (Liu, Bienkowska et al. 1995; Xiao, Smerdon et al. 1995). Dimerisation results in a large negatively charged channel, approximately 35 Å long, 35 Å wide and 20 Å deep, lined with those regions of the 14-3-3 protein that are conserved across all isoforms (Fig. 1-3). Indeed, the residues that form the phosphopeptide binding site are invariant in all known 14-3-3 proteins. Residues that vary between isoforms are generally found on the surface of the protein (Bridges and Moorhead 2005). Dimerisation is mediated by the N-terminus of the protein, via several residues conserved across most isoforms. Variability in this region may therefore control the extent of heterodimerisation between 14-3-3 isoforms. The structural basis of phosphopeptide binding was uncovered in 1997, when recombinant 14-3-3 was crystallised in complex with a phosphopeptide derived from the binding motif of polyomavirus middle T antigen (Yaffe, Rittinger et al. 1997). This study defined the canonical mode I and mode II 14-3-3 binding motifs, and also showed it was possible for each 14-3-3 molecule to simultaneously bind two phosphoserine residues (Fig 1-3). Indeed, the affinity of a peptide with two phosphoserines is 30 times greater than a singly phosphorylated peptide (Yaffe, Rittinger et al. 1997). This ability to bind two phosphorylated residues is critical in allowing 14-3-3 to regulate many of its targets. The structure of 14-3-3 in complex with a bona fide substrate, serotonin N- acetyltransferase (arylalkylamine N-acetyltransferase; AANAT), was first reported in 2001 (Obsil, Ghirlando et al. 2001). Not only did this study validate previous structural studies by demonstrating that 14-3-3 binding occurred via the same amphipathic groove as a phosphopeptide, but it also demonstrated that 14-3-3 binding affected the function of the substrate, altering AANAT’s substrate specificity and activity. While the majority of 14-3-3’s interactions with its substrates occur via phosphorylated residues, it must also be noted that some 14-3-3/target interactions occur in the absence of phosphorylation. In many of these cases, the presence of several negatively charged glutamic acid residues is thought to “substitute” for the negatively charged phosphate group in the canonical binding motifs. This has been demonstrated in the case of 14-3-3/R18 binding. R18 is a synthetic peptide sequence that was identified through phage display as a high affinity 14-3-3 substrate (Wang, Yang et al. 1999). The crystal

27 structure of the R18/14-3-3 complex shows that R18 occupies the same amphipathic groove as a phosphorylated ligand, with various acidic residues in the R18 sequence adjacent to basic residues on 14-3-3 (Petosa, Masters et al. 1998). Other interactions with non-phosphorylated substrates are reported to occur outside of the conserved amphipathic binding groove, mainly via the C-terminal region of 14-3-3. For example, a C-terminal fragment of 14-3-3 is capable of binding tryptophan hydroxylase (Ichimura, Uchiyama et al. 1995), Raf-1, and Bcr (Ichimura, Ito et al. 1997) in vitro, although the structural basis of this interaction remains unknown. As the C-terminal region of 14-3-3 is more variable than the conserved amphipathic groove, it is possible that this binding mode is responsible for some of the reported isoform-specific interactions. More recent work has indicated that the C-terminal region plays an important regulatory role in controlling substrate binding. Its deletion results in higher binding affinities to several substrates in vivo (Truong, Masters et al. 2002). This region is highly disordered, it is not solved in either of the 14-3-3 crystal structures reported above (Liu, Bienkowska et al. 1995; Xiao, Smerdon et al. 1995). This C-terminal region is now thought to occupy the phosphopeptide binding groove in the absence of bound ligand (Silhan, Obsilova et al. 2004), suggesting that it functions to prevent binding to inappropriate substrates.

Figure 1-3: The Structure of 14-3-3. 14-3-3 preferentially exists as a dimer. Here, the crystal structure of a 14-3-3 homodimer in complex with a phosphopeptide is shown. The two 14-3-3 monomers are depicted in red and blue, and the phosphopeptide in green. The two views are rotated 90° to each other. This image is based on the 2B05 structure in the Protein Data Bank (Berman, Westbrook et al. 2000) and was generated using the PDB Protein Workshop tool (Moreland, Gramada et al. 2005). 29 Regulation of 14-3-3

As 14-3-3 has no intrinsic enzymatic activity, regulation is understood to refer to post- translational modifications that alter 14-3-3’s binding to its substrates. Phosphorylation of the target protein is a well-established mechanism by which the binding of 14-3-3 is controlled. However, 14-3-3 itself can also be phosphorylated, and this has an important regulatory role. Varying isoforms of 14-3-3 have long been known to be phosphorylated (Aitken, Howell et al. 1995), but the first functional outcome of phosphorylation was only reported in 2003 when phosphorylation of 14-3-3 on Ser58 was shown to inhibit dimerisation both in vitro and in vivo (Woodcock, Murphy et al. 2003), although the physiological significance of this remains unclear. Phosphorylation of 14-3-3 can also prevent binding to target proteins. For example, DNA damage causes phosphorylation of 14-3-3 by c-Jun N-terminal kinase (JNK) on Ser184 dissociating 14-3-3 from c-Abl and allowing c-Abl to be targeted to the nucleus (Yoshida, Yamaguchi et al. 2005). JNK-mediated phosphorylation of 14-3-3 at Ser184 also induces dissociation from Bax, allowing Bax translocation to the mitochondria causing cytochrome C release and thus apoptosis (Tsuruta, Sunayama et al. 2004). Raf-1 interaction with 14-3-3 is inhibited by phosphorylation of 14-3-3 at Thr232 by casein kinase 1 (Dubois, Rommel et al. 1997). A structural basis for phosphorylation-mediated inhibition of substrate binding was described by Obsilova et al, who suggested that phosphorylation of 14-3-3 at Thr232 reduces the extent of conformational change in the C-terminal region of 14-3-3 that normally occurs after phosphopeptide binding (Obsilova, Herman et al. 2004). The Thr232 residue forms part of a sub-optimal 14-3-3 binding motif that may compete with other low affinity motifs once phosphorylated, but can presumably be out-competed for binding to the phosphopeptide binding groove by high-affinity motifs (Bridges and Moorhead 2005). Phosphorylation may represent a mechanism for isoform-dependent regulation. None of the three phosphorylation sites discussed above are conserved between all human 14-3-3 isoforms. For example, only the and isoforms have a phosphorylation site corresponding to Thr232, and has been reported to be phosphorylated at this site in vitro (Dubois, Rommel et al. 1997).

30 Finally, for the purposes of this study it is worth noting that Akt has been reported to phosphorylate 14-3-3 at Ser58 in vitro, although this had no effect on the dimerisation of 14-3-3 in that study (Powell, Rane et al. 2002).

Functions of 14-3-3

14-3-3 proteins have no enzymatic activity of their own, but rather transduce and integrate intracellular signals by binding to various target proteins. The structure of 14-3-3 is closely related to its ability to alter the activity and/or localisation of its substrates. The secondary structure of 14-3-3 is rich in helices, interactions between which result in a very rigid tertiary structure (Liu, Bienkowska et al. 1995; Xiao, Smerdon et al. 1995). Co-crystallisation experiments have shown that the conformation of 14-3-3 does not change significantly when bound to a substrate; rather the substrate re-shapes to bind 14-3-3 (Obsil, Ghirlando et al. 2001). Indeed, 14-3-3 has been described as a molecular “anvil” upon which other proteins are re-shaped (Yaffe 2002). 14-3-3 proteins form homo- or heterodimers. These dimers bind to either two substrate proteins or to two binding motifs on the one substrate. It has been proposed (Bridges and Moorhead 2005) that there are four major possible outcomes of 14-3-3 binding to a substrate (Fig. 1-4). In the first instance, which I have already alluded to, 14-3-3 binding may alter the conformation of the substrate, resulting in a change in the enzymatic activity or specificity of the substrate. Structural studies have demonstrated this mechanism in the case of AANAT, the substrate affinity of which is increased by phosphorylation and subsequent 14-3-3 binding (Obsil, Ghirlando et al. 2001). In this case, the binding of 14-3-3 results in stabilisation of the active conformation of the enzyme. Secondly, 14-3-3 binding may mask important regulatory elements of the substrate. A classic example is the binding of 14-3-3 to phosphorylated members of the FOXO class of transcription factors in the nucleus, leading to the termination of DNA binding and FOXO’s nuclear export (Brunet, Bonni et al. 1999). In this case, the binding of 14-3-3 to FOXO is thought to be in a 1:1 stoichiometric ratio (Obsil, Ghirlando et al. 2003); a single 14-3-3 dimer therefore binds across the DNA binding domain and prevents transcriptional activity. This re-localisation of substrates by 14-3-3 binding has also been reported in a Rho GTPase activating protein, DLC-1. Here, it was suggested that the binding of 14-3-3 results in an inhibition of GAP activity and a block in nuclear

31 import by masking a nuclear import signal adjacent to the 14-3-3 binding site (Scholz, Regner et al. 2009). Possibly the simplest motif that 14-3-3 binding masks is the phospho-serine/ threonine at the actual 14-3-3 binding site. This has been demonstrated to protect the phospho- residue from attack by protein phosphatases (Chiang, Harris et al. 2001), implying that termination of signalling is a two-step process; requiring the removal of 14-3-3 before dephosphorylation (Margolis, Walsh et al. 2003). The final proposed function of 14-3-3 is to act as a scaffolding molecule during signalling. This was originally described as the mechanism of interaction of the GTPase activating protein Bcr with Raf, a MAPKKK (Braselmann and McCormick 1995). Bcr does not bind Raf directly in a yeast two-hybrid system, but co-expression of 14-3-3 allows complex formation. The possibility of using 14-3-3 as a scaffolding molecule is appealing. As 14-3-3 binds predominantly to phosphorylated proteins, this would allow several levels of regulation of the one signalling process. In this model each substrate must be independently phosphorylated before 14-3-3 binding can occur; leading to transduction of the signal. 14-3-3 has also been reported to bring a kinase into close proximity of its substrate, allowing phosphorylation. In brain, GSK3 forms a complex with both 14-3-3 and its substrate, the tau protein (Agarwal-Mawal, Qureshi et al. 2003). This study also demonstrated that addition of exogenous 14-3-3 in an in vivo model dose-dependently increased tau phosphorylation by GSK3.

Figure 1-4: Functional Outcomes of 14-3-3 Binding. Binding of 14-3-3 to phosphoproteins has many outcomes. 14-3-3 can cause a conformational change in the bound protein (A); mask localisation signals and cause a change in protein localisation (B); protect proteins from further modification, such as dephosphorylation as shown in (C) or control the formation of protein complexes (D).

33 The “14-3-3 Interactome”

The interaction of 14-3-3 with a substrate protein has usually been described by searching for proteins that specifically interact with the substrate, rather than searching for specific substrates of 14-3-3 (For example, see (Ramm, Larance et al. 2006)). The majority of these studies identified 14-3-3 as co-purifying with the substrate from tissue lysate, or via an interaction in a yeast two-hybrid system where the substrate protein was used as the bait. Large scale genomic (Tong, Lesage et al. 2004) and proteomic (Gavin, Bosche et al. 2002) studies in S. cerevisiae uncovered novel substrates of the yeast 14-3-3 homologues BMH1 and BMH2, although the reported interactions differed between studies (Bridges and Moorhead 2005). Several large-scale studies searching for novel 14-3-3 interacting proteins have now been reported. These studies have used a variety of starting materials and affinity purification procedures, resulting in wide variation in the proteins identified. It has been estimated that as little as 25% of proteins are in common between these studies (Bridges and Moorhead 2005). Generally, an affinity purification step is followed by protein identification using tandem mass spectrometry. Meek et al searched for 14-3-3 substrates involved in cell cycle regulation by 14-3-3 affinity purification and R18 peptide elution from Hela cells in both interphase and undergoing mitosis (Meek, Lane et al. 2004). Jin et al searched for global 14-3-3 interaction using FLAG-14-3-3 immunoprecipitation and elution with excess FLAG peptide (Jin, Smith et al. 2004). Benzinger et al used tandem affinity tag (TAP) purification to search for novel 14-3-3 interacting proteins (Benzinger, Muster et al. 2005). As the isoform is significantly different in both expression and structure from other 14-3-3 isoforms, the set of proteins identified in this study are probably quite distinct from other studies. One group has performed two large-scale 14-3-3 interaction studies in the last six years. The first study used 14-3-3 affinity chromatography of Hela cell lysate coupled with competitive peptide elution to try and discover novel human proteins that would interact with yeast 14-3-3 (Pozuelo Rubio, Geraghty et al. 2004). The second study is perhaps the most interesting and was published during the course of the present studies. Instead of looking at global 14-3-3 binding, Dubois et al attempted to make some inroads into understanding regulated 14-3-3 binding in Hela and HEK293 cells that had been treated with or without insulin (used as an IGF-1 substitute) (Dubois, Vandermoere et al. 2009). This study attempted to determine the change in 14-3-3

34 binding by employing isotope differential dimethyl labelling to compare proteins from basal and insulin-stimulated states. This is a quantitative mass spectrometry method that allows comparison between samples. Although this study produced some interesting results, the use of the dimethyl labelling technique could possibly introduce bias, which the authors acknowledge. The main drawback with this technique is that the labelling is done after most of the handling steps, meaning that each sample must be processed individually, introducing error. To avoid this bias, a mass spectrometry technique is needed that allows all samples to be processed simultaneously, eliminating handling errors. Fortunately, such a technique exists; SILAC.

Using 14-3-3 as a Tool.

As Akt and 14-3-3 share the same recognition motif, Akt phosphorylation of proteins creates new sites for 14-3-3 binding. This has been demonstrated for several well- characterised substrates of Akt, as described above. In each of these cases, binding of 14-3-3 has a functional outcome, regulating the enzymatic activity of the substrates whether by direct conformational change or changing their localisation. It should therefore be possible to use 14-3-3 as a tool to discover new Akt substrates. By using 14-3-3 as an affinity matrix, it is possible to purify phosphorylated proteins from a complex mixture, such as a cell lysate. The identification of these 14-3-3 binding proteins can then be determined using tandem mass spectrometry. Indeed, this technique has already been used to identify novel 14-3-3 binding proteins (see above). Novel proteins identified by this method can then be studied in depth to determine whether they are bona fide Akt substrates.

However, using 14-3-3 binding as a marker for regulated Akt phosphorylation requires one to compare between basal and stimulated states. Taking a single “snapshot” of the phosphorylation state of the cell is not as informative as determining how phosphorylation changes in response to external stimuli. Akt substrate phosphorylation should correlate with Akt activity, which is induced by growth factors such as PDGF and insulin as described above. Furthermore, by inhibiting Akt activity in conjunction with stimulation, the Akt dependent-branch of the growth factor stimulated

35 phosphorylation event can be isolated. This would allow discrimination between proteins phosphorylated by Akt and those phosphorylated by other kinases.

Aims.

1. To identify novel Akt substrates using 14-3-3 as a marker for Akt-mediated phosphorylation. To do this, I will use 14-3-3 affinity chromatography in conjunction with tandem mass spectrometry. 2. To characterise the biology of these novel proteins identified in Aim 1, with an eye towards discovering the 14-3-3 binding site and confirmation that they are bona fide Akt substrates. 3. To elucidate the functional outcome of 14-3-3’s interaction with these novel proteins.

36 CHAPTER 2

Materials and Methods.

Materials

Dulbecco’s Modified Eagle Medium (D-MEM), minimal essential medium (alpha) (MEM) and F-12 nutrient mixture (Ham) was obtained from Invitrogen (Carlsbad, CA), myoclone-Plus foetal calf serum (FCS) from Trace Scientific (Melbourne, Australia) and antibiotics were from Gibco BRL (Paisley, UK). Insulin was obtained from Calbiochem (San Diego, CA) and bovine serum albumin (BSA) from USB (Cleveland, OH). Bicinchoninic acid (BCA) reagent, Supersignal West Pico chemiluminescent substrate, Triscarboxyethylphosphine (Bond-breaker TCEP neutral pH solution) and protein G agarose beads were from Pierce (Rockford, IL). Lipofectamine LTX, Lipofectamine 2000, Plus Reagent and Sypro Ruby were from Invitrogen (Carlsbad, CA). PVDF membrane was from Millipore (Billerica, MA). Trypsin was from Promega (Madison, WI). Complete protease inhibitor cocktail tablets were from Roche (Indianapolis, IN). Magic C18 material was from Alltech (Deerfield, IL). Gateway cloning reagents including pDONR221, pDEST vectors, BP and LR clonases were from Invitrogen. Unlabelled and stable isotope labelled Arginine and Lysine (>95% pure) were from Cambridge Isotope Laboratories (Andover, MA). All other materials were obtained from Sigma (St. Louis, MO). Pan 14-3-3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Total Akt, Akt pSer473, GST antibodies and recombinant Akt were purchased from Cell Signalling Technology (Danvers, MA) FLAG antibody (clone M2) was purchased from Sigma (St. Louis, MO). Alexa Fluor 680-conjugated secondary antibodies were obtained from Invitrogen. IrDye 800-conjugated secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA). HRP-conjugated secondary antibodies and CNBR sepharose were from GE Healthcare (Buckinghamshire, UK). ATP-32P was from MP Biomedicals (Solon, OH).

37 Plasmids and Constructs

Human Rho GAP 22 cDNA was purchased from Open Biosystems (clone #8992160, corresponding to GenBank accession number AAI26445.1). This construct was amplified using PCR to attach an N-terminal FLAG tag and 5’ and 3’ attB sites, allowing Gateway BP recombination into the pDONR221 vector (Invitrogen). FLAG- Rho GAP 22 was inserted into Gateway converted pMIG IRES GFP retroviral vector (Brummer, Larance et al. 2008) using LR recombination. The S16A, R211A, S395A and S476A mutants were created using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The S16A/S395A and S16A/S395A/R211A mutants were generated by restriction cloning using PstI/XmaI and XbaI/AgeI respectively. Rho GAP 22 fragments corresponding to amino acids 1-147 and 126-714 were amplified from the pDONR221 plasmid using PCR and inserted into pDEST53 (Invitrogen) to create N-terminal GFP fusions. Mouse Rho GAP 22 cDNA was purchased from Open Biosystems (clone #4237331, corresponding to GenBank accession number AAH38272.1). This construct was amplified using PCR to create a fragment corresponding to amino acids 355-609 tagged with attB sites at the 5’ and 3’ ends. The PCR product was inserted into pDONR221 using BP recombination and cloned into pDEST15 and pDEST17 (Invitrogen) by LR recombination to generate N-terminal GST and 6xHis fusions respectively. Myc-tagged p68RacGAP was a gift from C. Patterson (Aitsebaomo, Wennerberg et al. 2004).

Cell Lines

NIH3T3 fibroblasts and L6 myoblasts were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Chinese hamster ovary (CHO) cells stably expressing the insulin receptor and IRS-1 (CHO IR/IRS-1) were a gift from Morris White. Platinum E (Plat-E) cells were a gift from T Kitamura (Morita, Kojima et al. 2000). Double Akt1/2 knockout MEFs and wild-type controls were a gift from Morris Birnbaum (Bae, Cho et al. 2003).

38 Methods

Cell Culture

NIH3T3 fibroblasts were grown in Dulbecco’s modified eagle medium (D-MEM) containing 10% (v/v) FCS and 2 mM L-glutamine, 100 U/L penicillin and 100 μg/L streptomycin (PSG). L6 myoblasts were grown in minimal essential medium alpha (MEM) supplemented with 10% (v/v) FCS and 1% (v/v) antibiotic/antimycotic solution (AA). Differentiation of confluent myoblasts was induced by reducing the amount of serum in the culture media to 2% (v/v) for 6 days. Myotubes were harvested for experimentation on day 7. CHO IR/IRS-1 cells were grown in F-12 nutrient mixture (Ham) containing 10% (v/v) FCS and PSG and G418 (800 g/ml). Plat-E cells were grown in D-MEM containing 10% (v/v) FCS, PSG, 10 g/ml blasticidine and 1 g/ml puromycin. Mouse embryonic fibroblasts derived from either wild type (WT) or double Akt1/2 knockout (DKO) mice were grown in D-MEM supplemented with 10% FCS and

PSG. All cell lines were cultured at 37 °C/10% CO2.

Production of Retrovirus Using Plat-E cells

Plat-E cells (Morita, Kojima et al. 2000) cultured in 10 cm dishes were transfected with 15 g pMIG plasmid DNA encoding various FLAG-Rho GAP 22 constructs using Lipofectamine 2000 according to the manufacturer’s instructions. At 3 h post- transfection, the transfection media was replaced with 8 ml fresh culture media (without blasticidine and puromycin) and cells were incubated at 37 °C/10% CO2 for 24 h. At 24 h post-transfection, culture media was replaced with 6 ml fresh culture media (without blasticidine and puromycin) and cells incubated a further 24 h. At 48 h post- transfection, media containing viral particles was removed and centrifuged at 2000 g for 5 min. Cleared supernatant was stored at -80 °C.

Retroviral Infection of NIH3T3 Fibroblasts and Generation of Stable Cell Lines

NIH3T3 fibroblasts cultured in 10 cm dishes were incubated with pMIG retroviral supernatant in the presence of 4 mg/ml polybrene. At 24 h post infection, cells were

39 sorted for GFP expression using a FACS Vantage SETM cell sorter (BD Biosciences). Cells expressing a moderate amount of GFP (median 20%) were collected and used to establish a polyclonal cell line.

Transient Transfection of CHO IR/IRS-1 Cells Using Lipofectamine LTX

CHO IR/IRS-1 cells were cultured 37 °C 10% CO2 in 10 cm dishes containing 8 ml F- 12 media supplemented with 10% (v/v) FCS and PSG and G418 (800 g/ml). Cells were split the day before to be 90% confluent at the time of transfection. 15 g of each plasmid and 7.5 l PLUS reagent was added to 3 ml F-12 media containing no FCS or antibiotics and incubated at room temperature for 5 min. 30 l Lipofectamine LTX (Invitrogen) was added and incubated for a further 25 min. Culture media was removed and replaced 5 ml F-12 media supplemented with 10% FCS after washing once with this media. The DNA/Lipofectamine/PLUS reagent mixture was added drop wise and mixed by gentle rocking. The following day, media was removed and replaced with 8 ml F-12 media containing 10% FCS. Cells were used 48 h after transfection.

SILAC Labelling of L6 Cells

For SILAC labelling of L6 cells arginine and lysine free MEM was used and supplemented with stable isotope labelled arginine and lysine in addition to dialysed FCS after the method of Olsen (Olsen, Blagoev et al. 2006). L6 myoblasts were cultured as above for at least 6 doublings in this media supplemented either with unlabelled 2 13 15 13 15 13 amino acids (light), H4-Lys and C6-Arg (medium) or N2- C6-Lys and N4- C6-Arg (heavy) labelled arginine and lysine. Three different cell populations were required to allow comparison between serum-starved cells and cells treated with insulin, or insulin and wortmannin. Differentiation was induced by serum withdrawal as described above using MEM supplemented with 2% dialysed FCS and the corresponding stable isotope labelled amino acid.

40 Production of GST Fusion Proteins

10 ml LB media supplemented with 100 μg/ml ampicillin was inoculated using a stab from a glycerol stock and incubated overnight at 37 °C with shaking. The following day, 5 ml was used to inoculate 500 ml LB media supplemented with 100 μg/ml ampicillin. This culture was grown at 37 °C/180 rpm until OD600=0.6. IPTG (final concentration of 1 mM) was deed to induce expression of the fusion protein. Induction was allowed to proceed for 3 h. Bacteria were harvested by centrifugation at 7500 g and the pellet was stored at -20 °C or used immediately. The bacterial pellet was resuspended in 50 μl PBS (supplemented with Complete protease inhibitors (Roche) and 2 mg/ml lysozyme (Sigma) and 40 μg/ml DNaseI) per millilitre of culture. After incubation for 20 min on ice, bacteria were lysed by sonication, Triton X-100 was added to a final concentration of 1% (v/v) and lysates were incubated on ice for 30 min. Cell debris were pelleted by centrifugation at 12,500 g for 30 min at 4 °C. Supernatant was transferred to a new tube and 2 ml of GST Sepharose that had been pre-equilibrated by washing 3 times in cold PBS was added. Lysate/bead slurry was incubated at 4 °C overnight with gentle rotation. Beads were collected by centrifugation for 2 min at 2,000 g, washed 3 times in cold PBS by resuspension and centrifugation, packed into a 5 ml disposable column (Pierce) for elution or thrombin cleavage. For elution as a GST- tagged fusion protein, the column was washed once with 30 ml PBS, 10 ml GST elution buffer was added (50 mM Tris-HCl pH 8.0, 3 mg/ml reduced Glutathione (Sigma)). Fractions (1 ml) were collected and those fractions containing eluted fusion protein were pooled and concentrated using a 15 ml Amicon filter (Millipore). At the same time, the buffer was exchanged for PBS. Purified protein was stored at 4 °C. For elution by thrombin cleavage, the column was first washed with 20 ml TBS followed by 20 ml thrombin cleavage buffer (50 mM Tris-HCl pH8.0, 10 mM CaCl2). 10 U of thrombin (Sigma) in 5 ml thrombin cleavage buffer was added to the capped column and incubated overnight at room temperature. Purified protein was eluted from the column by addition of excess thrombin cleavage buffer. Fractions were collected and dialysed against PBS as described above.

41 Production of 6xHis Fusion Proteins

Preparation of bacterial cultures, protein expression and bacterial lysis were performed as described above for GST fusion protein expression, except that bacteria were lysed in 50 mM Sodium phosphate pH 7.0, 300 mM NaCl, 10 mM Imidazole (Sigma) and EDTA-free Complete protease inhibitors (Roche) . Cleared lysates were combined with 2 ml of pre-equilibrated TALON resin (BD) and incubated at 4 °C overnight with gentle rotation. The following day, beads were collected by centrifugation for 2 min at 2,000 g, washed 3 times in lysis buffer by resuspension and centrifugation and packed into a 5 ml disposable column (Pierce). The column was washed once with lysis buffer and proteins eluted in 10 ml elution buffer (50 mM Sodium phosphate pH 7.0, 300 mM NaCl, 150 mM Imidazole). 1 ml fractions were collected and dialysed against PBS as described above.

Coupling GST or 6xHis Fusion Proteins to CNBR Sepharose

Recombinant fusion proteins were produced as described above and coupled to Cyanogen Bromide (CNBR) beads for pulldown experiments. Purified protein was buffer exchanged into CNBR coupling buffer (0.1 M NaHCO3 pH 8.3, 0.5 M NaCl) using an Amicon filter. Lyophilised CNBR sepharose (GE) was washed for 15 min in 1 mM HCl. Approximately 57 mg of CNBR sepharose was used per milligram of protein. Equilibrated beads were combined with 14-3-3 ligand and incubated for 1 h at RT with rotation. The beads were then washed with 20 ml coupling buffer, and remaining reactive groups blocked by overnight incubation in 100 mM Tris-HCl, pH 8.0. The following day, beads were washed with PBS and stored at 4 °C as 50% slurry.

Production of Rho GAP 22 Antibody

Rho GAP 22 specific antisera were produced by IMVS Veterinary Services Division (Adelaide, SA). Briefly, mouse Rho GAP 22 was amplified by PCR to produce a fragment corresponding to amino acids 355-609 and cloned into pDEST53 as described above to produce an N-terminally tagged GST fusion protein. Rabbits were immunised with 1 mg of antigen emulsified in Freud’s complete adjuvant given subcutaneously at 42 day 0. Subsequent immunisations consisted of 1 mg antigen emulsified in Freud’s incomplete adjuvant given subcutaneously every 3 weeks for a total of 4 immunisations. Rabbits were exsanguinated at week 10.5 and serum collected.

Production of the pSer22 and pSer397 Rho GAP 22 Antibodies

Antibodies were raised against peptides corresponding to amino acids 16-29 (Ac- 22 YFTRSK[pS]LVMGEQSC-NH2 and Ac-CYFTRSK[pS]LVMGEQS-NH2) for Ser or

389-404 (Ac-LPTHRTS[pS]LDGPAAC-NH2 and Ac-CLPTHRTS[pS]LDGPAAA- 397 st NH2) for Ser , of mouse Rho GAP 22 were produced by 21 Century Biochemicals (Malboro, MA). Rabbits were inoculated with Rho GAP 22 pSer22 or pSer397 peptides conjugated to KLH carrier protein five times over a 72 day period. Subsequently, the phospho-specific antibody was affinity-purified by removal of antibodies, which bind the non-phosphorylated peptide and then affinity purification using the phosphorylated peptide.

14-3-3 Pulldown from L6 Myotubes or Mouse Quadriceps or MEF cells

Confluent L6 myotubes cultured in 10 cm dishes were washed 1x with 5 ml of basal media (MEM with no FCS or AA), and cells were serum starved in 5 ml of this media for 2 h. Cells were stimulated by addition of 100 nM Insulin for 30 min. After stimulation, cells were immediately placed on ice, media was aspirated and cells washed 3x5 ml with ice-cold TBS. All liquid was removed and cells were lysed in 1 ml/dish IP buffer (1% (v/v) Igepal CA-630, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% Glycerol (v/v) and complete protease and phosphatase inhibitors). Lysates were passed 10 times through a 22 gauge needle and then incubated on ice for 20 min, followed by centrifugation for 30 min at 18,000 g at 4 °C to pellet insoluble material. The supernatant was reserved and transferred to a new tube. Protein concentration was determined using a BCA assay (Pierce) as per manufacturers’ instructions. Frozen mouse quadriceps (isolated under the approval of the Garvan/St Vincent's animal ethics committee) were lysed using a Polytron homogeniser (Kinematica, Lucerne, Switzerland) for 30 s in IP buffer and incubated for 30 min at 4 °C. Insoluble material was pelleted by centrifugation at 18,000 g for 30 min at 4 °C. Mouse embryonic 43 fibroblasts (MEFs) from wild type (WT) or double Akt1/2 knockouts (DKO) were cultured in 10 cm dishes, washed 1x with 5 ml of basal media (D-MEM with no FCS or PSG) and cells were serum starved in 5 ml of this media for 2 h. Cells were then stimulated by addition of 100 nM Insulin for 30 min, and lysates prepared as for L6 myotubes. Equal protein amounts of each sample were then incubated with 14-3-3- cyanogen bromide (CNBR) beads which had been washed once in IP buffer (50 g of 14-3-3 per 1 mg of lysate, beads have 5 g of 14-3-3 per l of packed bead volume) and then incubated overnight at 4 °C on a rotating wheel. Beads were washed once in cold IP buffer by resuspension and centrifugation at 2000 g for 2 min at 4 °C. Beads were then washed once with IP buffer containing 500 mM NaCl, followed by two washes with IP buffer. Beads were then resuspended in 200 l cold TBS and transferred to a spin column (Pierce). Beads were dried by centrifugation at 500 g for 1 min at 4 °C. Spin columns were transferred to new tubes and proteins eluted by addition of 50 l hot (65 °C) SDS-PAGE buffer containing 50 mM TCEP and incubated at 65 °C for 5 min. Eluate was collected by centrifugation at 500 g for 1 min at room temperature and stored at -20 °C.

Immunoprecipitation (non FLAG)

After serum starvation and stimulation, cells were washed 3 times on ice with ice-cold TBS. All liquid was removed and cells were lysed in 1 ml/dish IP buffer (1% (v/v) Igepal CA-630, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% Glycerol (v/v) and complete protease and phosphatase inhibitors). Lysates were passed 10 times through a 22 gauge needle and then incubated on ice for 20 min, followed by centrifugation for 30 min at 18,000 g at 4 °C to pellet insoluble material. The supernatant was reserved and transferred to a new tube. Protein concentration was determined using a BCA assay (Pierce) as per manufacturers’ instructions. Equal protein amounts of each sample were then incubated with 20 l Protein G Sepharose, which had been washed once in IP buffer and the appropriate antibody. Samples were incubated overnight at 4 °C on a rotating wheel. Beads were washed 3 times in cold IP buffer by resuspension and centrifugation at 2000 g for 2 min at 4 °C. Beads were then resuspended in 200 l cold TBS and transferred to a spin column. Beads were dried by centrifugation at 500 g for 1 min at 4 °C. Spin columns were transferred to new tubes and proteins eluted by addition 44 of 50 l hot (65 °C) SDS-PAGE buffer containing 50 mM TCEP and incubated at 65 °C for 5 min. Eluate was collected by centrifugation at 500 g for 1 min at room temperature and stored at -20 °C.

Immunoprecipitation Using FLAG Antibody

After serum starvation and stimulation, cells were washed 3 times on ice with ice-cold TBS. All liquid was removed and cells were lysed in 1 ml/dish IP buffer (1% (v/v) Igepal CA-630, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% (v/v) Glycerol and complete protease and phosphatase inhibitors). Lysates were passed 10 times through a 22 gauge needle and then incubated on ice for 20 min, followed by centrifugation for 30 min at 18,000 g at 4 °C to pellet insoluble material. The supernatant was reserved and transferred to a new tube. Protein concentration was determined using a BCA assay as per manufacturers’ instructions. Equal protein amounts of each sample were then incubated with 20 l Protein G Sepharose which had been washed once in IP buffer and 1 l of FLAG antibody. Samples were incubated overnight at 4 °C on a rotating wheel. Beads were washed 3 times in cold IP buffer by resuspension and centrifugation at 2000 g for 2 min at 4 °C. Beads were washed once in ice-cold TBS and all liquid removed by aspiration with a microloader tip. FLAG tagged proteins were eluted by addition of 50 μl TBS containing 200 g/ml 3xFLAG peptide. Samples were incubated on ice for one h, with gentle agitation every 20 min. Following incubation, samples were centrifuged at 2000 g for 2 min at 4 °C and 40 l of eluate removed. Samples were prepared for SDS-PAGE by addition of 4x SDS-PAGE buffer and TCEP to a final concentration of 50 mM.

Western Blotting and SDS-PAGE

SDS-PAGE analysis was performed according to the method of Laemilli (Laemilli 1970) on 10% or 7.5% resolving gels with the addition of 50 mM TCEP in the sample buffer. Equal amounts of protein were loaded for each sample in a single experiment, typically 10 g per lane. For mass spectrometric identification SYPRO Ruby staining was performed as per manufacturer’s instructions (Invitrogen). For western blotting proteins were electrophoretically transferred to PVDF membrane, and the membrane 45 was blocked with 5% non-fat skim milk in 0.1% (v/v) Tween20 in TBS (TBST) and incubated with primary antibody in 5% BSA in TBST overnight at 4 °C. After incubation, membranes were washed three times in TBST and incubated with HRP- labelled or Alexa fluor 680/IrDye 800 labelled secondary antibodies in 5% non-fat skim milk in TBST for HRP conjugated secondaries or TBST with 0.01% SDS (w/v) for fluorescent secondaries. Proteins were visualised using Supersignal West Pico chemiluminescent substrate and imaged with X-ray film (Fuji) for HRP-labelled secondary antibodies or a Licor Odyssey imager for Alexa fluor 680/IrDye 800 labelled secondary antibodies.

Peptide Competition Assays

Affinity purified phospho-specific Rho GAP 22 antibodies in 5% BSA in TBST were incubated with 50-fold molar excess of both cognate phosphopeptides dissolved in TBS, 100-fold molar excess of the cognate non-phosphorylated peptide dissolved in TBS or TBS alone for 2 h at room temperature. This antibody solution was used to western blot FLAG immunoprecipitated Rho GAP 22 that had been over-expressed in CHO IR/IRS- 1 cells as described above. Membranes were then incubated with anti-FLAG antisera for 1 h at room temperature and proteins visualised using the Licor Odyssey imager as described above.

In-gel Tryptic Digest for Peptide Identification by LC MS/MS.

FLAG-Rho GAP 22 was transiently expressed in CHO IR/IRS-1 cells as described above, immunoprecipitated using FLAG antibody from either basal or insulin stimulated cells (100 nM 30 min), subjected to 10% SDS-PAGE and stained with SYPRO ruby. Protein bands of interest were excised and de-stained in 1 ml of 50% acetonitrile, 250 mM NH4HCO3 at RT for 45 min with shaking. The gel slice was dehydrated by incubation in 1 ml of 100% acetonitrile for 10 min at RT. All solution was carefully removed using a microloader tip prior to the addition of modified trypsin

(12.5 ng/μl) in 100 mM NH4HCO3 and incubation overnight at 37 °C. The following day peptides were extracted by the addition of 0.1 ml of 5% formic acid and incubation at 37 °C for 1 h. Peptides were further extracted by the addition of 0.1 ml of 100% 46 acetonitrile and incubation at 37 °C for 1 h. The gel slice was completely dehydrated by the addition of 0.5 ml of 100% acetonitrile and incubation at 37 °C for 10 min. The entire supernatant was then removed, transferred to a new tube and vacuum dried. Peptides were re-dissolved in 20 l of 5% formic acid for LC-MS/MS analysis.

Peptide Identification by LC MS/MS

Peptide solutions (5 l) prepared as described above were resolved on a 100 mm x 75 m C18 Magic reverse phase analytical column using a Dionex Ultimate 3000 liquid chromatography system over a 1 h organic gradient with a flow rate of 250 nl/min. Peptides were ionised by nano-electrospray ionisation at 1.8 kV from the end of the column which was pulled to an internal diameter of 5 m by a P-2000 laser puller (Sutter Instruments Co). Tandem mass spectrometry analysis was carried out on a Thermo Scientific (San Jose, CA) LTQ-FT Ultra mass spectrometer. The data dependent acquisition method used was the FT10 protocol as described by Haas (Haas, Faherty et al. 2006). Data were processed, searched and quantified using the Maxquant software version 1.0.13.13 package as described by Cox (Cox and Mann 2008) using the default settings. Searches were performed using Mascot server version 2.2 against the entire Maxquant mouse version 3.52 (with 111130 sequences searched) or human version 3.52 (with 148380 sequences searched) with databases. The settings used for the Mascot search were: 2 missed cleavages allowed; enzyme was Trypsin cleaving after arginine and lysine; variable modifications were methionine oxidation, proprionimide cysteine, phosphorylation of serine, threonine or tyrosine; no fixed modifications were used; a mass tolerance of 7 ppm was used for precursor ions and a tolerance of 0.5 Da was used for fragment ions. Using the default Maxquant settings a maximum false positive rate of 1% is allowed for both peptide and protein identification, this is used as a cut-off score for accepting individual spectra as well as whole proteins in the combined search and quantitation output. This threshold has previously been shown to be a rigorous method of identifying true positive matches (Cox and Mann 2008). Protein quantitation data was always derived from two or more peptides per protein. For the 14-3-3 SILAC screen proteins were included in the study if their ratio had a significance (B) <0.05 as described previously (Cox and Mann 2008) and were detected in both experiments.

47 In-vitro Phosphorylation using Recombinant Akt.

Various FLAG-GFP-tagged Rho GAP 22 constructs were expressed in HEK cells using Lipofectamine 2000 as per manufacturer’s instructions. Cells were cultured for 48 h, then treated with 100 nM wortmannin for 30 min. Cells were lysed on ice and proteins immunoprecipitated using FLAG antibody as above but using RIPA Buffer (0.1% (v/v) SDS, 0.5% (w/v) Sodium Deoxycholate, 1% (v/v) Igepal CA-630, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, EDTA-free complete protease inhibitors and phosphatase inhibitors) and natively eluted using 3xFLAG peptide. Nine microlitres of the eluate was transferred to a new tube and combined with 5 l of 3xAssay buffer (75 mM Tris-HCl 32 pH 7.4, 6 mM DTT, 30 mM MgCl2), 5 l of ATP mix (14.8 kBq/l ATP- P in 800 M ATP dissolved in 1x Assay Buffer) and 1 l diluted Akt kinase (200 ng/l in 1x Assay Buffer). Reactions were incubated at room temperature for 10, 30 or 60 min and stopped by addition of 4x SDS-PAGE buffer to 1x concentration and TCEP to 50 mM and heated at 65 °C for 5 min. Entire sample was subjected to 10% SDS-PAGE and then fixed and stained with Coomassie brilliant blue dye for 30 min. The gel was destained and then equilibrated using MilliQ water and dried in a vacuum gel drier onto filter paper. The dried gel was exposed to a phosphor imager plate overnight and the plate was imaged using a Fujifilm FLA-500 imaging system (Fujifilm, Tokyo, Japan).

Cell Motility Assays

NIH3T3 fibroblasts stably overexpressing various Rho GAP 22 mutants by pMIG-GFP infection were mixed in equal amounts with NIH3T3 fibroblasts infected with a pMIG- mCherry construct and seeded into fibronectin coated (10 μg/ml in PBS for 60min) 24- well plates at a final concentration of 5000 cells/ml. Cells were cultured overnight at 37

°C /10% CO2 using a heated CO2 incubator attachment to a Zeiss Observer inverted microscope (Carl Zeiss, Germany). Epifluorescent images in both the green and red channels were taken every 10 min for about 14 h using a 10x objective. Image stacks were minimally processed in Image J (Abramoff, Magelhaes et al. 2004) using a Gaussian blur and rolling ball algorithm to reduce background. Processed images were analysed using Imaris software (Bit Plane AG, Zurich, Switzerland). Cells were assigned spots in each frame of the image stack, spots were then tracked over time using 48 the Imaris particle tracking module. To qualify for analysis, particles required a minimum size of 9.5 μm, a quality >10 (as defined by the Imaris software) and had to be present for at least 30 consecutive frames (5 h). The total displacement of each cell in the stack over time was calculated and expressed as the mean ratio of the displacement of Rho GAP 22 expressing cells (green) over the displacement of pMIG mCherry control cells (red).

49 CHAPTER 3

Identifying Novel Akt Substrates Using 14-3-3 Affinity Chromatography and SILAC.

Introduction.

The Serine/Threonine kinase Akt acts as a central regulator of insulin’s many effects on cellular metabolism. Phosphorylation of Akt’s many substrates leads to changes in their enzymatic activity and/or localization, leading to alterations in function. Protein phosphorylation is a general mechanism by which protein function is regulated. It is eminently suitable for transduction of transient intracellular signals, such as growth factor signalling, as it is rapid and reversible. There are over 500 kinases encoded in the human genome, with about one third as many protein phosphatases (Cohen 2001). The abundance of these regulators underscores just how important phosphorylation is as a signalling mechanism. It is often said that over 30% (Cohen 2001) of human proteins undergo phosphorylation, however a recent study now indicates that the true figure may be closer to 75% (Olsen, Vermeulen et al.).

Although there are over 100 identified Akt substrates in humans (Table 1-1), it is clear that not all insulin-responsive Akt substrates have been identified, as many signalling pathways activated by insulin remain poorly characterised. Most insulin signalling pathways have been elucidated using a reverse discovery process. Having identified the metabolic actions of insulin, proteins could be assigned to the insulin signalling pathway if their perturbation resulted in defective insulin action. However, this does not allow discovery of new actions of insulin, as it is necessary to have a known endpoint to refer to. The advent of phosphoproteomics allows an alternative method to be employed. Protein phosphorylation in response to an insulin stimulus can be identified using mass spectrometry to detect the mass difference between a phosphorylated and non-phosphorylated peptide. This allows the use of a reverse discovery process, a protein that is phosphorylated in response to an insulin stimulus by definition insulin-

50 regulated. Moreover, this approach would allow new actions of insulin to be discovered, based on the functions of the new substrates that are identified. However, classical mass spectrometry is limited as it cannot provide quantitative information. This is due to the complexity of the sample being analysed. Proteins vary widely in their chemistry, fragmentation fingerprints, and their susceptibility to cleavage by tryptic digestion. This leads to varying ionisation yields for different proteins. Coupled with varying detector responses between runs, these problems make it impossible to compare the results of one mass spectrometry run with another. This fundamental problem has inspired many researchers to invent novel methods to circumvent these issues, or at least to reduce their impact.

One of the most successful approaches is the use of isotopic labelling. By introducing heavier isotopes of elements into the mass spectrometry sample, it becomes possible to compare the relative abundance of peptides containing the normal isotope versus the heavier isotope. Incorporation of the heavier isotope results in a detectable mass shift. As there is no chemical difference between the isotopic peptide species, and they are analysed in the same experiment, quantitation of the differences in their abundance becomes possible. Isotopic labelling has long been used in pharmacological research, whereby deuterated drug analogues serve as a useful reference samples (Browne, Van Langenhove et al. 1981).

Isotope Labelling and Quantitative Mass Spectrometry.

One of the first methods to employ isotope labelling for quantitative mass spectrometry used 15N substituted media. Microorganisms were grown in media where the nitrogen was the heavier 15N isotope, allowing for quantitative differences in protein expression to be compared across yeast strains, as well as differences in protein phosphorylation (Oda, Huang et al. 1999). While useful, this technique was limited in its application to mammalian systems. As the amount of nitrogen atoms varies from protein to protein, it becomes difficult to interpret the resulting mass spectra, especially as 15N incorporation may be less than 100%. Additionally, 15N substituted media are expensive, and difficult to make for mammalian cell culture systems (Ong, Blagoev et al. 2002).

51 The next advance in isotope labelling technology came with the advent of the isotope- coded affinity tag (ICAT) (Gygi, Rist et al. 1999), which has since evolved into the iTRAQ system (Ross, Huang et al. 2004). This technology employs an affinity reagent that is isotope tagged and attached to particular amino acids in all proteins in the sample. Protein samples from differently treated cell populations are tagged with isotopically different affinity tags. After peptides are generated using proteolytic cleavage, the tagged peptides are purified using the affinity tag. Purified peptides are then identified by mass spectrometry. ICAT relies on two assumptions. Firstly, although labelling with the affinity reagent only labels a portion of the available protein, enough unique sequence information may be present to enable protein identification. Secondly, pairs of peptides tagged with the different affinity tags are chemically identical and can therefore be used as mutual standards for accurate quantitation (Gygi, Rist et al. 1999). The iTRAQ system expands on ICAT by increasing the number of treatment groups that can be analysed, and by incorporation of synthetic peptide standards that can be used for absolute quantitation (Ross, Huang et al. 2004). The original ICAT system suffered from non-specific interactions between peptides and the affinity matrix, and was unsuitable for the identification of post-translationally modified proteins (Ross, Huang et al. 2004). The newer iTRAQ system has improved upon these areas.

A competing approach relies on the incorporation of the isotope tag into the protein itself. This approach is known as Stable Isotope Labelling of Amino acids in Cell culture (SILAC). Ong et al recognised that mammalian cells cannot synthesise certain amino acids. These essential amino acids must be supplied externally in the diet when considering the whole organism, or in the growth media when considering cultured cells. By substituting isotopically labelled analogues of these essential amino acids into growth media lacking the “normal” amino acid, proteins synthesised from this media will contain the isotope tag (Ong, Blagoev et al. 2002). Growing the cells in this media for several generations ensures that all cellular proteins are labelled. Furthermore, cell populations can be labelled differently, for example the control group may be grown in media containing “light” amino acids, whereas the treatment group can be grown in “heavy” media. After treatment, the cell populations can be harvested and directly combined, as all proteins will be isotopically tagged, either as “heavy” or “light”. The ability to combine protein samples in this fashion is very powerful; it eliminates any

52 error that may arise downstream as a result of differences in the handling of two separate samples. Proteins can be identified as being from the control or treatment population during mass spectrometry, based on the differences in their mass induced by the difference in isotope that is incorporated into the peptide chain (Fig. 3-1). One advantage of SILAC over ICAT is that the peptide labelling efficiency is greater than 50%, whereas ICAT will label only 20% (Ong, Blagoev et al. 2002). The SILAC method was developed further and improved upon by Olsen et al, who used it to study the temporal regulation of the phosphoproteome (Olsen, Blagoev et al. 2006). SILAC has also been used to study differences in protein expression between normal and knock-out mice (Krüger, Moser et al. 2008), and recently, to study the global regulation of protein phosphorylation during mitosis (Olsen, Vermeulen et al.).

Figure 3-1: A sample SILAC spectra showing the isotope-induced mass difference of the same peptide from three different cell populations. L6 myoblasts were grown in media supplemented either with unlabelled amino acids (Basal), 2H4-Lys and 13C6-Arg (Insulin) or 15N2-13C6-Lys and 15N4-13C6-Arg (Wortmannin + Insulin) labelled arginine and lysine. After being grown in this media for 6 doublings, cells were differentiated by serum withdrawal as described in Materials and Methods, and treated either with (Insulin) or without (Basal) 100 nM insulin for 30 min, or with 100 nM Wortmannin for 30 min, followed by 100 nM insulin for 30 min (Wortmannin + Insulin). Cells were lysed and subjected to 14-3-3 Pulldown as described in Materials and Methods. Shown in this figure is a representative mass spectrum of a phosphopeptide from the TSC2 protein. The same peptide has three different masses, due to incorporation of the three different isotope labels. The increased peak height of the peptide from the insulin-treated cells indicates increased abundance. As all three peptides are analysed in the same experiment, differences in their relative abundance can be calculated. Each peptide gives numerous peaks due to incorporation of other naturally occurring heavy isotopes, namely 13C.

54 Combining SILAC and 14-3-3

With the recognition that Akt phosphorylation of its substrates often creates a 14-3-3 binding site, I hypothesised that I could use 14-3-3 to identify novel targets of Akt that are phosphorylated in response to insulin treatment. By using 14-3-3 as an affinity matrix, it was possible to isolate those proteins that undergo insulin-induced 14-3-3 binding. Furthermore, by using the PI3K inhibitor Wortmannin, it was possible to distinguish Akt-mediated phosphorylation from other insulin-responsive kinases that are not downstream of PI3K. Wortmannin binds to the catalytic subunit of PI3K and irreversibly inhibits its activity (Norman, Shih et al. 1996). When this approach was combined with SILAC, it became possible to quantitatively measure changes in protein abundance between cells treated with or without insulin, or with wortmannin and insulin. As these proteins had previously been purified using 14-3-3, any change in their abundance indicated a change in 14-3-3 affinity, and thus phosphorylation. The experimental approach I used is summarised in Figure 3-2. I chose to use the L6 skeletal muscle cell line, an insulin-responsive cell line originally derived from rat thigh muscle {Yaffe, 1968 #418}. Although it would have been more physiologically relevant to use primary muscle cells, it would have been difficult to SILAC label primary cells in culture. To ensure complete labelling of the cell’s amino acids, it is necessary to use dialysed fetal bovine serum. This medium would therefore lack other growth factors necessary for primary cell culture. L6 cells are therefore an ideal compromise between physiological relevance and the technical requirements of the experimental system.

Figure 3-2: Summary of Experimental Procedure for the SILAC screen for novel 14-3-3 binding proteins. L6 myoblasts are grown in isotopically labelled growth media for 6 doublings to label cellular protein (1). After differentiation (2), cells are treated as indicated and lysed (3). Cell lysates are mixed (4) and subjected to 14-3-3 pulldown (5) and mass spectrometry (6). 56 Results

Akt Substrates Identified in this Study.

By combining 14-3-3 affinity chromatography and SILAC, I have identified 29 proteins that undergo a significant change in 14-3-3 binding state with insulin treatment (Table 3-1). As 14-3-3 predominantly binds to phosphoproteins, it can be assumed that these identified proteins undergo phosphorylation in response to insulin stimulus. Over 400 proteins were identified as binding to 14-3-3 (Supplemental table S3-1) although only the aforementioned 29 had a significant change. The majority of these proteins were also sensitive to wortmannin, indicating that they are downstream of PI3K. There were three exceptions to this general trend. Butyrate response factors 1 and 2 were wortmannin insensitive, as well as the PI3K regulatory subunit . Five of these 29 proteins have previously been identified as Akt substrates. Foxo3a is a member of the Forkhead family of transcription factors by which Akt controls cell survival (Brunet, Bonni et al. 1999). The tuberous sclerosis protein (TSC2) forms a heterodimeric complex with TSC1 and acts as a GTPase activating protein to suppress the activity of the small GTPase Rheb (Tee, Fingar et al. 2002). Phosphorylation of TSC2 by Akt and subsequent 14-3-3 binding dissociates the complex, inhibiting the GAP activity and relieving the repression of Rheb activity (Manning, Tee et al. 2002). Rheb then activates the mTORC1 complex, leading to increased protein synthesis. The proline-rich Akt substrate of 40 kDa (PRAS40) is also involved in the regulation of protein synthesis. PRAS40 binds to the mTORC1 complex and negatively regulates its activity (Sancak, Thoreen et al. 2007). The phosphorylation of PRAS40 by Akt induces 14-3-3 binding, dissociating PRAS40 from mTORC1, allowing activation (Vander Haar, Lee et al. 2007). The Grb2 associated binder 2 (Gab2) functions as a scaffolding protein in many intracellular signalling pathways by recruiting various SH2-domian containing proteins to the PM (Gu and Neel 2003). Phosphorylation of Gab2 by Akt leads to 14-3-3 binding and subsequent termination of signalling (Brummer, Larance et al. 2008). Finally, the Butyrate Response Factor 1 (BRF-1) belongs to the Tis11 family of CCCH zinc-finger proteins. This family of proteins binds mRNAs that contain an AU-rich element (ARE) in their 3 untranslated region. This promotes deadenylation of the mRNA and leads to its degradation (Lai, Kennington et al. 2003). BRF-1 is able to

57 promote mRNA degradation in an in vitro system, and this activity is reduced by phosphorylation of BRF-1 by Akt. It is suggested that this occurs via Akt-induced 14-3-3 binding to BRF-1, which sequesters BRF-1 from other factors of the mRNA degradation machinery (Schmidlin, Lu et al. 2004). Ten proteins included on this list underwent only a minor change (less than 2-fold) in phosphorylation state in response to insulin stimulus (LARP1, LMO7, PI4KB, AHNAK MTSS1L RABGEF1 ABI2 KIF13B SLAIN2 and FOXK1) indicating that insulin probably played only a minor role in the regulation of their phosphorylation. This study also identified five proteins that had not previously been reported as insulin sensitive (EDC3, Rho GAP 22, TRIP12, Beclin-1 and MYCBP2). Many of these proteins are poorly described in the literature, making them very interesting for future study. Phosphorylation of these proteins was wortmannin-sensitive, indicating that they lie downstream of PI3K in the insulin signalling cascade. The enhancer of mRNA decapping protein 3 (EDC3), as its name suggests, is involved in post-transcriptional regulation of mRNA stability (Eulalio, Rehwinkel et al. 2007). EDC3, along with several other proteins and mRNA, forms large structures known as processing (p) bodies, which serve as an inert storage structure for mRNA in the cytosol (Parker and Sheth 2007). The thyroid receptor interacting protein 12 (TRIP12) is a probable E3 ubiquitin-protein ligase and interacts with the ligase domain of the thyroid hormone receptor (Lee, Choi et al. 1995). Beclin-1 plays a central role in regulating autophagy, the process by which a cell recycles organelles into nutrients in response to starvation. Beclin-1 is involved in the formation of the autophagosome, a double membrane structure that surrounds organelles destined for degradation in the lysosome (Liang, Jackson et al. 1999). Myc-binding protein 2 (also known as Pam/highwire/rpm-1 protein, Phr) is a probable E3 ubiquitin-protein ligase that has been reported to play a role in synaptogenesis via its association with the transcription factor Myc (Burgess, Peterson et al. 2004). Finally, the protein Rho GAP 22 has been reported as a GTPase activating protein (GAP) for Rac1, and to play a role in endothelial cell capillary tube formation via an interaction with VEZF-1 (Aitsebaomo, Wennerberg et al. 2004). In the course of this study, Rho GAP 22 was reported as being involved in the regulation of cell migration (Sanz-Moreno, Gadea et al. 2008). Akt had previously been implicated in regulating cell migration, but its role was poorly described.

58 Uniprot Gene Mean SD Mean SD Protein Descriptions Accession Names (I/B) (I/B) (W+I/B) (W+I/B) Q8K2D3 EDC3 Enhancer of mRNA-decapping protein 3 5.96 0.80 1.99 0.52 O88380 TRIP12 thyroid hormone receptor interactor 12 5.35 0.19 0.88 0.15 Q9WVH4 FOXO3 Forkhead box O3a 4.83 0.96 1.22 0.59 Q9D1F4 PRAS Proline-rich AKT1 substrate 1 4.41 0.40 1.25 0.04 Q62433 NDRG1 Protein NDRG1 4.20 0.07 1.23 0.16 Q8BL80 ARHGAP22 Rho GTPase activating protein 22 3.33 0.20 0.69 0.07 Q9EP53 TSC1 Tuberous sclerosis 1 2.93 0.56 1.32 0.15 Q8C570 MRNP41 mRNA export factor 2.83 0.32 1.52 0.15 EH domain-binding protein 1-like Q99MS7 EHBP1L1 2.80 0.29 0.97 0.04 protein 1 P23949 BRF2 Butyrate response factor 2 2.77 0.35 2.03 0.58 Growth factor receptor bound protein 2- Q9Z1S8 GAB2 2.63 1.08 1.02 0.11 associated protein 2 P23950 BRF1 Butyrate response factor 1 2.55 0.38 2.08 0.58 Q61037 TSC2 tuberous sclerosis 2 2.54 0.52 1.10 0.18 Probable E3 ubiquitin-protein ligase Q7TPH6 MYCBP2 2.49 0.00 1.62 0.23 MYCBP2 Phosphatidylinositol 3-kinase regulatory O08908 PIK3R2 2.43 0.20 2.32 0.30 subunit beta Q99J03 BECN1 Beclin 1 2.35 0.12 1.17 0.10 Q148W8 DUSP27 Inactive dual specificity phosphatase 27 2.21 0.08 1.38 0.02 Q9WTX5 SKP1 S-phase kinase-associated protein 1 2.16 0.58 1.52 0.41 Pleckstrin homology-like domain family Q6PDH0 PHLDB1 2.10 0.08 1.03 0.25 B member 1 Q6ZQ58 LARP1 la related protein 1.98 0.08 1.03 0.05 A0T1J8 LMO7 LIM domain only 7 1.83 0.28 1.46 0.13 Q8BKC8 PI4KB Phosphatidylinositol 4-kinase beta 0.61 0.01 0.86 0.16 A0JLR7 AHNAK AHNAK nucleoprotein isoform 1 0.59 0.14 0.80 0.08 Q6P9S0 MTSS1L MTSS1-like protein 0.58 0.04 0.58 0.11 Q9JM13 RABGEF1 Rab5 GDP/GTP exchange factor 0.56 0.13 0.83 0.18 Q6AXD2 ABI2 Abl interactor 2 0.54 0.02 0.59 0.02 O35063 KIF13B kinesin family member 13B 0.54 0.17 1.09 0.10 Q8CI08 SLAIN2 SLAIN motif-containing protein 2 0.52 0.02 0.54 0.07 P42128 FOXK1 Forkhead box protein K1 0.38 0.01 0.81 0.05

Table 3-1: Insulin-responsive proteins identified by SILAC and 14-3-3 affinity chromatography. Twenty-nine proteins had a significant change in phosphorylation state after insulin treatment, as assayed by binding to 14-3-3. Results are expressed as mean ratio of fold change in 14-3-3 binding with insulin stimulation (I) or wortmannin and insulin stimulation (W) compared to basal (B). S.D = standard deviation. 59 Bioinformatic Analysis of Novel Akt Substrates

To determine whether the novel proteins identified in this screen were Akt substrates, I undertook bioinformatic analysis to determine probable Akt phosphorylation sites and 14-3-3 binding motifs. Potential Akt phosphorylation sites and 14-3-3 binding sites were predicted using the Scansite (Obenauer, Cantley et al. 2003) or Eukaryotic Linear Motifs tool (Puntervoll, Linding et al. 2003). As 14-3-3 generally binds disordered regions of proteins (Bustos and Iglesias 2006), the GlobPlot algorithm (part of the ELM resource) was used to predict protein disorder around putative 14-3-3 binding sites. This analysis showed that all five novel insulin-regulated proteins were predicted to contain 14-3-3 binding sites that corresponded to putative Akt phosphorylation sites (Table 3.2). Moreover, all but one of these predicted 14-3-3 binding sites occurred within regions of predicted protein disorder. All predicted Akt and 14-3-3 sites fell into the top 1% of all sites in the Scansite database, indicating that they were likely to be real sites. Mode 3 14-3-3 binding sites were predicted using the ELM resource, which does not rank sites. These data indicated that the five novel insulin-regulated proteins were probable Akt substrates, and that phosphorylation by Akt would probably generate a binding site for 14-3-3.

60 Binding Predicted UNIPROT Akt Sites and site within Name 14-3-3 Sites Accession Scores disordered and Scores region? S161 0.451% S161 0.008% Y Q8K2D3 EDC3 S231 0.652% S231 0.924% Y T262 0.273% T262 0.217% N T18 0.735 % S22 0.838 % S22 0.699 % Y Q8BL80 Rho GAP 22 T431 0.350 % S397 Mode 3 S397 0.664 % Y S36 0.393% S36 0.848% Y T152 0.224% S236 0.033% S236 0.325% Y O88380 TRIP12 S238 0.028% S238 0.141% Y S312 0.154% S312 0.667% Y T328 0.700% T1421 0.033% T1421 0.067% Y S29 Mode 3 Q99J03 Beclin 1 S232 0.851 % S293 0.460 % S293 0.977 % N S89 0.198% S586 0.673% S861 0.126% S2440 0.098% Q7TPH6 MYCBP2 S2817 0.618% S2905 0.393% S2905 0.102% Y S2930 0.548% S3460 0.036% S3460 0.341% Y

Table 3-2: Bioinformatic analysis of novel 14-3-3 binding proteins identified in this study. Five proteins that had not previously been reported to bind 14-3-3 or be insulin responsive were analysed using Scansite and ELM for the presence of putative Akt phosphorylation sites and 14-3-3 binding sites. Protein disorder was predicted using the GlobPlot algorithm.

Novel Proteins Identified in this Study Bind 14-3-3 in vivo

To confirm that this SILAC screen had indeed identified novel 14-3-3 binding proteins, antisera specific for several of the novel proteins were obtained. These were used to immunoblot a 14-3-3 pulldown. Mouse quadriceps were prepared and used in a 14-3-3 pulldown as described in Materials and Methods. Immunoblotting the pulldowns with antisera specific for the novel insulin-regulated proteins revealed that both Edc3 and Beclin-1 bound 14-3-3 in both the fed state and in muscle that had been acutely stimulated with insulin (Fig. 3-3). AS160 and GEF-H1, known 14-3-3 binding proteins, were immunoblotted as a control AS160 was found to bind 14-3-3 in quadriceps that had been acutely insulin-treated and to a lesser extent in the fed state. GEF-H1 bound

61 14-3-3 in the fed state, and in the insulin stimulated state, albeit with some variability between mice.

Figure 3-3: Confirmation that proteins identified by SILAC are true insulin - responsive 14-3-3 binding proteins. Twenty week old male C57BL/6 mice were either fasted overnight followed by mock IP injection (FAST), ad libitum fed overnight followed by mock IP injection (FED) or fasted overnight followed by an IP injection of 1U/kg insulin (FAST+I). Three individual mice were used in each group. Mice were sacrificed 10min after injection, quadriceps excised and subjected to 14-3-3 pulldown as described previously. Novel 14-3-3 substrates such as Edc3 and Beclin-1 bind 14-3-3 in both the fed and insulin- stimulated state. AS160, a known 14-3-3 substrate is included as a control.

63 Discussion

By combining SILAC, a method to quantitatively measure changes in protein abundance in mass spectrometry, with 14-3-3 affinity chromatography, this study has identified 29 proteins that undergo a significant change in 14-3-3 binding after insulin treatment. As 14-3-3 generally only binds phosphoproteins, it can be assumed that insulin stimulation causes a change in the phosphorylation state of these proteins. Ten proteins underwent only a minor change in 14-3-3 binding state after insulin stimulation, indicating that insulin probably plays only a minor role in their regulation. Nevertheless, the criteria used when analysing the mass spectrometry data ensure that though minor, these changes were significant. Of the 19 remaining proteins, the binding of 14-3-3 to 16 was significantly inhibited by prior treatment with the PI3K inhibitor wortmannin, indicating that that are downstream of PI3K in the insulin signalling cascade. This excludes the possibility that these proteins are regulated by pathways, other than Akt, that are also activated by insulin, such as the MAPK or c-Cbl/TC10 pathways. This study identified five proteins that had previously been shown to be Akt substrates and 14-3-3 binding proteins. The presence of known proteins in the cohort indicates that the experiment was successful in identifying Akt/14-3-3 regulated proteins. Of the eleven remaining proteins, five were selected for further study, based on the degree of change in their 14-3-3 binding state, as well as their novelty. Bioinformatic analysis demonstrated that these proteins are probable substrates for Akt and 14-3-3. The remainder of this study will focus on one of these proteins, Rho GAP 22.

Notably, several known Akt substrates are absent from the list. One of the most obvious absentees is glycogen synthase kinase 3, a well known Akt substrate (Cross, Alessi et al. 1995). Phosphorylation of GSK-3 by Akt is known to cause 14-3-3 binding at Ser9 (Agarwal-Mawal, Qureshi et al. 2003). The majority of glucose that is taken up by skeletal muscle is metabolised into glycogen, a process that is controlled GSK-3. Not only is GSK-3 absent from Table 3.1, indicating that it did not undergo a significant change in 14-3-4 binding, it was not identified as binding to 14-3-3 at all (Supplemental table S3-1). The failure of this study to identify GSK-3 is puzzling, and GSK-3 can therefore serve as a surrogate for other known 14-3-3 substrates that should have been

64 identified. Some previously identified Akt substrates (such as AS160) are expressed at low levels in L6 cells, thus they were unlikely to be identified by this screen (Chavez, Roach et al. 2008). However, GSK-3 is known to be expressed in L6 cells and expression is up-regulated during differentiation into myotubes {MacAulay, 2005 #419}. There are several reasons why this study failed to identify proteins such as GSK-3. The first and most obvious explanation is one of timing. It is possible that only a small proportion of proteins such as GSK-3 are phosphorylated at the 30 min time point used in this experiment. This would to a relatively small amount of GSK-3 binding to the 14-3-3 column, making the relative abundance of GSK-3 peptides very low. As the mass spectrometer has a limited analytical capacity, only the 10 most abundant peptides eluting from the LC column at any one time are selected for fragmentation and identification. It is entirely possible that peptides from proteins such as GSK-3 were always less abundant than other peptides eluting at the same time, thus they would never be identified. The second possibility concerns the affinity of GSK-3 for 14-3-3. If the affinity of GSK for 14-3-3 is low compared to other proteins, it may be possible that GSK-3 is out- competed for 14-3-3 binding by other higher-affinity substrates. GSK-3 has only one 14-3-3 binding site, and there is structural evidence to suggest the affinity of 14-3-3 for proteins with two binding motifs is 30-fold higher than that for substrates with only one binding motif (Yaffe, Rittinger et al. 1997). Finally, it is possible that the GSK-3/14-3-3 interaction is isoform dependent. Several reports have indicated that isoform-specific binding is possible (see Chapter 1) although the fact that 14-3-3 can heterodimerise makes the biological relevance of this unclear. Nevertheless, GSK-3 was first identified as binding to the isoform (Agarwal-Mawal, Qureshi et al. 2003). The human isoform was used as the affinity matrix for the screen described in this chapter and so it is possible that GSK-3 was not detected as it preferentially binds the isoform. However, it should be noted that a number of 14-3-3 proteins were identified in the SILAC screen by mass spectrometry (Supplemental Table S3-1), indicating that significant heterodimerisation occurred between the affinity matrix and cellular 14-3-3 protein. Thus, the affinity matrix could be thought of as a mixed population of 14-3-3 isoforms, making isoform specific binding unlikely.

65 To enable identification of non-abundant proteins, it may be beneficial to subject the protein sample to further enrichment steps prior to 14-3-3 pulldown and tryptic digest. For example, by performing a subcellular fractionation of the crude lysate, it would be possible to enrich for 14-3-3 substrates in different subcellular compartments such as the cytosol, nucleus and plasma membrane. This approach would also allow identification of proteins that undergo a 14-3-3-induced change in their localisation.

Interesting, several proteins identified in this screen appear to undergo induction of 14- 3-3 binding in mouse muscle despite only minimal activation of Akt in the fed state (Fig. 3-3). This binding probably reflects the fact that a very small amount of Akt phosphorylation is required for robust activity in vivo {Ng, 2008 #433}. The results of the experiment using fed mice would therefore illustrate the more physiological role of Akt rather than the supra-physiological stimulation that is typically seen in cell culture models.

Using a combination of 14-3-3 affinity chromatography and SILAC, this study has identified several promising novel 14-3-3 binding proteins. These proteins are presumably Akt substrates as their 14-3-3 binding is down-regulated by inhibition of PI3K. Confirming the Akt-mediated interaction of these proteins will serve as the basis for future study, as well as investigation of the role they play in the regulation of insulin’s many metabolic effects.

66 CHAPTER 4

Rho GAP 22 is an Insulin-Responsive 14-3-3 Binding Protein

Introduction

One of the most interesting proteins identified in the SILAC screen was Rho GAP 22. This protein is a GTPase activating protein with specific activity for Rac1, a master regulator of the actin cytoskeleton and cell motility. The binding of Rho GAP 22 to 14-3-3 was increased 4-fold in response to insulin stimulation, and this interaction was dependent on PI3K signalling. Rho GAP 22 demonstrated the third highest fold change in 14-3-3 binding, after TRIP12 and Edc3. Binding of 14-3-3 has been shown to regulate the GAP activity and function of other GAP proteins; most notably the Rab GAP AS160 (Ramm, Larance et al. 2006), and recently the Rho GAP DLC-1 (Scholz, Regner et al. 2009). However, the involvement of 14-3-3 in regulating the activity of Rac1 has not yet been reported. The remainder of my studies will focus on the biology of Rho GAP 22, with an emphasis on the role of 14-3-3. This chapter will confirm that Rho GAP 22 binds to 14-3-3 in an insulin responsive manner; characterise a Rho GAP 22 antibody; delineate the basic biology of Rho GAP 22 and demonstrate that it binds directly to 14-3-3.

Rho GAP 22 in the Literature

At the commencement of this study, little was known about the biology of Rho GAP 22, and it had not previously been reported to bind 14-3-3. Rho GAP 22 was originally identified as a novel VEZF-1 binding protein in a yeast 2-hybrid assay (Aitsebaomo, Wennerberg et al. 2004). Bioinformatic analysis revealed the presence of a Rho-GAP domain, indicating that this protein, originally named p68RacGAP by Aitsebaomo et al, was a GTPase activating protein. The same study demonstrated that p68RacGAP had GAP activity specifically towards Rac1, and not RhoA or Cdc42, the other best-

67 characterised members of the Rho family of small GTPases (Wherlock and Mellor 2002). Subsequent large-scale genomic analyses revealed that p68RacGAP has multiple isoforms, generated by alternate splicing in both Homo sapiens and Mus musculus. Full length Rho GAP 22, the product of the ARHGAP22 gene was first cloned by Katoh and Katoh (Katoh 2004). The full-length isoforms were given the systemic name of Rho GAP 22 based on their similarity to Rho GTPase activating protein 1, the product of the ARHGAP1 gene. Therefore, p68RacGAP is an alternate name for Rho GAP 22 isoform 2 in mouse. Aitsebaomo et al demonstrated that p68RacGAP co-localises with the VEZF-1 transcription factor and over-expression of p68RacGAP dose-dependently represses endothelin-1 promoter activity. Furthermore, adenoviral-mediated over-expression of p68RacGAP in HUVECs inhibited the formation of capillary tube-like structures in vitro. It was proposed that p68RacGAP inhibits Rac1, leading to an inhibition of lamellipodia formation and a defect in coordinated migration of endothelial cells. However, the authors failed to show a specific role for the GAP activity of p68RacGAP in this process, leaving the molecular mechanism unclear. A recent study by Sanz-Moreno et al, examining the switch from mesenchymal to amoeboid cell migration, implicated Rho GAP 22 as a Rac1 suppressor, stimulating cells to migrate in an amoeboid fashion (Sanz-Moreno, Gadea et al. 2008). The authors suggested that Rho GAP 22 suppresses Rac1 activity in response to intracellular signalling through RhoA and its effector ROCK. While the authors demonstrated that ROCK activation was sufficient to suppress Rac1 activity, this was not mediated by direct action of ROCK on Rho GAP 22. Instead, the authors suggested an indirect mechanism mediated by actomyosin contractility. While there are 68 members of the Rho GAP family (see below), only knockdown of Rho GAP 22 in A375M2 cells, led to increased Rac-GTP levels. Knockdown of the closely related family members Rho GAP 24 and Rho GAP 25 had no effect on Rac-GTP levels in the same cell line. This result demonstrated the importance of Rho GAP 22 in regulating Rac activity and cell migration. However, these effects may also be cell-type dependent, and so it will be of interest to determine if this dominant effect of Rho GAP 22 knockdown holds true in other cell lines, aside from melanoma-derived tumour cell lines.

68 The Rho GAP Family of Proteins

The presence of a Rho-GAP domain defines Rho GAP 22 as a member of the Rho GAP family of proteins. There are 68 members of this protein family in Homo sapiens, all either possessing or being predicted to have the ability to stimulate the GTPase activity of members of the Rho family of small GTPases. Thus, Rho GAP proteins have an important role in regulating the activity of Rho GTPases, as the majority of Rho GTPase/effector interactions are GTP-dependent. Rho family GTPases play many roles in diverse biological processes, such as focal complex and focal adhesion assembly, cell cycle progression, membrane trafficking, cell adhesion and cell polarity (Hall 1998). Many of these processes are dependent on remodelling of the actin cytoskeleton, providing a common mechanism of control by Rho GTPases. There are at least 21 members of the Rho small GTPase family in humans, with RhoA, Cdc42 and Rac1 being the most well-studied (Peck, Douglas et al. 2002). In contrast, there are at least 68 predicted Rho GAP proteins in the human genome. The reason for this abundance of GAPs is not understood and remains an active area of research. Each Rho GAP may possess GTPase activating activity towards several Rho GTPases, although this can be difficult to determine because the specificity of GAP activity can differ depending on whether an in vivo or in vitro assay is employed. For example, Rho GAP p190A, which possesses equal GAP activity for RhoA, Cdc42 and Rac1 in vitro (Settleman J, Albright CF et al. 1992), exhibits a preference for RhoA in vivo (Ridley, Self et al. 1993). Other GAPs such as -Chimaerin (Diekmann, Brill et al. 1991) and Rho GAP 22, would seem to possess GAP activity towards only one Rho GTPase. How the specificity of GAP activity is controlled is unclear, and the preferential specificity of different GAPs for RhoA, Cdc42 or Rac1 cannot be predicted from the amino acid sequence of the Rho GAP domain (Peck, Douglas et al. 2002). It should be noted that the presence of a predicted Rho GAP domain does not always correlate with GAP activity. For example, OCRL-1, 5PTase and the PI3K regulatory subunits p85 and all possess putative GAP domains, but lack a crucial invariant arginine residue in the GAP domain that is required for GAP activity. PI3K subunits p85- and have been demonstrated to be catalytically inactive (Zheng, Hart et al. 1993), and OCRL-1 and 5Ptase are also predicted to lack GAP activity (Peck, Douglas et al. 2002). Interestingly, p85 and can bind GTP-loaded Cdc42 in vitro (Zheng, Bagrodia et al. 1994) and p85 has been

69 reported to act as a scaffold during cytokinesis by binding to both Cdc42 and septin 2 (Garcia, Silio et al. 2006). To investigate where Rho GAP 22 lies within this family, a multiple sequence alignment was performed and a phylogenetic tree was constructed. The sequence of all 68 human proteins containing Rho GAP domains were obtained from the UNIPROT database (The UniProt Consortium 2009) and aligned using the Clustal W algorithm (Larkin, Blackshields et al. 2007) using default options. Figure 4-1 demonstrates the evolutionary relationships between family members. Based on this alignment, the closest homologs to Rho GAP 22 are FilGAP (Rho GAP 24) and Rho GAP 25. Both of these proteins have the same domain organisation as Rho GAP 22 and both are predicted to have multiple 14-3-3 binding sites by sequence homology, although this has not been demonstrated experimentally. FilGAP was originally identified as a Filamin A binding protein in a yeast 2-hybrid assay (Ohta, Hartwig et al. 2006). Ohta et al also demonstrated that FilGAP had GAP activity towards Rac1 and Cdc42 both in vitro and in vivo, with a slight preference for Rac1. This, and other results, led the authors to propose that the interaction of FilGAP with Filamin-A acts to antagonise Rac1 activity in response to ROCK activation by RhoA. In contrast to FilGAP, the ARHGAP25 gene (encoding Rho GAP 25) was cloned based on its homology to ARHGAP22 (Katoh 2004) but has not otherwise been reported in the literature. Rho GAP 22, FilGAP and Rho GAP 25 thus constitute a subfamily of Rho GAPs that remains poorly characterised.

Figure 4-1: Phylogenetic structure of the Rho GAP family of proteins. Sequences of all 68 human proteins containing a Rho GAP domain were retrieved from UNIPROT and aligned using the Clustal W algorithm. See text for details. Rho GAP 22 is highlighted in red. 71 Domain Organisation of Rho GAP 22

Rho GAP 22 contains 3 conserved protein domains as predicted by sequence homology using the SMART tool (Letunic, Doerks et al. 2009); an N-terminal Pleckstrin Homology (PH) domain (residues 38-147), a Rho GAP domain, defining it as a member of the Rho GTPase activating protein family (residues 140-376), and a C-terminal coiled-coil domain (residues 607-687), thought to be important for protein-protein interactions. This coiled-coil domain in FilGAP (Rho GAP 24) is necessary for dimerisation and binding to Filamin A (Nakamura, Heikkinen et al. 2009). Figure 2a shows the domain organisation of Rho GAP 22, with putative 14-3-3 binding sites indicated by red circles.

Figure 4-2: Bioinformatic analysis of Rho GAP 22 (A) Schematic Representation of the domain organisation of Rho GAP 22 isoform 1. PH = Pleckstrin Homology domain, RhoGAP = Rho GTPase activating domain, CC = Coiled Coil domain. Red circles indicate putative 14-3-3 binding sites. (B) Conservation of 14-3-3 binding sites of Rho GAP 22 across various species. Red text indicates a putative 14-3-3 binding site. Numbering refers to human Rho GAP 22 isoform 1.

73 Results

Rho GAP 22 Binds 14-3-3 in an Insulin-Responsive Manner

To confirm our initial results from the SILAC experiment, myc-tagged p68RacGAP (murine Rho GAP 22 isoform 2) was transiently over-expressed in insulin-responsive CHO cells (CHO IR/IRS-1). This cell line is amenable to transient transfection using lipid-based transfection reagents and is highly insulin-responsive, making it ideal for these experiments. Lysates from these cells were subjected to 14-3-3 pulldown. Figure 3a shows that p68RacGAP binds 14-3-3 in response to insulin stimulation, in a PI3K dependent fashion. This validates the SILAC screen that was used to identify insulin- responsive 14-3-3 binding proteins. To demonstrate that p68RacGAP binds 14-3-3 directly and not via an intermediary protein, myc-p68RacGAP was immunoprecipitated using anti-myc antisera from CHO IR/IRS-1 cells. No myc-p68RacGAP was immunoprecipitated with normal rabbit IgG. The 14-3-3 binding potential of immunoprecipitated myc-p68RacGAP was determined in a far-western analysis using GST-14-3-3, (Fig. 4-3b). A specific interaction was observed between GST-14-3-3 and myc-p68RacGAP. This interaction was significantly higher when p68RacGAP was immunoprecipitated from insulin-stimulated cells and it was dependent on PI3K activity. This confirms that 14-3-3 interacts directly with p68RacGAP in an insulin responsive manner, as seen in Figure 3a. This indicates that Rho GAP 22 is phosphorylated with insulin treatment as 14-3-3 only binds to phosphorylated serine or threonine residues (Obata, Yaffe et al. 2000). The identification of the 14-3-3 binding site will be the focus of the next chapter. Having demonstrated that exogenous p68RacGAP binds 14-3-3 in an insulin-dependent manner, I sought to recapitulate this result using endogenous Rho GAP 22.

Figure 4-3: Rho GAP 22 binds 14-3-3 in response to insulin stimulus (A) Myc-p68RacGAP (Rho GAP 22 isoform 2) or empty vector (V) was expressed in CHO IR/IRS-1 cells. Cells were serum starved and treated as basal (B), with100 nM insulin for 30 min (I) or 100 nM Wortmannin for 30 min and then 100 nM insulin for 30 min (Wm). Cell lysates were subjected to 14-3-3 pulldown and blotted with indicated antibodies. (B) Myc-p68RacGAP (Rho GAP 22 isoform 2) was expressed in CHO IR/IRS-1 cells. Cells were serum starved and treated as basal (IgG and B), with 100 nm insulin for 30 min (I) or 100 nM Wortmannin for 30 min and then 100 nM insulin for 30 min (Wm). Cells lysates were immunoprecipitated with either normal IgG as control (IgG) or anti- myc antibody. Phospho-Rho GAP 22 was detected using GST 14-3-3 in a far-western blot.

75 Production of a Rho GAP 22 Antibody

As there were no commercially available Rho GAP 22 antibodies at the commencement of this project, it was necessary to produce one. This antibody was directed against the disordered region between the Rho GAP and coiled coil domains, as this region has the least homology to other Rho GAP proteins. This region is also common to all isoforms of Rho GAP 22, so this antibody will allow detection of all predicted isoforms. Accordingly, a fragment of murine Rho GAP 22 corresponding to amino acids 126-714 was fused to an N-terminal GST tag and specific anti sera against this fusion protein were produced and affinity purified as described in Materials and Methods. Figure 4a shows that this antibody specifically recognises recombinant myc-tagged murine Rho GAP 22 isoform 2 (p68RacGAP) as well as FLAG-tagged human Rho GAP 22 isoform 1, when over-expressed in CHO IR/IRS-1 cells. This was confirmed by concurrent blotting with anti-sera specific for myc and FLAG as indicated. The Rho GAP 22 antibody appeared quite specific in that only one major band of the appropriate molecular weight was detected. The slight cross-reactivity seen in cells over-expressing FLAG Rho GAP 22 may indicate a breakdown product of this protein, especially as there was no cross-reactivity in cells overexpressing myc-p68RacGAP. The absence of a band of the appropriate molecular weight in the vector control lane may indicate that this antibody is incapable of detecting hamster Rho GAP 22 or that the expression of endogenous Rho GAP 22 in these cells is below the limit of detection. To show that this antibody is capable of detecting endogenous Rho GAP 22, various mouse tissue lysates were immunoblotted (Fig. 4-4b). Interestingly, the highest expression was seen in testis, liver and brown adipose tissue. Rho GAP 22 was not detected in brain or eye. The presence of multiple bands in the testis and brown adipose tissue could indicate the expression of multiple isoforms in these tissues. Rho GAP 22 was also expressed in quadriceps, albeit at lower levels, validating the original identification of this protein in skeletal muscle cells. As our laboratory is primarily concerned with studying the effects of insulin on peripheral tissues such as skeletal muscle and adipose tissue, I sought to uncover a role for Rho GAP 22 in skeletal muscle using the L6 cell line as a model.

Figure 4-4: Characterisation of the Rho GAP 22-specific antibody and tissue distribution of Rho GAP 22. (A) CHO IR/IRS-1 cells were transfected with the indicated Rho GAP 22 constructs or empty vector. Cell lysates were blotted with the indicated antibodies. (B) Various mouse tissue lysates (10 μg per lane) were blotted with the indicated antibodies. BAT = brown adipose tissue, WAT = white adipose tissue, Quad = quadriceps.

77 Endogenous Rho GAP 22 Binds 14-3-3 in an Insulin-Responsive Manner

To demonstrate that endogenous Rho GAP 22 binds 14-3-3, serum-starved L6 myotubes were incubated with or without 100 nM insulin for 30 min or insulin and wortmannin. Cells were lysed and subjected to 14-3-3 pulldown. The 14-3-3 pulldowns were immunoblotted with the Rho GAP 22 specific antibody described above. Rho GAP 22 bound 14-3-3 in response to insulin stimulation in a PI3K-dependent manner (Fig. 4-5a). This result is in complete agreement with the experiments using over- expressed myc-p68RacGAP. To further investigate the dynamics of Rho GAP 22/14-3-3 binding, 14-3-3 pulldowns were performed using L6 myotubes that had been treated with insulin for varying times. Figure 4-5b shows that the binding of 14-3-3 to Rho GAP 22 was time-dependent. The binding of Rho GAP 22 to 14-3-3 could be detected after one minute of insulin stimulation. Maximum binding occurred after 30 min of insulin treatment and the amount of bound 14-3-3 returned to basal levels after 120 min. Rho GAP 22 therefore binds 14-3-3 rapidly after insulin stimulation, but this binding is slow to increase to a maximal level and is sustained for a relatively long period. It should also be noted that this Rho GAP 22 antibody detects a non-specific band in L6 myotubes, visible just below the insulin responsive band (Fig. 4-5b). Having demonstrated that endogenous Rho GAP 22 binds 14-3-3 in a cell line, it was decided to repeat this result using a more physiologically relevant approach.

Figure 4-5: Endogenous Rho GAP 22 binds 14-3-3 in response to insulin treatment. (A). Endogenous Rho GAP 22 binds 14-3-3 in an insulin and PI3K dependent manner. L6 myotubes were serum starved (B), and treated with 100nM insulin for30min (I) or insulin and 100nM wortmannin (Wm). Cell lysates were subjected to14-3-3 pulldown and immunoblotted with indicated antibodies. (B). Binding of Rho GAP 22 to 14-3-3 is time dependent. L6 myotubes were serum starved and treated with 100nM insulin as indicated. 14-3-3 pulldowns were performed as above. Maximum 14-3-3 binding is attained at 60min.

79 Rho GAP 22 Binds 14-3-3 in Isolated Mouse Quadriceps

Mouse quadriceps were prepared and used in a 14-3-3 pulldown as described in Materials and Methods. Immunoblotting the pulldowns with the Rho GAP 22 antibody revealed binding of Rho GAP 22 to 14-3-3 in the fed state, but not in the fasted state. Surprisingly, an increase in binding of Rho GAP 22 to 14-3-3 was not detected in mice acutely treated with insulin (Fig. 4-6), despite robust activation of Akt. GSK3, an Akt substrate (Cross, Alessi et al. 1995), that has previously been shown to bind 14-3-3 (Yuan, Agarwal-Mawal et al. 2004), was present in 14-3-3 pulldowns from both fed and insulin treated animals. Regardless, based upon the results observed in the fasting to fed transition it is clear that a similar regulation of Rho GAP 22 to that observed in vitro is also observed in vivo

Full-length Rho GAP 22 Binds 14-3-3 Directly

As previously mentioned, there are multiple isoforms of Rho GAP 22. The overexpression experiments described above utilised myc-p68RacGAP, which is analogous to mouse Rho GAP 22 isoform 2. This isoform lacks the N-terminal PH domain and a well-conserved 14-3-3 binding site. This isoform was originally detected in endothelial cells, and is predicted to be 68kDa. However, the insulin-responsive protein detected in 14-3-3 pulldowns from L6 cells and isolated quadriceps has an apparent molecular weight greater than this and probably represents the full-length protein, also known as isoform 1. To demonstrate that this isoform also binds 14-3-3, full-length human Rho GAP 22 was obtained and engineered with an N-terminal FLAG tag. FLAG-tagged proteins can be natively eluted from FLAG antibody using an excess of FLAG peptide. This allows for the detection of endogenous 14-3-3 bound to FLAG-Rho GAP 22. This protein was exogenously over-expressed in CHO IR/IRS-1 cells and 14-3-3 pulldowns and immunoprecipitations were performed as in Figure 4-3. As opposed to previous results using myc-p68RacGAP (Fig. 4-3) there was a significant interaction between FLAG-Rho GAP 22 and 14-3-3 in serum-starved cells but this interaction was increased with insulin stimulation in a PI3K dependent manner (Fig 4-7). This can be seen in both the far western using GST-14-3-3 and in the co-immunoprecipitation of endogenous

80 14-3-3. These results indicate that there is significant constitutive phosphorylation of FLAG-Rho GAP 22.

Figure 4-6: Rho GAP 22 binds 14-3-3 in isolated mouse quadriceps. Twenty week old male C57BL/6 mice were either fasted overnight followed by mock IP injection (FAST), ad libitum fed overnight followed by mock IP injection (FED) or fasted overnight followed by an IP injection of 1 U/kg insulin (FAST+I). Three individual mice were used in each group. Mice were sacrificed 10 min after injection, quadriceps excised and subjected to 14-3-3 pulldown as described previously. Rho GAP 22 binds 14-3-3 in the fed state, but not in the fasted state and only partially with acute insulin stimulation. GSK3, a known 14-3-3 substrate is included for comparison.

Figure 4-7: Full-length Rho GAP 22 binds 14-3-3. FLAG-Rho GAP 22 isoform 1 was expressed in CHO IR/IRS-1 cells. Cells were serum starved and treated as basal (IgG and B), with 100 nm insulin for 30 min (I) or 100 nM Wortmannin for 30 min and then 100 nM insulin for 30 min (Wm). Cells lysates were immunoprecipitated with either normal IgG as control (IgG) or anti-FLAG antibody.

83 Discussion.

In this chapter, I have demonstrated that both Rho GAP 22 isoforms 1 and 2 bind 14-3-3 in response to insulin treatment and this interaction is dependent on PI3K activity. Most interestingly, Rho GAP 22 isoform 1 bound a significant amount of 14-3-3 in serum- starved cells as opposed to isoform 2, which exhibited very little 14-3-3 binding without insulin stimulation. Moreover, it appeared that isoform 1 had a higher level of constitutive phosphorylation than isoform 2. This can be seen in the far-western blot in Figure 4-7. Bioinformatic analysis using Scansite (Obenauer, Cantley et al. 2003) and Eukaryotic Linear Motifs (ELM) (Puntervoll, Linding et al. 2003) software indicated that there are 8 putative 14-3-3 binding sites in Rho GAP 22. Multiple sequence alignment using Clustal W revealed that several of these sites are well conserved across species, as indicated in Figure 4-2b. Seven of these potential 14-3-3 binding sites are common to isoforms 1 and 2. However, isoform 2 lacks the most N-terminal of these motifs, which is N-terminal to the Pleckstrin homology domain. It is possible that this site is constitutively phosphorylated in isoform 1, leading to constitutive 14-3-3 binding. The insulin-responsive 14-3-3 binding could be mediated by phosphorylation at another site that is common to both isoforms. It remains unclear whether endogenous Rho GAP 22 behaves in this fashion. Endogenous Rho GAP 22 certainly binds 14-3-3 in an insulin-responsive fashion, but this does not preclude the possibility that two 14-3-3 dimers are binding to one Rho GAP 22 protein, with only one doing so in an insulin- responsive fashion. One way of investigating this would be to immunoprecipitate endogenous Rho GAP 22 from L6 cells and look for GST-14-3-3 binding as a marker of phosphorylation, comparing this to the amount of endogenous 14-3-3 that co- immunoprecipitates. Another approach would be the use of size-exclusion gel chromatography to determine the molecular weight of the Rho GAP 22/ 14-3-3 complex although this could be complicated by the presence of other proteins in the complex. The published rat Rho GAP 22 sequence lacks one of the highly conserved 14-3-3 binding sites (S16 in the human sequence). The longest published rat protein sequence in the UNIPROT and GenBank databases differs from that of other species in that it lacks the N-terminal PH domain and it is conceivable that this represents a misannotation of the rat genome. An analysis of the latest release of the sequence databases used by the MaxQuant search software shows that the human proteome is the

84 most complete (174214 sequences), followed by mouse (114106 sequences) with rat a distant third (79878 sequences). While the disparity in sequence number between human and mouse can be attributed to the large genetic distance between these two species, this is not likely the case for rat and mouse. Hence it is quite likely that rat does express other isoforms of Rho GAP 22. Indeed, there is a transcript of the ARHGAP22 gene in the Ensemble rat genome database that has the N-terminal predicted 14-3-3 binding site and PH domain (Transcript: IPI00358035.3), although this is not yet in curated sequence databases such as UNIPROT. The data described here are consistent with Akt being the Rho GAP 22 kinase in as far as the interaction is PI3K dependent, and Akt activity correlates with 14-3-3 binding to Rho GAP 22. Subsequent experiments have shown that the 14-3-3/ Rho GAP 22 interaction is Akt dependent and that Akt can phosphorylate Rho GAP 22 in vitro (Chapter 5). However, the inability of in vivo administration of insulin to mice to activate 14-3-3 binding to Rho GAP 22 was surprising, particularly since robust activation of alternate Akt substrates was observed in the same samples. Several possibilities may explain these findings. Firstly, the amount of Rho GAP 22 in the 14-3- 3 pulldown from the three fed samples may be under-represented compared to the other conditions, as the amount of 14-3-3 in the pulldown was apparently reduced. This was probably an artefact of the Western blotting procedure, as the same amount of GST-14- 3-3 beads were used in each pulldown. Secondly, there was a significant amount of phosphorylated (indicated by 14-3-3 binding) Rho GAP 22 present in fasted mice, and relative to the experiments performed in L6 cells, only a modest increase in Rho GAP 22 binding to 14-3-3 was observed in vivo even with feeding. Hence, the sensitivity of detection of Rho GAP 22 phosphorylation in vivo may be much less than in vitro. It should be noted that even in vitro the kinetics of Rho GAP 22 phosphorylation was rather slow and so it may be that in the studies shown here where only one time point was examined the peak of Rho GAP 22 phosphorylation may have been missed. Finally, it is quite possible that the majority of immunoreactive Rho GAP 22 detected in quadriceps lysate is not expressed in myotubes but rather in endothelial cells. This would support other studies showing high expression of Rho GAP 22 in these cells (Aitsebaomo, Wennerberg et al. 2004). If this were the case it could be that the majority of 14-3-3 binding to Akt substrates like GSK3 is due to phosphorylation by Akt in

85 muscle cells, while the regulation by Akt in endothelial cells may be somewhat different. Further study is needed to resolve this. Having demonstrated that Rho GAP 22 is a 14-3-3 binding protein, the next chapter will focus on the identification of the 14-3-3 binding site and determine which kinase phosphorylates Rho GAP 22.

86 CHAPTER 5

Mapping the 14-3-3 Binding Site and its Kinase

Introduction

Having demonstrated that Rho GAP 22 is an insulin-responsive 14-3-3 binding protein, this chapter will focus on identifying the 14-3-3 binding site and the kinase responsible for its phosphorylation. Based on sequence homology, there are 8 potential 14-3-3 binding sites in Rho GAP 22. There are three main modes of 14-3-3 binding, the recognition sequences of which are shown in Table 1, as well as the sequences of the predicted 14-3-3 binding sites in Rho GAP 22 and the Akt recognition sequence.

Position -5 -4-3 -2 -1 0 +1 +2 +3

14-3-3 Mode 1 X XR SFWY X S/T X P X

14-3-3 Mode 2 X R X SYFWTQAD X S/T X PLM X

14-3-3 Mode 3* X X RHK STALV X S/T X PERSDIF X

Ser16 R A R S K S L V M

Ser395 A H R T S S L D G

Thr410 S R T A P T G P G

Thr424 G K K V Q T L P S

Ser439 Q P R S L S G S P

Ser476 H R R A S S G D R

Thr491 V Q R L S T Y D N

Thr615 L C R Q R T E Y E

Akt Consensus R X R X X S/T X X X

Table 5-1: Sequences of predicted 14-3-3 binding sites in Rho GAP 22 compared to known 14-3-3 binding motifs.

The mode 1 14-3-3 consensus sequence was originally derived from screening a library of phosphopeptides based on the 14-3-3 binding site in Raf1 (Muslin, Tanner et al. 1996) . Subsequent structural studies performed by Yaffe et al uncovered the basis of the 14-3-3/substrate interaction and identified a novel 14-3-3 recognition motif, subsequently referred to as the mode 2 site (Yaffe, Rittinger et al. 1997). However, the stringency of these motifs remains unclear. Various non-canonical interactions have been shown, leading to the proposal of an alternate mode 3 binding motif, based on the sequence similarities in these novel interactions (Puntervoll, Linding et al. 2003). Notably, the mode 3 motif is similar to mode 1, and so the mode 3 motif may simply reflect plasticity in 14-3-3/substrate recognition. Hence, this may represent the “true” mode 1 14-3-3 binding motif. I have marked this sequence with an asterisk to highlight this uncertainty. The “Mode 3” nomenclature was originally given to a C-terminal sequence identified by Coblitz et al, using a genetic screen to identify C-terminal signal motifs that override endoplasmic reticulum localisation (Coblitz, Shikano et al. 2005). This motif was distinct from the mode 1 and 2 motifs, having the form SWpTX-COOH. Other studies revealed other novel 14-3-3 binding motifs, one of which is the so-called “Serine-rich” motif in the adaptor protein Cbl (Liu, Liu et al. 1997). This sequence contains multiple serine residues and depending on which residue is phosphorylated is analogous to the mode 1 or mode 2 motifs described above. Another atypical motif was identified in keratin. This site, RPVSSAApSVY, is distinct from the mode 1 and 2 motifs (Ku, Liao et al. 1998). Various other non-canonical interaction motifs have been reported; such as YpTV in the plant H(+)-ATPase (Fuglsang, Visconti et al. 1999) and KGQSTpSRG in human p53 (Waterman, Stavridi et al. 1998). In the case of p53, double serine phosphorylation in the interaction motif prevents 14-3-3 association; dephosphorylation at Ser376 is required to allow 14-3-3 binding at Ser378. This demonstrates that the specificity of the 14-3-3/ substrate interaction is more complex than initially proposed. While 14-3-3 proteins were originally described as binding solely to phosphopeptides, it is becoming clear that this is not the case. 14-3-3 binds to the motif DALDL in ADP- ribosyltransferase exoenzyme S from Pseudomonas aeruginosa (Henriksson, Francis et al. 2002), the synthetic R18 sequence that was identified through phage display (Wang, Yang et al. 1999) and the 43 kDa Inositol Polyphosphate 5-Phosphatase (Campbell,

88 Gurung et al. 1997). In this latter case, the presence of multiple negatively charged glutamate residues in the identified recognition motif (RSESEE) is speculated to substitute for the negatively charged phosphate group in the more classical motif. Rho GAP 22 appears to be another non-canonical 14-3-3 substrate. Of the two 14-3-3 binding sites (see below), only one (Ser395) has the classical recognition sequence (mode 3), while the other, Ser16 lacks the appropriate residue in the +2 position. The second part of this chapter concerns the search for the kinase that phosphorylates the 14-3-3 recognition motif. Ser16 and Ser395 are predicted to be high stringency Akt sites (Table 1), although as described below this may be erroneous.

Results

Phosphopeptide Identification using Semi-Quantitative Mass Spectrometry

To identify potential phosphorylation sites in Rho GAP 22 that could encode insulin- regulated 14-3-3 binding sites, I used semi-quantitative mass spectrometry. The advantage of this method is that it can identify all potential phosphorylation sites in a protein in an unbiased way. In addition, this approach potentially overcomes the need to mutate each of the 8 potential 14-3-3 binding sites individually. FLAG-Rho GAP 22 was immunoprecipitated from CHO IR/IRS-1 cells that had been serum starved and treated with or without 100 nM insulin for 30 min, as described in Materials and Methods. Samples were subjected to SDS-PAGE and the FLAG-Rho GAP 22 band was excised and peptides were extracted via in-gel tryptic digestion. Extracted peptides were identified by Liquid chromatography tandem mass spectrometry. By comparing the abundance of each phosphopeptide with its cognate non-phosphorylated peptide in each experiment, a relative measure of the fold change with insulin was obtained. The annotated results of this analysis are shown in supplemental table S5-1, as well as sample mass spectra for selected phosphopeptides of putative 14-3-3 binding sites (Supplemental Fig. S5-1 and S5-2). Figure 5-1 shows a summary of these data, displayed as the mean relative fold change in phospho versus non-phosphorylated peptides. All sequence numbering refers to isoform 1 of the human Rho GAP 22 protein. Of the 5 phosphopeptides identified, only Ser16, Ser359 and Ser395 exhibited a change in phosphorylation with insulin treatment. However, only Ser16 and Ser395 are

89 predicted to form part of a potential 14-3-3 binding site. Phosphopeptides comprising a third potential 14-3-3 binding site at Ser476 were identified. This peptide was not included in the above analysis, as it was miscleaved by trypsin due to the presence of an N-terminal arginine close to the phospho-serine. This gave rise to alternate peptide species, rendering it unsuitable for this kind of analysis. Subsequent experiments showed that Ser476 is not a significant contributor to Rho GAP 22’s ability to bind 14-3-3 (Fig. 5-2).

Figure 5-1: Insulin stimulation increases phosphorylation at Ser16, Ser359 and Ser395. The phosphorylation of FLAG-Rho GAP 22 under basal and insulin-stimulated conditions was analysed by semi-quantitative mass spectrometry. Results are shown as the mean increase in phosphopeptide abundance expressed as fold change over basal. The mean of three experiments is shown, error bars indicate standard deviation.

91 14-3-3 Binds Rho GAP 22 at Ser16 and Ser395

To confirm that Ser16 and Ser395 were the insulin-responsive 14-3-3 binding sites, I used site-directed mutagenesis. Each mutant, as well as the wild type protein, was FLAG- tagged and immunoprecipitated from CHO IR/IRS-1 cells that had been serum starved and treated with or without 100 nM insulin for 30 min, or with 100 nM wortmannin for 60 min and 100 nM insulin for 30 min. The ability of each protein to bind 14-3-3 in vivo and in vitro was determined by western blotting for co-immunoprecipitated 14-3-3 and by far-western blotting with GST 14-3-3, respectively. Figure 5-2a shows that the S16A mutation greatly reduced insulin-responsive in vivo binding to 14-3-3, but did not totally ablate it. The S16A mutant was still competent to bind 14-3-3 in vitro, although this binding appeared to be insulin and wortmannin insensitive. The S395A mutant displayed reduced in vivo binding to 14-3-3, but not to the same extent as the S16A mutant. This mutant also bound 14-3-3 in vitro in an insulin- and wortmannin- dependent fashion. The S476A mutant had only slightly reduced in vivo binding to 14-3-3 and yet this mutation had a greater effect on 14-3-3 binding in vitro than the S395A mutation. All three sites contributed to 14-3-3 binding in vitro, as indicated by the reduction in binding compared to the wild type protein. These results suggested that more than one phospho site is responsible for the observed insulin-responsive 14-3-3 binding in vivo. To test this hypothesis, a FLAG-tagged S16A/S395A mutant was generated by site- directed mutagenesis and immunoprecipitated from CHO IR/IRS-1 cells that had been serum starved and treated with or without insulin (100 nM) for 30 min. This mutant was unable to bind 14-3-3 in vivo (Fig. 5-2b) and bound only trace amounts of 14-3-3 in vitro. This suggests that both Ser16 and Ser395 are required for insulin-responsive 14-3-3 binding, although the stoichiometry of the interaction is unclear. The residual amount of 14-3-3 that bound in vitro was probably bound to the Ser476 site, although this site did not contribute to binding in vivo. As this site did not bind 14-3-3 in vivo, and only bound a marginal amount in vitro, it was not deemed of major significance.

Figure 5-2: Insulin stimulation increases 14-3-3 binding to Ser16 and Ser395. Residues on Rho GAP 22 were mutated either singly (A) or in combination (B) to determine the insulin-responsive 14-3-3 binding site. FLAG-tagged proteins were over- expressed in CHO IR/IRS-1 cells. Cells were serum starved and treated with no additions (B), with 100 nM insulin for 30 min (I) or 100 nM Wortmannin for 30 min and then 100 nM insulin for 30 min (W). Cell lysates were subjected to FLAG immunoprecipitation and immunoblotted with indicated antibodies, or used in a far- western with GST 14-3-3 as a probe.

93 Production of Phospho-Rho GAP 22 Antibodies

To further investigate the regulation of phosphorylation at Ser16 and Ser395, I generated phospho-specific antibodies against peptides containing these phospho-serines (see Materials and Methods). These antibodies were directed against the sequence of murine Rho GAP 22, where Ser22 is equivalent to Ser16 and Ser397 is equivalent to Ser395 in the human protein. To demonstrate the specificity of these antibodies, they were used to western blot immunoprecipitates and lysates from CHO IR/IRS-1 cells expressing wild type and mutant Rho GAP 22 (Fig. 5-3). Each phospho-antibody was specific for its cognate phospho-serine, as mutation of that serine totally ablated binding. Use of these antibodies also confirms that both Ser16 and Ser395 were phosphorylated with insulin treatment. This increase was most apparent when the cell lysate was western blotted, probably because the signal from the FLAG immunoprecipitate was approaching saturation. However, a slight increase was still detectable. It is unclear why both the phospho-specific and FLAG antibodies detect a doublet instead of a single band. To demonstrate that these antibodies specifically recognise only the phosphorylated protein, a peptide competition assay was employed. Each phospho-antibody was pre- incubated with either the cognate phospho or non-phosphopeptide at 100 fold molar excess, before being used in a western blot of FLAG-immunoprecipitated protein, as described in Materials and Methods. Each phospho-antibody only bound to its cognate phosphopeptide, as indicated by reduced binding to the immunoprecipitated protein (Fig. 5-4). Labelling with the phospho-specific Ser397 antibody was almost totally out- competed using a 100 fold molar excess of the cognate phosphopeptide. However, the avidity of the phospho-specific Ser22 antibody was greater and would probably need a greater excess of antigen to totally abolish binding. Taken together, these results indicated that these antibodies only recognised the appropriate phosphorylated residues on Rho GAP 22, and did not bind the protein at other non-specific sites. Unfortunately, subsequent experiments blotting cell lysates for endogenous Rho GAP 22 showed that these antibodies recognised other proteins non- specifically, limiting their use for immunoblotting whole cell lysates. I was unable to circumvent this issue by immunoprecipitating endogenous Rho GAP 22 using the total antibody and immunoblotting the immunoprecipitate. This was not a problem when blotting exogenously expressed protein, presumably because it was expressed at higher

94 levels than the endogenous protein. Use of these antibodies was limited to blotting exogenously expressed Rho GAP 22.

Figure 5-3: Both phospho-specific Ser22 and Ser397 antibodies are specific for their cognate sites in Rho GAP 22. Various FLAG-Rho GAP 22 constructs were expressed in CHO IR/IRS-1 cells that were treated with or without 100 nM insulin for 30 min as indicated. Cell lysates were immunoprecipitated with FLAG antibody and western blotted as indicated.

Figure 5-4: The phospho-specific Rho GAP 22 antibodies bind only their cognate phosphopeptide. FLAG-Rho GAP 22 was expressed in CHO IR/IRS-1 cells that were treated with or without 100 nm insulin for 30 min as indicated, before lysis and FLAG immunoprecipitation. The immunoprecipitate was subjected to SDS-PAGE and western blotted with phospho-specific antibody that had been pre-incubated with either TBS or 100-fold Molar excess of the cognate phosphorylated or non-phosphorylated peptide as indicated.

97 Insulin Treatment Causes Phosphorylation at Ser16 and Ser395

Having demonstrated that the phospho-specific Rho GAP 22 antibodies specifically recognise phosphorylated Rho GAP 22, they were used to confirm that Ser16 and Ser395 were phosphorylated in response to insulin stimulation. Accordingly, NIH3T3 fibroblasts stably overexpressing FLAG-Rho GAP 22 (see Materials and Methods) were treated with 20 ng/ml PDGF for various times and cell lysates were FLAG- immunoprecipitated and western blotted with the phospho-specific antibodies (Fig. 5-5). PDGF was used in this instance, as NIH-3T3 fibroblasts are more sensitive to PDGF than insulin (Ridley and Hall 1992) and the signalling pathways are very similar, both converging at PI3K to signal via Akt and being susceptible to inhibition by wortmannin, although I have not demonstrated that PDGF-stimulated Rho GAP 22 phosphorylation is inhibited by wortmannin in this system. In accordance with previous results, the amount of 14-3-3 that co-immunoprecipitated was greatest at 30 min, indicating maximum phosphorylation. This was mirrored by the phospho-Ser397 antibody, which also reached a maximum at 30 min before decaying back to basal levels at 120 min. However, there was a small amount of phosphorylation in the basal state, which was not reflected in the amount of 14-3-3 that co-immunoprecipitated possibly because it was below the level of detection. The Ser16 site behaved slightly differently. The amount of basal phosphorylation was much higher and while the maximum amount of phosphorylation was also reached at 30 min, it did not decay as rapidly. It is tempting to conclude that Ser395 was responsible for the majority of growth factor- stimulated 14-3-3 binding, as phosphorylation at this site closely paralleled the amount of 14-3-3 that co-immunoprecipitated. However, previous data (Fig. 5-2) indicated that Ser16 is the site that binds the majority of 14-3-3. This disparity indicates that the regulation of Rho GAP 22/14-3-3 binding may be more complex than previously thought.

Figure 5-5: PDGF stimulation in NIH3T3 fibroblasts causes phosphorylation at Ser16 and Ser395. NIH3T3 fibroblasts stably expressing FLAG-Rho GAP 22 were serum-starved and treated with 20 ng/ml PDGF for the indicated amounts of time. Cells lysates were FLAG-immunoprecipitated and blotted with the indicated antibodies.

99 Phosphorylation at Ser16 and Ser395 is Reduced by Inhibition of Akt

A major aim was to identify the kinase(s) that regulate phosphorylation and 14-3-3 binding of Rho GAP 22. To this end, I used several kinase inhibitors in conjunction with the phospho-specific Rho GAP 22 antibodies described earlier. Kinases selected for inhibition were all involved in the insulin signalling pathway (wortmannin, Akti, rapamycin, PD98059 and Gö6983), or in the control of Rac1 signalling (Y27632). Akti is a specific Akt inhibitor that binds to the PH domain of Akt preventing its association with the plasma membrane and thus activation by PDK-1 {Green, 2008 #430}. These inhibitors were used at concentrations that had previously been shown to inhibit their targets. Accordingly, FLAG-Rho GAP 22 was immunoprecipitated using FLAG antibody from CHO IR/IRS-1 cells that had been serum starved and incubated with or without 100 nM insulin for 30 min, or with 100 nM insulin for 30 min and various kinase inhibitors. The immunoprecipitate was western blotted using the phospho-Rho GAP 22 antibodies (Fig. 5-6). Phosphorylation at Ser16 was reduced when cells were pre-treated with wortmannin, as expected. A reduction in phosphorylation at Ser16 was also seen when cells were exposed to Akti, a selective Akt inhibitor, although to a lesser extent than wortmannin. Pre-treatment with wortmannin and Akti also reduced phosphorylation at Ser395. Neither Ser16 phosphorylation nor Ser395 phosphorylation were inhibited by pre-treatment with an mTOR inhibitor (rapamycin), an inhibitor of the ROCK family of kinases (Y27632), an inhibitor of MEK1, also known as MAPK and Erk (PD98059) or an inhibitor of various members of the PKC family (Gö6983). While it has already been shown that the kinase responsible for regulating the binding of 14-3-3 to Rho GAP 22 was downstream of PI3K via the use of wortmannin, this new result places the kinase at the level of Akt or a kinase downstream of Akt.

100

Figure 5-6: Insulin-stimulated phosphorylation of Ser16 and Ser395 is inhibited by Wortmannin and Akti. CHO IR/IRS-1 cells expressing FLAG-Rho GAP 22 were serum-starved and treated with (I) or without (B) 100 nM for 30 min insulin as indicated, or with 100 nM insulin for 30 min after 100 nM Wortmannin for 30 min (Wm); 5 M Akti for 30 min (Akti); 20 nM Rapamycin for 30 min; 10 M Y27632 for 30 min (ROCKi); 50 M PD98059 for 30 min (MEKi) or 100 nm Gö6983 for 30 min (Gö6983) before lysis and western blotting with indicated antibodies.

101 Inhibition of Akt Reduces 14-3-3 Binding to Endogenous Rho GAP 22

The previous result demonstrated that the specific inhibition of Akt is sufficient to prevent phosphorylation of Rho GAP 22 at Ser16 and Ser395, the residues responsible for insulin-stimulated 14-3-3 binding. However, this was via the use of over-expressed Rho GAP 22, which is not an ideal experimental system. To recapitulate this result in a more physiologically relevant way requires the use of endogenous Rho GAP 22. Although the phospho-specific antibodies are unsuitable for western blotting of the endogenous protein, the ability to bind 14-3-3 in a 14-3-3 pulldown assay can be used as an indirect marker of phosphorylation. I have already demonstrated that Ser16 and Ser395 are the major 14-3-3 binding sites; it can therefore be assumed that 14-3-3 binding indicates phosphorylation of these two residues. Cell lysates of L6 myotubes that had been serum starved and treated with or without insulin, or with insulin and various kinase inhibitors, were subjected to 14-3-3 pulldown as described in Materials and Methods. Only wortmannin and Akti were able to prevent the insulin-stimulated binding of 14-3-3 to Rho GAP 22 (Fig. 5-7), while the other kinase inhibitors had no effect. Phosphorylation of Rho GAP 22 correlated reasonably well with the activity of Akt, as measured by Ser473 phosphorylation. There was however some residual 14-3-3 binding in cells treated with Akti where Akt activity was not totally inhibited, and 14-3-3 binding appeared to be increased with rapamycin treatment. Notably, Akt Ser473 was also up- regulated with rapamycin treatment. These data further implicate Akt as the kinase responsible for Rho GAP 22 phosphorylation at Ser16 and Ser395.

102

Figure 5-7: Insulin-stimulated 14-3-3 binding to endogenous Rho GAP 22 is inhibited by Wortmannin and Akti. Six day old L6 myotubes were serum starved and treated with (I) or without (B) 100 nM for 30 min insulin as indicated, or with 100 nM insulin for 30 min after 100 nM Wortmannin for 30 min (Wm); 5 M Akti for 30 min (Akti); 20 nM Rapamycin for 30 min; 10 M Y27632 for 30 min (ROCKi); 50 M PD98059 for 30 min (MEKi) or 100 nm Gö6983 for 30 min (Gö6983) before lysis and 14-3-3 pulldown. Cell lysates and 14-3-3 pulldowns were western blotted with the indicated antibodies.

103 Akt is Capable of Phosphorylating Rho GAP 22 in vitro

While my previous data are sufficient to implicate Akt as the kinase that phosphorylates Ser16 and Ser395 of Rho GAP 22, they do not rule out the possibility that a kinase downstream of Akt is involved. To confirm that Akt is indeed the kinase, I sought to demonstrate that Rho GAP 22 is an Akt substrate. Analysis of the sequence of human Rho GAP 22 using Scansite (Obenauer, Cantley et al. 2003) indicates that there are 3 potential Akt sites in Rho GAP 22. These sites, Ser16, Thr189 and Ser395, are within the top 1% of Akt sites in the Scansite database, indicating that they are very good Akt sites. As I have identified both Ser16 and Ser395 to be 14-3-3 binding sites, there is a high likelihood that the binding of 14-3-3 to Rho GAP 22 is regulated by Akt. To confirm that Rho GAP 22 is indeed an Akt substrate, an in vitro kinase assay was used, as described in Materials and Methods. To simplify the interpretation of the results, I elected to analyse each of the 14-3-3 binding sites individually by not using the full- length protein. Instead fragments corresponding to residues 1-147 and 126-714 were used. The N terminal Rho GAP 22 truncation mutant (1-147) was phosphorylated by Akt in vitro, in a time-dependent manner. The phosphorylation of the 1-147 N-terminal fragment was substantially, although not completely, reduced when Akt was omitted from the assay. This residual phosphorylation may reflect the presence of a small amount of Akt co-immunoprecipitating with the 1-147 N-terminal fragment. Phosphorylation was totally absent in the S16A mutant, indicating that this was the only site of phosphorylation in this polypeptide.

104

Figure 5-8: Recombinant Akt2 can phosphorylate Ser16 of Rho GAP 22 in vitro. FLAG-GFP-tagged Rho GAP 22 constructs were expressed in HEK cells by transfection with Lipofectamine 2000. Cells were treated with 100 nM wortmannin for 30 min, lysed and subject to FLAG-immunoprecipitation. Eluted proteins were diluted in reaction buffer and incubated with 74 kBq ATP-32P with or without 200 ng of recombinant Akt2 as indicated and incubated for various time points. Reactions were run on a 10% SDS-PAGE gel and dried to filter paper. Incorporation of 32P was measured using a phospho-imager (Auto-rad). The gel was stained with Coomassie to show total protein loading (Coomassie). The bottom panel shows expression of the various FLAG-GFP-Rho GAP 22 constructs in HEK cells.

105 The FLAG-GST-126-714 construct also exhibited a time-dependent increase in phosphorylation (Fig. 5-8). However, phosphorylation of this mutant was not reduced by the S395A mutation, indicating that phosphorylation likely occurs at an alternate site. As with the 1-147 N-terminal fragment, residual phosphorylation was again seen in the 126-714 fragment when Akt was omitted from the reaction. Again, this is probably due to the presence of a small amount of co-immunoprecipitating Akt. Thus, it is concluded that Ser395 is not phosphorylated by Akt in vitro, but this peptide contains an alternate Akt site, possibly at Thr189. This was surprising, as I had previously used myc- tagged p68RacGAP (murine Rho GAP 22) in a kinase inhibitor screen similar to that shown in Figure 5-7. This construct lacks the N-terminal portion of the full protein, including the Ser16 14-3-3 binding site. Using this construct, it was apparent that only wortmannin and Akti inhibited insulin-stimulated 14-3-3 binding (Fig. 5-9). The reduction in 14-3-3 binding with Gö6983 was not reproducible. This result indicates that inhibition of Akt prevents phosphorylation at Ser395, as well as 14-3-3 binding to this site. It would seem that this site is not phosphorylated by Akt, although active Akt is required for its phosphorylation. These data illustrate that Rho GAP 22 can be phosphorylated by Akt at one of the 14-3-3 binding sites and inhibition of Akt prevents phosphorylation at both sites.

106

Figure 5-9: Wortmannin and Akti inhibit insulin-stimulated 14-3-3 binding to myc-p68RacGAP. CHO IR/IRS-1 cells were transfected with myc-tagged p68RacGAP (murine Rho GAP 22 isoform 2) or vector (V) and serum starved. Cells were stimulated with (I) or without (B) with 100 nM insulin for 30 min, or 100 nM wortmannin (Wm); 5 M Akti (Akti); 100 nM Gö6983 (Gö6983); 5 M KN-62 (KN-62); or 20 nM rapamycin for 30 min followed by 100 nM insulin for 30 min. Cells were lysed and lysates used in a 14-3-3 pulldown and immunoblotted with the indicated antibodies.

107 Akt is Necessary for Insulin-Stimulated 14-3-3 Binding to Rho GAP 22

Although kinase inhibitors are useful tools, there is still an inherent risk in their use. It is possible that their target kinases will not be completely inactivated, as seen with the use of Akti in Figure 5-7. Many kinase inhibitors also have off-target effects, inhibiting various other kinases at different levels of efficiency. To avoid these pitfalls, and to confirm that Akt was indeed required for phosphorylation of Rho GAP 22, the 14-3-3 pulldown experiments were recapitulated in Akt 1/2 knock-out mouse embryonic fibroblasts (Akt DKO MEF) (Bae, Cho et al. 2003). These cells were serum-starved and then treated with or without insulin or with insulin and wortmannin and cell lysates were subjected to 14-3-3 pulldown as described in Materials and Methods. Insulin- stimulated 14-3-3 binding to Rho GAP 22 in the absence of Akt 1 and 2 was almost totally absent (Fig. 5-10). There was still a very small amount of binding, probably caused by Akt 3, the presence of which could be inferred from the trace amount of Ser473 phosphorylated Akt in these cells. This result further confirmed the requirement for Akt in the insulin-stimulated phosphorylation of, and 14-3-3 binding, to Rho GAP 22.

108

Figure 5-10: Akt is required for insulin-stimulated 14-3-3 binding to Rho GAP 22. Embryonic fibroblasts from either wild-type (WT MEF) or Akt 1/2 knockout mice (Akt DKO MEF) were serum starved and treated with (Ins) or without (B) 100 nm insulin for 30 min or with 100 nM wortmannin for 30 min and then 100 nM insulin (Wm). Cells were lysed and lysates used in a 14-3-3 pulldown, before being immunoblotted with indicated antibodies.

109 Discussion

These data indicate that activation of the PI3K/Akt pathway results in a marked increase in phosphorylation of Rho GAP 22 at Ser16 and Ser395 and phosphorylation at these sites is required for regulated 14-3-3 binding. The activity of Akt is required for phosphorylation at both sites although only the S16 site could be verified as a bona fide Akt site in vitro. Further study is required to fully elucidate the regulation of 14-3-3 binding to the Ser395 site.

Phosphorylation of Rho GAP 22 by Akt

An intriguing aspect of these data concerns the involvement of Akt as the Rho GAP 22 kinase. Phosphorylation of the 14-3-3 binding residues Ser16 and Ser395 is dependent on Akt, and Akt directly phosphorylates one of these sites in vitro. The fact that Akt only phosphorylated one of these sites yet it was required for insulin-stimulated 14-3-3 binding to both sites was interesting. While there may be many possibilities for this finding, I would like to describe two putative models. In the first, phosphorylation at Ser395 is dependent on phosphorylation at Ser16. Thus, the absence of Akt activity, whether by chemical inhibition or knock-out, would prevent 14-3-3 binding to Ser16 and thus Ser395. It is possible that the phosphorylation of, and 14-3-3 binding to the Ser395 site is regulated via a conformational change in Rho GAP 22. In this model, Rho GAP 22 in the unstimulated state would exist in a closed conformation, with the N-terminus blocking access to the Ser395 site. As a consequence of insulin or other growth factor stimulation, Ser16 would be phosphorylated and bind 14-3-3. Either the phosphorylation event itself or the binding of 14-3-3 would trigger a conformational change, unfolding the protein and allowing access to Ser395 by its kinase, resulting in phosphorylation and 14-3-3 binding. This model is depicted in Figure 5-11. This model can also explain why inhibition of Akt reduces insulin-stimulated 14-3-3 binding to Ser395 in a Rho GAP 22 construct that lacks the Ser16 site (Fig. 5-9). In this truncated form of the protein, the entire N-terminus, including Ser16 is missing. This would relieve the conformational control on Ser395 phosphorylation and 14-3-3 binding, allowing direct access by its kinase. The one confounding piece of data is the inability of Akt to phosphorylate Ser395 in vitro on a Rho GAP 22 fragment that lacks the Ser16 site (Fig 8). While Ser395 is

110 predicted to be a good Akt site (see above), it is not canonical as it lacks an arginine residue in the -5 position (Table 1). It is possible that this is enough to prevent recognition by Akt in vitro, but it may still be recognised in vivo. The other possibility is that Akt does not directly phosphorylate the Ser395 site but rather this function is regulated by an alternate kinase. If this hypothesis is correct the alternate kinase is likely downstream of Akt because inhibition of Akt blocks insulin-stimulated 14-3-3 binding at the Ser395 site (Fig. 5-9). I have tested the possible role of a number of likely kinases that might be considered to be downstream of Akt using chemical inhibitors. However none of these were able to inhibit insulin stimulated 14-3-3 binding to the Ser395 site. Therefore, the putative Ser395 kinase must lie in the insulin signalling cascade, distal to Akt, but cannot be downstream of mTOR, MEK, PKC,,,,, and isoforms, ROCK or CAMKII. It is intriguing that it has previously been proposed that Rho GAP 22 acts to suppress Rac1 activity in response to intracellular signalling through RhoA and its effector ROCK (Sanz-Moreno, Gadea et al. 2008), although the authors could find no evidence that ROCK phosphorylates Rho GAP 22 directly. Consistent with this I could find no evidence that 14-3-3 binding to Rho GAP 22 is dependent on ROCK activity. It is of course possible that the 14-3-3 binding to Rho GAP 22 is independent of its effects on Rac1, although my other results would indirectly suggest that this is not the case (Chapter 6). I am therefore unable to reconcile my results with the previously published data in this case. The absence of insulin-induced 14-3-3 binding to Rho GAP 22 in the double Akt knockout MEFs indicates that Akt is necessary for this interaction. There is however, one caveat to using such an experimental system. Although these cells undergo normal differentiation into pre-adipocytes {Bae, 2003 #9}, it is possible that they have undergone other genetic changes to allow continued survival in the absence of an important molecule such as Akt. One way to examine this possibility would be to re- introduce Akt expression into the knockout cells, and determine whether this rescues the Rho GAP 22/ 14-3-3 interaction. It would also be of interest to discover if other 14-3-3/ substrate interactions that are not mediated by Akt remain unaffected.

111

Figure 5-11: The regulation of dual 14-3-3 binding to Rho GAP 22 may be under conformational control. It is proposed that under serum-starved conditions, the protein exists in a closed conformation, blocking access to the Ser395 by its kinase. Only after Ser16 phosphorylation and/or 14-3-3 binding, does the protein undergo a conformational change, allowing access to the Ser395 site.

112 The Gatekeeper Hypothesis

The fact that there are two 14-3-3 binding sites in Rho GAP 22 raises several interesting possibilities. Firstly, one Rho GAP 22 molecule may bind two 14-3-3 dimers, one dimer binding to Ser16 and the other to Ser395. This was the originally proposed method of 14-3-3/ substrate interaction (Yaffe, Rittinger et al. 1997) and is seen in 14-3-3 mediated coordination of multimeric protein complexes (Ottmann, Marco et al. 2007). The other possibility is that each phospho-serine may bind separate halves of a single 14-3-3 dimer. This mode of binding has been demonstrated to occur in the 14-3-3- mediated activation of PKC (Kostelecky, Saurin et al. 2009), and opens up exciting possibilities for the conformational regulation of 14-3-3 substrates. Another possibility is that one 14-3-3 dimer may remain bound to either Ser16 or Ser395 even in the basal state and insulin may cause either a second dimer to bind at the other site, or Rho GAP 22 may undergo a conformational change to allow one 14-3-3 dimer to bind at both sites. One of these sites would therefore act as a “gatekeeper site” (Yaffe 2002). In this model, 14-3-3 binding to the second site would be dependent upon its initial interaction with the first site. The data shown in Figure 5-2 supports this model. The S16A mutant interacted only slightly with 14-3-3 in vivo. In contrast, the S395A mutant displayed a reduced in vivo interaction when compared to the wild-type protein, but bound more 14-3-3 than the S16A mutant. If each site bound 14-3-3 independently, it would be expected that mutation of either site would reduce the 14-3-3 interaction to the same extent. It is also interesting to consider the far western blotting data looking at 14-3-3 binding to Rho GAP 22 in vitro. While mutation of S395A reduced the amount of 14-3-3 that bound to Rho GAP 22, mutation of S16A not only reduced the binding to a greater extent, but rendered the interaction insulin-insensitive. This indicates that Ser16 controls the insulin-sensitivity of the Rho GAP 22/ 14-3-3 interaction, making it the more important site, or the so called “gatekeeper”. The difference between the effects of the S16A mutation in the in vivo versus in vitro interaction may be due to the much higher concentration of available 14-3-3 ligand in the in vitro assay, leading to saturable binding. As the protein has been denatured by SDS-PAGE and only partially re-natures on the PVDF membrane, the Ser395 site may be able to bind 14-3-3 independently of the Ser16 site’s control in this particular assay. An important aspect of the gatekeeper hypothesis is that the second 14-3-3 site is cryptic (Kostelecky, Saurin et al. 2009). When the gatekeeper site is not 113 phosphorylated, the affinity of the second site for 14-3-3 is not strong enough to enable binding. It was originally proposed that binding to the second site will only occur when the concentration of 14-3-3 in the local environment is artificially increased by the binding of one half of the 14-3-3 dimer to the gatekeeper site, in effect tethering it to the substrate (Yaffe 2002). Is there any evidence for this in Rho GAP 22? In Rho GAP 22, both the Ser16 and Ser395 sites are mode 3 binding sites, although they both lack the semi-conserved residue in the +2 position. Analysis with Scansite shows that Ser16 is within the best 0.712% of all sites in its database, whereas the Ser395 site is only within the top 2.190% of all sites. Although Ser16 is a marginally better site, both sites have the same recognition sequence. This would seem to preclude regulation of 14-3-3 binding solely on the level of the primary structure. It is therefore more likely that the regulation of phosphorylation at Ser395 is controlled by the conformation of Rho GAP 22, as described above. To confirm this hypothesis, further experiments are required. As mentioned previously in Chapter 4, the stoichiometry of the Rho GAP 22/ 14-3-3 complex could be elucidated via the use of analytical gel filtration. Furthermore, if there was no apparent size shift when comparing Rho GAP 22/ 14-3-3 complexes prepared from serum starved or insulin-stimulated cell lysates, it could be concluded that only one 14-3-3 dimer binds to Rho GAP 22, and simply binds at both sites when stimulated by insulin. An alternative approach would be to study the in vitro phosphorylation of full-length Rho GAP 22 versus Rho GAP 22 possessing the S16A mutation. Immobilising immunoprecipitated protein on PVDF membrane, and performing an in vitro kinase experiment using recombinant Akt and 32P-labelled ATP would achieve this. As the immobilised protein would be denatured, this would relieve the conformational regulation of Ser395. However, to reduce non-specific phosphorylation, these experiments may need to be done in a T189A background, as I suspect this site undergoes phosphorylation in vitro (see above). If the efficiency of phosphorylation is great enough, it may even be possible to show that the Ser395 site is phosphorylated using the phospho-specific antibody described in this chapter. Finally, this approach may allow dissection of the role of phosphorylation of Ser16 in regulating Ser395 as distinct from 14-3-3 binding to Ser16. This would be done via the use of a phospho- mimetic mutation at Ser16, versus the introduction of an R18 peptide that causes constitutive 14-3-3 binding (Wang, Yang et al. 1999). Further investigation of this mode

114 of interaction would require structural studies, possibly even the crystallisation of the Rho GAP 22/ 14-3-3 complex from serum-starved versus insulin-stimulated cells.

In summary, I have shown that 14-3-3 binds to both Ser16 and Ser395 of Rho GAP 22, with Ser16 binding the majority of 14-3-3 and possibly acting to control 14-3-3 binding at the Ser395 site. I have also identified Akt as the probable kinase responsible for phosphorylating Ser16. The regulation of Ser395 is less clear, but I have identified the kinase for the more important 14-3-3 binding site.

115 Chapter 6

A Functional Role for the Rho GAP 22/ 14-3-3 Interaction

Introduction

Having identified Rho GAP 22 as an insulin-responsive 14-3-3 binding protein, I was interested in identifying the functional outcome of this interaction particularly since little was known about the function of Rho GAP 22 at the commencement of this study. An isoform of Rho GAP 22 (p68RacGAP) had been shown to be involved in the regulation of the endothelin-1 promoter in endothelial cells. However, it was unclear if this effect was mediated by the GAP activity of the protein. Another study showed that Rho GAP 22 plays an important role in regulating the switch between the mesenchymal and amoeboid modes of cell migration, presumably by regulating the activity of Rac1. Neither of these studies identified Rho GAP 22 as a 14-3-3 binding protein, yet based on my work this seemed like an obvious connection.

Functions of 14-3-3

As discussed previously (Chapter 1), there are four main functional outcomes that follow the binding of 14-3-3 to a substrate protein. Briefly, these can be summarised as; a conformational change in the substrate induced by 14-3-3 binding, relocalisation of the substrate caused by 14-3-3 masking localisation signals, protection from further posttranslational modification and assembly/disassembly of a multimeric protein complex. It is interesting to speculate which of these three general categories the 14-3-3/ Rho GAP 22 interaction falls into, with a view to explaining the regulation of the GTPase activity of Rho GAP 22. Rho GAP 22 does not possess any obvious localisation signals such as a Nuclear Localisation Signal (NLS), so it is unlikely that 14-3-3 regulates the spatial distribution of Rho GAP 22 in a similar fashion to DLC-1. Rho GAP 22 does possess a lipid binding PH domain, which plays a role in localising proteins to specific regions of membranes. It is possible therefore that 14-3-3 binding 116 controls the membrane association of Rho GAP 22. Rho GAP 22 is found in the cytosol and in the nucleus in unstimulated cells (Aitsebaomo, Wennerberg et al. 2004) and I have demonstrated that it is not bound to 14-3-3 under these conditions. Insulin stimulation could possibly cause a relocalisation of Rho GAP 22 by 14-3-3 binding across the PH domain, preventing membrane association. This would be an interesting area to investigate further. It is also possible that 14-3-3 acts as a scaffold to bring Rho GAP 22 and Rac1 into close proximity, allowing Rho GAP 22 to activate the GTPase activity of Rac1. Rac1 has not been reported to bind 14-3-3 directly, although it is predicted to contain a Mode 1 14-3-3 binding motif at Ser71. Interestingly, this site is also predicted to be an Akt phosphorylation motif. It is tempting to speculate that Akt could phosphorylate both Rac1 and Rho GAP 22, allowing 14-3-3 to act as a scaffold and bind both molecules, leading to Rac1 inactivation. However, the biology of Rac1 has been extensively studied and no such phosphorylation event has been found. The most probable outcome of 14-3-3 binding to Rho GAP 22 is direct regulation of its GAP activity by a conformational change. This is an attractive hypothesis, especially as I have demonstrated that 14-3-3 binds to two sites on Rho GAP 22. This double interaction could force a conformational change in Rho GAP 22, as discussed above. However, the only way to verify this experimentally would be by crystallising the Rho GAP 22/14-3-3 complex.

Rho GTPase Biology.

To understand the role that 14-3-3 plays in regulating Rac1 via its binding to Rho GAP 22, it is necessary to investigate the biology of Rac1 itself. Rac1, Rac2, Rac3 and Rho G form a sub-family (Boureux, Vignal et al. 2007) among the Rho family of small GTPases, itself a member of the Ras super-family of GTPases. Although all four members of the family are involved in regulating the formation of lamellipodia and membrane ruffles, they differ in their pattern of expression. Rac1, the best characterised member of the family, is ubiquitously expressed whereas Rac2 is restricted to cells of the hematopoietic lineage and Rac3 is most abundant in brain (Didsbury, Weber et al. 1989; Shirsat, Pignolo et al. 1990; Haataja, Groffen et al. 1997). RhoG, which has the lowest sequence homology to Rac1 is also widely expressed (Vincent, Jeanteur et al. 1992). The activity of most small GTPases is dependent on their nucleotide binding

117 state, and the Rac family is no exception. When bound to GTP, the GTPase is able to interact with and activate effector proteins and thus engage in intracellular signal transduction. The most common mechanism of effector activation appears to be a conformational change induced by GTPase binding. In the case of Cdc42 and its effector Pak (also a Rac effector), binding of the GTPase displaces an auto-inhibitory domain of Pak and exposes the kinase domain (Tu and Wigler 1999). Small GTPase signalling is eventually terminated by the intrinsic GTPase activity, returning the protein to its inactive state. Small GTPases can therefore be thought of as molecular switches. The nucleotide binding state of the GTPase is regulated by three classes of proteins. Guanine nucleotide exchange factors (GEFs) serve to load the GTPase with GTP and thus activate it. This activation is achieved by a conformational change in the GTPase induced by binding of the GEF, resulting release of the nucleotide from the GTPase (Rossman, Worthylake et al. 2002). As the cytosolic concentration of GTP is higher than GDP, the GTPase is preferentially loaded with GTP (Rossman, Der et al. 2005). Interestingly, 14-3-3 has previously been implicated in the control of Rac1 activation at this level; binding of 14-3-3 to the GEF 1Pix inhibits its activity and consequently down regulates Rac1 activity (Chahdi and Sorokin 2008). GTPase activating proteins (GAPs) such as Rho GAP 22 activate the intrinsic GTPase activity of the GTPase, returning it to the inactive GDP-bound form. The GAP/GTPase interaction results in rearrangement of the GTPase, stabilising the catalytic pocket. A conserved arginine residue in the GAP domain also enters the nucleotide binding site to stabilise the transition state and drive the hydrolysis reaction to completion (Scheffzek, Ahmadian et al. 1997). Finally, guanine nucleotide dissociation inhibitors (GDIs) control the localisation of small GTPases by binding to their C-terminal prenyl groups and thus sequestering them in the cytosol away from their regulators and targets. This interaction also inhibits nucleotide exchange and retards hydrolysis, locking the GTPase in its nucleotide bound state (Dovas and Couchman 2005). These interactions are summarised in Figure 6-1. Rac has been reported to be involved in the regulation of multiple cellular processes, such as cell cycle regulation (Olson, Ashworth et al. 1995), transcriptional control (Hill, Wynne et al. 1995; Perona, Montaner et al. 1997), phagocytosis via the NADPH oxidase complex (Abo, Pick et al. 1991), and secretion (Norman, Price et al. 1996). However, its originally described and best understood role

118 is in the regulation of the actin cytoskeleton, specifically the formation of lamellipodia and the control of cell migration (Hall 1998).

119

Figure 6-1: Regulation of small GTPases such as Rac1. Small GTPases exist in an inactive GDP bound state, and an active GTP-bound state. The activation is mediated by guanine nucleotide exchange factors (GEFs) which promote the release of GDP and the binding of GTP. GTP binding probably causes a conformation change, allowing the small GTPase to bind to and activate effector proteins. Signal termination is catalysed by GTPase activating proteins (GAPs) such as Rho GAP 22, which activate the intrinsic hydrolytic activity of the GTPase, converting GTP to GDP and releasing phosphate (Pi). Inactive GTPases can also be sequestered away from their regulators and targets by guanine nucleotide-dissociation inhibitors (GDIs) which bind to their C-terminal prenyl groups. See text for detail.

120 Rho GTPases and the Regulation of the Cytoskeleton in Cell Migration

Cell migration is important in wound healing, development and immune surveillance. It can be envisaged as a cyclical process; a quiescent cell first becomes polarised, perhaps in response to an extracellular cue, a process known as chemotaxis. By sensing signalling molecules in the extracellular environment, membrane receptors trigger intracellular signalling pathways that result in the production of second messenger molecules, such as PIP3, localised to the leading edge of the cell. The leading edge of the cell then begins to protrude, a process driven by actin polymerisation. Indeed, experiments in fragments of fish skin keratinocytes, devoid of most organelles and microtubules, demonstrated that in response to a mechanical stimulus actin polymerisation and contractile asymmetries in the actin cytoskeleton can drive continuous cell motility (Verkhovsky, Svitkina et al. 1999). Cells form both filopodia and lamellipodia. Filopodia are small finger-like protrusions that contain bundles of filamentous F-actin that play a role in probing the extracellular environment (Kater and Rehder 1995). Lamellipodia are broad protrusions that are characterised by an actin “meshwork”, the polymerisation of which is thought to provide the motive force to push the lamellipodium forwards. To allow the cell to pull itself forward, it must attach to the extracellular matrix (ECM). This requires the formation of focal adhesions and focal adhesion complexes, which serve to link the actin cytoskeleton to the ECM, via integrin proteins. These structures, and the role that Rho GTPases play in regulating them, will be discussed later. The disassembly of adhesions at the rear of the cell, and the retraction of the trailing edge result in a net forward movement of the cell body and represent the end of the migration cycle. The retraction of the trailing edge is mediated by the contractile force generated by the motor protein Myosin II, creating a tensile force across the actin cytoskeleton via its anchorage to the ECM (Mitchison and Cramer 1996). Regulation of the actin cytoskeleton is crucial to coordinated cell migration. Therefore, it is not surprising that Rho family GTPases control cell migration, as they are known to control the assembly of the actin cytoskeleton. At the leading edge of the cell, both CDC42 and Rac regulate the activity of the Arp2/3 complex, a heptameric protein complex that nucleates new actin filaments either from the sides (Mullins, Heuser et al. 1998) or at barbed ends (Pantaloni, Boujemaa et al. 2000) of existing actin filaments. Arp2/3 thus regulates the branching of the actin cytoskeleton. The regulation of Arp2/3 121 by CDC42 and Rac is indirect and mediated by members of the Wiskott-Aldrich syndrome (WASP) family of proteins. There are five members of this protein family in mammals, all containing a C-terminal module that binds the Arp2/3 complex (Machesky, Mullins et al. 1999). The most-well studied member of this family is N- WASP, which was originally described in brain but is widely expressed (Miki, Miura et al. 1996). GTP-loaded CDC42 interacts directly with N-WASP in vitro via its CRIB domain, causing a conformational change and exposing an Arp2/3 binding site (Rohatgi, Ma et al. 1999). However, the regulation of N-WASP activity in vivo is believed to be more complicated, as the majority of N-WASP exists in a complex with a protein that suppresses activation by CDC42 (Martinez-Quiles, Rohatgi et al. 2001). The regulation of the Arp2/3 complex by Rac occurs through the WAVE protein (WASP family verprolin homologous protein), also known as Scar, a member of the WASP family (Miki, Suetsugu et al. 1998). Interestingly, WAVE does not possess a CRIB domain, but rather it is thought that active Rac controls WAVE activity by regulating its association with a complex of inhibitory proteins (Eden, Rohatgi et al. 2002). Rac and CDC42 also control actin filament nucleation via the regulation of the actin filament severing protein cofilin (Ghosh, Song et al. 2004). The binding of cofilin to an actin filament results in deformation and strain on the filament, leading to severing (McGough, Pope et al. 1997). This promotes actin filament nucleation and branching by providing free barbed filament ends that can be extended. Phosphorylation of cofilin by LIM kinases provides a major layer of regulation of cofilin activity (Arber, Barbayannis et al. 1998), LIM kinases are activated by PAK (Edwards, Sanders et al. 1999), an effector of Rac and CDC42. It is unclear how the requirement for active Rac and active (unphosphorylated) cofilin at the leading edge of the cell can be reconciled, although it has been suggested that spatial regulation plays a role (Dawe, Minamide et al. 2003). Rac and CDC42 can also generate new unbranched actin filaments by activating mDia1 and mDia2, members of the formin family of actin capping proteins (Peng, Wallar et al. 2003). The action of Rac at the leading edge promotes membrane protrusion, while the retraction of the trailing edge of the cell is mediated by Rho (Allen, Jones et al. 1997). While CDC42 and Rac promote branching of the actin cytoskeleton, Rho promotes the formation of stress fibres, large bundles of actin and myosin that traverse the cell. The organisation of individual actins filaments into these large bundles is mediated by the

122 Rho effector, ROCK (Leung, Chen et al. 1996) via the phosphorylation-induced inactivation of myosin light chain phosphatase (MLCP). ROCK phosphorylates the myosin binding subunit (MBS) of MLCP inducing its dissociation from myosin (Velasco, Armstrong et al. 2002). This results in increased phosphorylation of the regulatory MLC of myosin II, inducing its interaction with actin. This activates the ATPase activity of myosin, resulting in enhanced cell contractility (Amano, Chihara et al. 1998). ROCK proteins can also phosphorylate MLC directly, although the physiological significance of this remains unclear (Iizuka, Yoshii et al. 1999). Like CDC42 and Rac, Rho can also affect the turnover of actin filaments and thus the availability of free actin. Once again, this is through the action of ROCKs which phosphorylate and activate LIM kinases, thus inactivating cofilin as described above (Maekawa, Ishizaki et al. 1999). The release of the trailing edge of the cell from the substrate is often the rate-limiting step in cell motility (Chen 1981). This process is not thought to be directly regulated by Rho, but by the protease calpain, which is activated when mechanosensitive Ca2+ channels in the membrane are opened as the plasma membrane at the trailing edge stretches [43]. Calpain is known to degrade many constituents of focal adhesions, thus its activity could release the cell from the substratum (Glading, Lauffenburger et al. 2002). An interesting aspect of the role Rho GTPases play in regulating cell migration is the large amount of cross-talk between Cdc42, Rac and Rho. Much of this inter-regulation occurs via the Par (partitioning defective) complex, consisting of Par 3, Par 6 and atypical protein kinase C (aPKC), which plays a crucial role in regulating cell polarity and directed migration (Kemphues, Priess et al. 1988). Activated CDC42 recruits the Par complex to the plasma membrane, where aPKC is activated (Etienne- Manneville and Hall 2001). CDC42 is thus a master regulator of cell polarity and its activity strongly regulates the direction of migration (Etienne-Manneville 2004). Active CDC42 can recruit the Rac GEF TIAM-1 to the Par complex, leading to localised Rac activation at the leading edge of the cell (Nishimura, Yamaguchi et al. 2005). Furthermore, the Rho effector ROCK can phosphorylate Par 3, disrupting the Par 3/TIAM-1 complex and preventing TIAM-1 mediated-activation of Rac (Nakayama, Goto et al. 2008). Rho, through its effector ROCK, also regulates Rac activity via FilGAP (Rho GAP 24) (Ohta, Hartwig et al. 2006). Rac can also regulate the activity of Rho; expression of activated Rac has been shown to inhibit Rho function (Sander, ten

123 Klooster et al. 1999). The mechanism of regulation remains unclear, although it has been suggested that PAK could play a role by phosphorylating and inactivating myosin light chain kinase, leading to decreased phosphorylation of MLC and thus reduced contractility (Sanders, Matsumura et al. 1999). The involvement of other components of the cytoskeleton, such as microtubules, in cell motility seems to be cell-type dependent. The migratory ability of fast moving cells such as neutrophils and keratinocytes is unaffected by nocodazole, a microtubule polymerisation inhibitor (Rich and Hoffstein 1981; Euteneuer and Schliwa 1984). In contrast, nocodazole inhibits the migration of epithelial cells and fibroblasts (Gotlieb, Subrahmanyan et al. 1983; Middleton, Brown et al. 1989). One scenario that explains these contradictory results suggests that microtubules control the turnover of focal adhesions in slow moving cells (Kaverina, Krylyshkina et al. 1999). This would also explain why microtubules are necessary for tail retraction (Ballestrem, Wehrle-Haller et al. 2000). Experiments using nocodazole provided the first link between microtubules and Rho family GTPases. Nocodazole treatment activated Rho, whereas removal of nocodazole led to Rac activation (Liu, Chrzanowska-Wodnicka et al. 1998). Rho was later shown to promote the stability of microtubules through its effector mDia1, a microtubule capping protein (Ishizaki, Morishima et al. 2001; Palazzo, Cook et al. 2001). It has been suggested that the reorganisation of the microtubule cytoskeleton in polarised cells is important in maintaining directed and efficient cell migration, but is not necessary for short-term migration (Raftopoulou and Hall 2004). The polarisation of the microtubule network to the front/rear axis of the migrating cell orients organelles such as the nucleus and Golgi apparatus in the direction of migration, facilitating sustained migration by directing transport pathways to the leading edge (Ma and Chisholm 2002). Cell polarisation is dependent on CDC42 activation of the Par complex as previously discussed; activated aPKC can indirectly activate the microtubule binding protein adenomatous polyposis coli (APC), which binds specifically to the plus ends of microtubules at the leading edge of the cell and induces microtubule reorganisation and centrosome reorientation (Etienne-Manneville and Hall 2003). Less is known about the role of intermediate filaments in cell migration, and how they are regulated by Rho family GTPases. Primary fibroblasts from vimentin-null mice show impaired migration in collagen gel culture (Eckes, Dogic et al. 1998) and the mice

124 have impaired wound healing, as their fibroblasts cannot efficiently migrate into the wound (Eckes, Colucci-Guyon et al. 2000). The exogenous expression of active Rho induces changes in intermediate filament organisation, perhaps via phosphorylation of vimentin by ROCK (Sin, Chen et al. 1998). In addition, expression of CDC42 or Rac can induce collapse of vimentin-containing intermediate filaments into the perinuclear region (Meriane, Mary et al. 2000). This process may be important in reorganising the intermediate filaments during cell migration. Considering all these data, an attractive and rather simple model for cell migration arises. CDC42, via its regulation of the Par complex, acts at the leading edge to influence cell polarity and thus the direction of migration. Rac acts at the leading edge to drive the cell forward via polymerisation of the actin cytoskeleton into a branched network. Rho acts at the trailing edge to mediate retraction. Each GTPase would regulate the activity of the other to ensure that there is only one lamellipodium and one trailing edge. However, this simplistic model may be misleading. For instance, GTP- loaded Rac has been found at both the front and rear of migrating neutrophils (Gardiner, Pestonjamasp et al. 2002). Furthermore, the activation of Rac alone has been demonstrated to be sufficient to promote migration in a directed manner (Wu, Frey et al. 2009). In contrast, Rho has been reported by be required for generating membrane protrusions, with Rac and CDC42 suggested to be involved in stabilising new protrusions (Machacek, Hodgson et al. 2009). Although Rho family GTPases were first linked to the control of cell migration 18 years ago (Ridley and Hall 1992; Ridley, Paterson et al. 1992), their biology and regulation are still not completely understood.

Rho GTPases and Cell Adhesion

In addition to extending and retracting its membrane, a migrating cell must anchor new membrane protrusions to the substratum, and dissolve adhesions at the trailing edge to allow membrane retraction. The adherence of cells to the substratum is mediated by the integrin family of proteins. These proteins are transmembrane proteins that bind ECM proteins and provide a link to the actin cytoskeleton. Integrins were first recognised as a distinct family of proteins in 1987 (Hynes 1987). It has since become clear that integrins are obligate heterodimers and their expression is restricted to metazoans. Integrins are capable of binding various proteins in the ECM such as fibronectin, vitronectin, laminin

125 and collagen. In mammals, there are 24 distinct integrins, formed by the dimerisation of 18 subunits and 8 subunits (Hynes 2002). They not only provide a physical link to the ECM, but also play an important role in sensing the extracellular environment. Integrins are linked to the cell’s actin cytoskeleton by multimeric protein complexes known as adhesions. The term “adhesion” refers to a broad range of attachment complexes, mostly defined based on their morphology and composition. For example, a cell may have focal adhesions, fibrillar adhesions, focal complexes (also known as focal adhesion complexes) and podosomes. Here, I will be discussing focal complexes, small adhesions that are formed in a Rac-dependent manner (Rottner, Hall et al. 1999) at the periphery of the cell and focal adhesions, a mature form of focal complexes. Focal adhesions were first identified by electron microscopy as electron dense regions of the plasma membrane that made contact with the substrate (Abercrombie, Heaysman et al. 1971). New focal complexes are preferentially formed at the periphery of the cell (Regen and Horwitz 1992). It is these small adhesions, undetectable in some cell types, that drive rapid cell migration (Beningo, Dembo et al. 2001). As the cell moves over the focal complex, other proteins are recruited to the complex and it matures into a focal adhesion (Webb, Parsons et al. 2002). Over 50 different proteins have been described in focal adhesions, including cytoskeletal proteins, kinases, phosphatases and other enzymes (Zamir and Geiger 2001). Not all of these proteins will be in each adhesion as some exhibit cell specific expression and not all adhesions are the same. In other cases involvement may be limited to a certain class of adhesion. The mature focal adhesion is disassembled at the trailing edge of the cell, although some components such as integrins, will remain associated with the substrate (Webb, Parsons et al. 2002). How the turnover of focal complexes and focal adhesions is regulated remains an ongoing and active field of study. As already mentioned, Rac is required for the formation of new adhesions, however the Rac and CDC42 effector Pak, has been implicated in the control of focal adhesion disassembly (Frost, Khokhlatchev et al. 1998). The disassembly of focal adhesions is dependent on Rho-ROCK signalling. Inhibition of Rho results in accumulation of integrins in the unretracted tail (Worthylake, Lemoine et al. 2001). The effect of Rho on adhesion disassembly appears to be mediated by MLCK, inhibition of which results in cells with elongated tails (Somlyo, Bradshaw et al. 2000). Interestingly, some components of focal adhesions can also regulate the activity of Rho GTPases. An example of this is Syndecan 4, which

126 binds fibronectin in association with 51 integrin (Saoncella, Echtermeyer et al. 1999) and signals through PKC to restrict Rac activity to the leading edge of the cell, promoting directional migration. Syndecan 4-null fibroblasts migrate in a random fashion, due to Rac being activated all around the cell periphery (Bass, Roach et al. 2007). It is perhaps not surprising that Rho GTPases are involved in the regulation of focal complexes and focal adhesions, as cell adhesion is intricately linked with cell migration.

In this chapter I characterise the functional role of the 14-3-3/Rho GAP 22 interaction, with a focus on modulation of Rac activity. In view of the essential role of Rac in the control of cell migration, I focused on this functional output.

Results

Generation of Polyclonal Cell Lines

To investigate the role of Rho GAP 22 in regulating cell migration, I generated stable cell lines expressing Rho GAP 22 wt and Rho Gap 22 mutants. NIH3T3 fibroblasts were infected with pMIG retrovirus encoding FLAG-Rho GAP 22 or FLAG-Rho GAP 22 mutants (see Materials and Methods). The pMIG retrovirus uses an internal ribosome entry sequence (IRES) to express GFP independently of the cDNA cloned into the multiple cloning site. I exploited this function to generate polyclonal cell lines using FACS. Infected NIH3T3 fibroblasts were sorted and the median 20% of GFP- expressing cells (as measured by intensity of GFP signal) were collected and used to establish polyclonal cell lines. As GFP expression correlated with FLAG-Rho GAP 22 expression, this provided an easy method for generating cell lines that were relatively well matched for the level of FLAG-Rho GAP 22 expression. Cell lines expressing empty pMIG vector (pMIG), wild-type Rho GAP 22 (WT), Rho GAP 22 where the critical catalytic residue in the GAP domain was mutated (R211A), a 14-3-3 binding mutant (S16A/S395A) and an S16A/S395A/R211A Rho GAP 22 mutant. As shown in Figure 6-2, the expression of each construct was similar in the different cell lines. It can also be seen from these studies that the level of expression achieved for recombinant Rho Gap 22 was significantly higher than endogenous Rho Gap 22. This is probably 127 due to low endogenous expression of Rho GAP 22 in NIH3T3 cells. The FLAG antibody detected a doublet in cells expressing wild type Rho GAP 22 of approximately 90 kDa, while only a single band was detected in the case of each of the mutants. Curiously the molecular weight of the R211A mutant corresponded to the lower band observed in the case of the wild type protein, while in the case of the other mutants the molecular weight of the observed band corresponded to the upper band seen for the wild type protein. This difference in molecular weight was not due to a mutation in one of the constructs as they were verified by sequencing. The apparent difference in molecular weight is likely due to a post-translational modification of the protein. These five cell lines were used for all experiments described in this chapter.

128

Figure 6-2: Over-expression of various Rho GAP 22 mutants in NIH3T3 cells. NIH3T3 fibroblasts were infected with pMIG retrovirus encoding empty vector (pMIG), FLAG-Rho GAP 22 (WT) or mutants of FLAG-Rho GAP 22 as indicated. The pMIG retrovirus also independently expresses eGFP using an IRES sequence. Polyclonal cell lines expressing each construct were generated by FACS sorting for the median 20% of GFP expressing cells of each line. Cells from each line were lysed and lysates immunoblotted with the indicated antibodies.

129 Cell Migration Studies

I next examined the role of Rho GAP 22 in regulation of cell migration, and the role of 14-3-3 in regulating the function of Rho GAP 22. To this end, I developed a novel migration assay using dual-wavelength time-lapse microscopy. This approach allowed direct measurement of the rate at which the various cell lines migrated in independent wells of a growth chamber as compared to an internal control that was common to each well of the chamber. Having the same control in each well allowed direct comparison across wells. In this experiment, the five NIH3T3 cell lines expressing various Rho GAP 22 constructs were combined in equal numbers with NIH3T3 cells stably expressing empty pMIG vector that also expressed mCherry under control of the IRES sequence. These cells were plated into a fibronectin-coated 24 well plate and imaged every 10 min for 14 h (see Materials and Methods). Representative time-lapse videos for each cell line are available in Supplemental Figure S6-1 on the disc attached to this thesis. Individual cells were tracked over the 14 h time course of the experiment using IMARIS software. These tracks can be seen in Figure 6-3A. The tracks for control cells expressing pMIG mCherry are represented by a red line; that of the cells expressing the FLAG-Rho GAP 22 constructs are represented by a green line. The abundance of shorter tracks for the S16A/S395A mutant indicated a defect in migration. However, while the total track length is of interest, a cell may either migrate in a straight line or move in a more random fashion and still produce a track of the same length. It is therefore more interesting to study the directionality of its motion by examining its total displacement (Gail 1973). The mean square displacement over 14 h was quantified using IMARIS software (see Materials and Methods), and results expressed as the mean ratio of the mean displacement of the experimental cells lines divided by the mean displacement of the control cells. This experiment showed that only cells overexpressing a mutant FLAG-Rho GAP 22 that was unable to bind 14-3-3 (S16A/S395A) had a significant reduction in displacement when compared to cells expressing wild type Rho GAP 22 (Fig. 6-3B). In contrast, cells over-expressing mutant FLAG-Rho GAP 22 that was unable to bind 14-3-3 and was also catalytically inactive (S16A S395A/R211A) did not differ significantly from the wild type, indicating that the effect of loss of 14-3-3 binding was dependent on the GAP activity of Rho GAP 22.

130

Figure 6-3: Inhibition of 14-3-3 binding to Rho GAP 22 causes a defect in cell motility. (A) NIH3T3 fibroblasts expressing various Rho GAP 22 constructs (green) or vector only (red) were imaged every 10min for 14 h. Tracks depict migration of individual cells. (B) Cell displacement over 14 h was calculated using IMARIS software and reported as mean ratio of Rho GAP 22 mutant-expressing cell displacement divided by the displacement of cells expressing empty vector. The mean of 3 experiments is shown, error bars show standard deviation. * = p < 0.05 131 Discussion

A moderate increase in the amount of active Rac1 stimulates the formation of multiple lamellipodia which promote random migration (Pankov, Endo et al. 2005). Thus, as cells expressing the S16A/S395A mutant migrated in a more random fashion, it is probable that they have higher levels of active Rac1-GTP as compared to cells expressing wild type Rho GAP 22 or catalytically inactive Rho GAP 22. This implies that the S16A/S395A mutant has a lower GAP activity, leading to higher amounts of Rac1-GTP. As this mutant is unable to bind 14-3-3, it can be inferred that 14-3-3 binding to Rho GAP 22 stimulates its GAP activity. This potentially also explains why over-expression of the wild type protein had no effect on cell motility. Even though FLAG-Rho GAP 22 is more much abundant than the endogenous protein (Fig. 6-1), it has not been activated by 14-3-3 binding and thus has no effect on cell motility. Although the S16A/S395A/R211A mutant cannot bind 14-3-3 in a similar fashion to the S16A/S395A mutant, it presumably had no effect on cell migration as it is catalytically inactive. The failure of the S16A/S395A/R211A to impair cell migration indicates that the effect of the S16A/S395A is at the level of Rac and not an interaction with a third protein.

While these experiments have shown that 14-3-3 has a role in regulating cell motility via its interaction with Rho GAP 22, I have only shown indirect evidence that 14-3-3’s association with Rho GAP 22 activated its GAP activity. In order to show a direct effect on Rac-GTP loading, an effector pulldown using the CRIB domain of Pak in a GST pulldown could be employed (Benard, Bohl et al. 1999). This experiment would provide a direct measure of the amount of active Rac in the cell, as only GTP-loaded Rac is able to bind Pak via its CRIB domain. It would also be interesting to study the effects of constitutive 14-3-3 binding to Rho GAP 22. As already described in the previous chapter, this could be achieved by inserting an R18 sequence into Rho GAP 22. R18 is a synthetic peptide that was first identified as having high affinity for 14-3-3 in a phage display experiment, and has no homologous sequence in mammals (Wang, Yang et al. 1999). By engineering R18 sequences into the S16A/S395A background, it would be possible to differentiate between the effects of 14-3-3 binding and phosphorylation in the experimental systems described in this chapter. Furthermore, this construct could be

132 used in the GST-Pak pulldown experiment described above to directly demonstrate the effect of 14-3-3 binding on Rac activity.

In conclusion, this chapter has shown that mutating Rho GAP 22 to inhibit 14-3-3 binding impairs cell migration. This effect is presumably mediated through Rho GAP 22 activity on Rac GTPase activity, implying that 14-3-3 binding activates the GAP activity of Rho GAP 22.

133 Chapter 7

General Discussion

Since the discovery of insulin in 1921, the elucidation of the mechanism by which it regulates its many effects has been slow and laborious. This has generally been carried out using a reverse discovery process. This approach involved firstly establishing a particular metabolic action of insulin such as glycogen synthesis or glucose transport and then working backwards in order to track the connection between this event and the activity of the insulin signalling pathway. In the case of glycogen synthesis and glucose transport the precise details still remain incompletely understood. Recent developments in mass spectrometry have provided the possibility of identifying novel actions of insulin using a forward discovery process. Because the canonical PI3K/Akt pathway plays a fundamental role in most of insulin’s actions and our understanding of Akt substrate phosphorylation is well understood it is conceivable to use this knowledge to identify new Akt substrates in insulin target tissues and then try to connect these substrates to a relevant metabolic action.

Akt and 14-3-3

The Ser/Thr kinase Akt is a major regulator of the diverse actions of insulin. Insulin stimulation causes Akt translocation to the plasma membrane, where it is activated by multiple phosphorylation events. Activated Akt sets in motion the diverse functions of insulin by phosphorylating multiple substrate proteins. The first Akt substrate was discovered in 1995 and there are now 111 proteins in the human proteome that are known to be phosphorylated by Akt. Interestingly, phosphorylation of several of these proteins by Akt creates a binding site for a third class of proteins, the 14-3-3 family. 14-3-3 comprises a unique family of proteins. They do not share significant sequence homology to other proteins in the mammalian genome. Found in almost all eukaryotic cells, they are highly acidic and exist preferentially as dimers. Mammals express seven isoforms of 14-3-3, which are capable of both homo and heterodimerisation. The most interesting aspect of 14-3-3 biology is that they preferentially bind phosphoproteins, 134 either a phospho-serine or phospho-threonine residue. Each half of the dimer binds a single phosphorylated residue, allowing the dimer to bind two phosphorylated residues. In some cases it appears that 14-3-3 binds to two phosphorylation sites within the same protein and in other cases it binds to two phosphorylation sites found in two separate proteins. The binding of 14-3-3 to a substrate protein generally has one of four functional outcomes. As the structure of 14-3-3 is quite rigid, the substrate protein may be forced to undergo a conformational change to bind 14-3-3, especially if 14-3-3 binds two phosphorylated residues on the one protein. 14-3-3 binding can also mask localisation signals on the substrate, leading to a change in the substrate’s localisation. 14-3-3 can act as a scaffold, bringing two substrates into close apposition. Conversely, 14-3-3 binding can also prevent a substrate from interacting with other proteins. Finally, binding of 14-3-3 can prevent the substrate protein from further post-translational modification. Thus, 14-3-3 binding can translate one intracellular signal, phosphorylation, into many functional outcomes. Akt phosphorylation creates a canonical 14-3-3 binding motif, and Akt phosphorylation is known to induce 14-3-3 binding to many of its substrate proteins. It is therefore reasonable to propose that 14-3-3 binding acts as a general mechanism by which Akt mediates many of insulin’s effects. It follows that new functions of Akt could therefore be discovered by looking for novel proteins that bind 14-3-3 in an insulin-responsive manner. To achieve this aim, this study has combined 14-3-3 affinity chromatography with a quantitative mass spectrometry method, allowing protein identification.

Novel Roles for Insulin Identified in this Study

This study identified 29 proteins that bound 14-3-3 in response to insulin stimulation in the L6 skeletal muscle cell line. Five of these proteins had previously been described as Akt substrates, indicating that the screen was robust. Proteins homologous to known Akt substrates were also identified. Of the remaining proteins, five stood out from the rest. These five proteins had not previously been described as Akt substrates, nor as insulin-regulated targets. These proteins, (EDC3, Rho GAP 22, TRIP12, Beclin-1 and MYCBP2) all underwent at least a 2-fold increase in 14-3-3 binding in response to insulin treatment. In all five cases, this increase in 14-3-3 binding was prevented by pre- treatment with wortmannin, indicating that they are all downstream of PI3K in the 135 insulin signalling pathway. Bioinformatic analysis revealed that all five proteins were predicted to be phosphorylated by Akt, and to bind 14-3-3. Recognition that insulin causes Akt-mediated phosphorylation of these proteins links insulin to new areas of cellular metabolism. These proteins will therefore serve as the starting point for new areas of investigation. EDC3 is known to regulate mRNA stability via decapping (Eulalio, Rehwinkel et al. 2007), and is also a component of structures known as p-bodies; large cytosolic complexes composed of both protein and RNA. Sequestration of mRNA in these structures serves as a readily reversible storage mechanism that has the added effect of protecting the stored mRNA from degradation (Parker and Sheth 2007). It is conceivable that insulin-induced 14-3-3 binding to EDC3 may dissociate the p-body, releasing the stored mRNA and allowing rapid protein translation in response to an insulin signal. This would then mean that the insulin-Akt axis would not only control protein production via transcription and translation, but also the ‘middle ground’ between these processes, regulation of mRNA turnover. Beclin-1, also known as Atg6, is involved in the cellular process known as autophagy. Autophagy is a process by which cells recycle organelles and cytosolic proteins to generate nutrients in response to starvation or to degrade functionally redundant or damaged organelles or proteins. Beclin-1 is essential for the initiation of the formation of double membrane structures known as autophagosomes, which encapsulate organelles destined for lysosomal degradation (Liang, Jackson et al. 1999). Insulin is released from the pancreas to catalyse glucose uptake after a meal. An insulin signal is therefore indicative of the fed state, and it is not surprising that insulin has been demonstrated to suppress autophagy. This is accomplished by two mechanisms. Transcription of autophagy-related genes is controlled by Foxo3a (Mammucari, Milan et al. 2007), which is phosphorylated and inhibited by Akt in response to an insulin stimulus. Secondly, insulin regulates autophagy via mTOR which phosphorylates and inhibits the protein kinase ULK1 (Blommaart, Luiken et al. 1995), which also plays a role in forming the autophagosomal membrane. Identification of Beclin-1 as an insulin- regulated protein possibly provides a third, more direct mechanism for insulin to regulate autophagy. Binding of 14-3-3 to Beclin-1 may inhibit formation of the autophagosomal membrane, suppressing autophagy.

136 TRIP12 and MYCBP2 are both probable E3 ubiquitin ligases. E3 ligases catalyse the transfer of activated ubiquitin from an E2 ubiquitin conjugating enzyme to a substrate protein. Classically, ubiquitination was thought to target the substrate for proteasomal degradation, however other outcomes are possible. As very little is known about these proteins, it is difficult to speculate what functions insulin-stimulated 14-3-3 binding could regulate. However, insulin has previously been implicated in regulating the activity of the 26S proteasome (Bennett, Hamel et al. 2000), which degrades polyubiquitinated proteins.

Notably, several known Akt substrates, such as GSK-3, were not identified in the screen. This may have been due to the low abundance of these proteins in L6 cells or to their low level of phosphorylation or transient phosphorylation. To enable identification of non-abundant proteins, it may be beneficial to subject the protein sample to further enrichment steps prior to 14-3-3 pulldown and tryptic digest. For example, by performing a subcellular fractionation of the crude lysate, it would be possible to enrich for 14-3-3 substrates in different subcellular compartments such as the cytosol, nucleus and plasma membrane. This approach would also allow identification of proteins that undergo a 14-3-3-induced change in their localisation.

The Novel Akt/14-3-3 Substrate Rho GAP 22.

One of the novel 14-3-3 binding proteins identified in this screen was Rho GAP 22, a GTPase activating protein that had previously been described as a GAP for Rac, and to play a role in endothelial cell capillary tube formation. Rho GAP 22 had not previously been described as an insulin-responsive protein, or as an Akt or 14-3-3 substrate. This study has determined that 14-3-3 binds Rho GAP 22 at two sites, and that binding of 14-3-3 to the Ser395 site is dependent on binding to the Ser16 site. I have also demonstrated that Akt is capable of phosphorylating the Ser16 site in vitro, and that insulin-responsive 14-3-3 binding to Rho GAP 22 is dependent on Akt in vivo. Overexpression of a mutant Rho GAP 22 that was unable to bind 14-3-3 resulted in significantly decreased cell motility when compared to overexpression of the wild type protein. This indicated that the interaction of 14-3-3 with Rho GAP 22 probably

137 regulates its GAP activity towards Rac, a small GTPases that is intimately associated with the control of cell motility. It is not clear how the binding of 14-3-3 regulates Rho GAP 22. The two most likely mechanisms are via direct regulation of the GAP activity of Rho GAP 22, or by controlling its localisation. If the stoichiometry of the interaction is one 14-3-3 dimer binding one molecule of Rho GAP 22, then both of these models depend on a conformational change of Rho GAP 22 that is induced by 14-3-3 binding. As 14-3-3 binds two residues on Rho GAP 22, it is likely that Rho GAP 22 would be forced to change conformation to suit the rigid structure of 14-3-3. In the first model, this would activate the GAP domain, allowing Rho GAP 22 to inactivate Rac. In the second model, the bound conformation would prevent the association of Rho GAP 22’s PH domain with the PM, sequestering it in the cytosol and preventing Rac inactivation at the PM. This would allow for precise spatial regulation of Rac activity. Rho GAP 22 has previously been described as being localised to the cytosol. It is possible that the proportion of Rho GAP 22 bound to the PM is too small to be seen via microscopy. It would be interesting to investigate whether the mutant Rho GAP 22 that is unable to bind 14-3-3 has a different localisation than that of the wild type protein. If so, this could indicate that 14-3-3 binding alters the localisation of Rho GAP 22 by masking the PH domain. Of course, the other way of determining which model is correct would be to crystallise Rho GAP 22 either on its own, or in complex with 14-3-3. By comparing the two structures, it would be easy to determine the effects of 14-3-3 binding on the conformation of Rho GAP 22. It is also possible that 14-3-3 does not interact with Rho GAP 22 in a 1:1 ratio. In this case, the binding of 14-3-3 could influence the association of Rho GAP 22 with other proteins, or even influence the possible dimerisation of Rho GAP 22. The closest homologue of Rho GAP 22, FilGAP, has previously been shown to dimerise through its C-terminal coiled-coil domain (Nakamura, Heikkinen et al. 2009). Rho GAP 22 has the same domain structure and organisation as FilGAP; therefore it is possible that Rho GAP 22 exists as dimer within the cell. One method to determine whether Rho GAP 22 dimerises would be to co-transfect cells with two Rho GAP 22 constructs, each having a different tag. It would then be possible to immunoprecipitate using one tag and to blot for the presence of the other. If the protein forms dimers, it would be interesting to determine if whether this is regulated by 14-3-3 binding.

138 The role of the PH domain of Rho GAP 22

As previously stated, the N-terminus of Rho GAP 22 contains a lipid-binding Pleckstrin Homology (PH) domain. These domains bind phospholipids with varying specificities, allowing PH domain-containing proteins to be sequestered to various membranous structures in the cell, including the cytosolic side of the plasma membrane. This spatial regulation is important for controlling the biological activity of numerous proteins, including Akt. One possible model for the role of 14-3-3 in regulating the GAP activity of Rho GAP 22 via the PH domain is discussed above. However, it would also be of interest to examine the function of the PH domain itself more rigorously. By determining which particular species of phospholipid binds to Rho GAP 22’s PH domain, it would be possible to determine which membrane Rho GAP 22 is likely to bind to. Furthermore, it is possible to mutate certain residues within the so-called core region of a PH domain to prevent lipid binding. Examining the phenotype of a mutant Rho GAP 22 that was unable to be sequestered to membranes but was also unable to bind 14-3-3, or had constitutive 14-3-3 binding, would shed light on the role of localisation in regulating the biological function of the Rho GAP 22/ 14-3-3 interaction.

Interaction of Rho GAP 22 with Other Proteins

This study has not investigated whether Rho GAP 22 binds proteins other than 14-3-3. The closest homologue of Rho GAP 22, FilGAP, is a Filamin-A binding protein (Ohta, Hartwig et al. 2006). This association is dependent on the C-terminal coiled-coil domain of FilGAP (Nakamura, Heikkinen et al. 2009), which is also responsible for dimerisation as described above. Filamin A is an actin filament binding protein that serves to cross-link actin filaments and has also been reported to bind members of the Rho GTPase family. Interestingly, both FilGAP, and small GTPases, bind Filamin A in the same region, the 23rd -sheet. Many other molecules involved in the regulation of the actin cytoskeleton have been reported to bind Filamin A in this region (Vadlamudi, Li et al. 2002; Ueda, Ohta et al. 2003; Ohta, Hartwig et al. 2006). Thus, Filamin A might serve as a scaffold to bring these signalling molecules into close apposition. It would therefore be interesting to determine whether Rho GAP 22 binds Filamin A via its coiled-coil domain, as this would serve as a way to spatially regulate its GAP

139 activity. However, it is not clear how the coiled-coil domain regulates both the dimerisation of FilGAP and its association with Filamin A. It is possible that dimerisation and binding to Filamin A is mutually exclusive. Moreover, as FilGAP is predicted to bind 14-3-3 in a similar fashion to Rho GAP 22, it is unclear if 14-3-3 binding would influence association with Filamin A. These question merit further study, both in Rho GAP 22 and FilGAP.

The Specificity of Rho GAP 22 for Rac.

Rho GAP 22 has previously been reported to have GAP activity for Rac only (Aitsebaomo, Wennerberg et al. 2004). However, these experiments were carried out using p68RacGAP, the murine isoform of Rho GAP 22 that lacks the PH domain. FilGAP has been reported as having GAP activity towards Rac1 and Cdc42 both in vitro and in vivo, with a slight preference for Rac1 (Ohta, Hartwig et al. 2006). Interestingly, an isoform of FilGAP that lacks the N-terminal PH domain (analogous to p68RacGAP) has previously been reported to have GAP activity specifically towards Rho but not Rac or CDC42 (Su, Hahn et al. 2004) and in a separate study, towards Rac and CDC42 but not Rho (Lavelin and Geiger 2005). It is therefore unclear whether the full-length protein has the same GAP specificity as the shorter isoform. Of course, the same could be true for Rho GAP 22. It would therefore be interesting to compare the GAP specificity of full-length Rho GAP 22 and p68RacGAP. It has also been shown that phosphorylation of a GAP can change its target GTPase. Phosphorylation of MgcRacGAP by Aurora B during cytokinesis alters the specificity of its GAP activity, allowing it to GAP Rho as well as Rac and CDC42 (Minoshima, Kawashima et al. 2003). Although Akt does not phosphorylate Rho GAP 22 in the GAP domain, as is the case for Aurora B and MgcRacGAP, it would nevertheless be interesting to determine whether phosphorylation and 14-3-3 binding change the GAP specificity of Rho GAP 22.

Rho GAP 22 and Other Functions of Rac.

This study has only investigated the role of Rho GAP 22 in one of Rac’s many functions, regulation of cell migration. This function was chosen after publication of a 140 study showing that knockdown of Rho GAP 22 results in impaired cell migration. The regulation of cell migration is also the best-characterised function of Rac. However, Rac also regulates other cellular processes, such as cell adhesion and the expression of some genes. In phagocytic cells such as macrophages and neutrophils, Rac-mediated actin polymerisation is required for phagocytosis (Heasman and Ridley 2008). Rac also forms a component of the NAPDH oxidase complex that generates superoxide to kill phagocytosed bacteria (Diekmann, Abo et al. 1994). It would be interesting to determine whether Rho GAP 22 is involved in the regulation of these processes, although this might go beyond the regulation of insulin signalling.

The Role of Rho GAP 22 in Insulin Signalling

Akt has previously been implicated in the regulation of cell motility. The Rac effector Pak is capable of targeting Akt to the PM where it is activated by PDK-1 (Higuchi, Onishi et al. 2008). However, it has not previously been demonstrated that Akt can directly control the activity of Rac by signalling through a GAP. Insulin has previously been shown to promote cell migration and membrane ruffling, both processes being dependent on Rac activity (Cheresh, Leng et al. 1999). Thus, Rho GAP 22 provides a link between insulin and the control of Rac. In order for directed migration to occur, Rac activity must be subject to precise regulation. Even a moderate increase in Rac- GTP levels promotes more random migration, via the generation of multiple competing lamellipodia (Gail 1973). Rho GAP 22 could therefore restrict Rac activity to allow directed cell migration in response to insulin stimulation.

Concluding Remarks

This study has identified 29 proteins that undergo a significant change in 14-3-3 binding after insulin stimulation. Five of these proteins were prioritised for further study based on the possibility that they regulate novel actions of insulin. Of these five, Rho GAP 22 was investigated in-depth and found to undergo 14-3-3 binding at two sites. This study has demonstrated a complex interplay between the 14-3-3 binding sites, with the binding of 14-3-3 to Ser395 being dependent on binding to Ser16. It was also demonstrated that 14-3-3 binding is dependent on Akt, and that Akt is capable of 141 phosphorylating Rho GAP 22 in vitro. Rho GAP 22 is thus a novel insulin-regulated Akt substrate. The binding of 14-3-3 to Rho GAP 22 was found to be important for regulation of Rho GAP 22 ability to control the activity of Rac. A mutant Rho GAP 22 that was unable to bind 14-3-3 caused a defect in cell migration, presumably via an inability to properly regulate Rac activity. However, the mechanism by which this occurs is not yet clear and further experiments are needed. It is possible that 14-3-3 binding causes an activation of the GAP activity of Rho GAP 22 by catalysing a conformational change when bound. Another explanation is that 14-3-3 regulates the localisation of Rho GAP 22, allowing for precise spatial regulation of Rac activity. Additional experiments are also required to determine what other proteins regulate the function of Rho GAP 22. FilGAP, the close homologue of Rho GAP 22, will serve as a model for these experiments.

142 REFERENCES

Abel, E. D., O. Peroni, et al. (2001). "Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver." Nature 409(6821): 729-733. Abercrombie, M., J. E. M. Heaysman, et al. (1971). "The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella." Experimental Cell Research 67(2): 359-367. Abo, A., E. Pick, et al. (1991). "Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1." Nature 353(6345): 668-670. Abramoff, M. D., P. J. Magelhaes, et al. (2004). "Image Processing with ImageJ." Biophotonics International 11(7): 36-42. Agarwal-Mawal, A., H. Y. Qureshi, et al. (2003). "14-3-3 Connects Glycogen Synthase Kinase-3 to Tau within a Brain Microtubule-associated Tau Phosphorylation Complex." Journal of Biological Chemistry 278(15): 12722-12728. Aitken, A. (2006). "14-3-3 proteins: A historic overview." Seminars in Cancer Biology 16(3): 162-172. Aitken, A., S. Howell, et al. (1995). "14-3-3 and are the Phosphorylated Forms of Raf-activating 14- 3-3 and ." Journal of Biological Chemistry 270(11): 5706-5709. Aitsebaomo, J., K. Wennerberg, et al. (2004). "p68RacGAP Is a Novel GTPase-activating Protein That Interacts with Vascular Endothelial Zinc Finger-1 and Modulates Endothelial Cell Capillary Formation." Journal of Biological Chemistry 279(17): 17963-17972. Alessi, D. R., M. Andjelkovic, et al. (1996). "Mechanism of activation of protein kinase B by insulin and IGF-1." EMBO J 15(23): 6541-6551. Alessi, D. R., M. Deak, et al. (1997). "3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase." 7(10): 776-789. Alessi, D. R., S. R. James, et al. (1997). "Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha." Curr Biol 7(4): 261-269. Allen, W., G. Jones, et al. (1997). "Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages." J Cell Sci 110(6): 707-720. Altomare, D. A., K. Guo, et al. (1995). "Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene." Oncogene 11(6): 1055-1060. Amano, M., K. Chihara, et al. (1998). "Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase." Genes Cells 3(3): 177-188. Arber, S., F. A. Barbayannis, et al. (1998). "Regulation of actin dynamics through phosphorylation of cofilin by LIM- kinase." Nature 393(6687): 805-809. Arrieumerlou, C. and T. Meyer (2005). "A Local Coupling Model and Compass Parameter for Eukaryotic Chemotaxis." Developmental Cell 8(2): 215-227. Bae, S. S., H. Cho, et al. (2003). "Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B." J Biol Chem 278(49): 49530-49536. Bae, S. S., H. Cho, et al. (2003). "Isoform-specific Regulation of Insulin-dependent Glucose Uptake by Akt/Protein Kinase B." Journal of Biological Chemistry 278(49): 49530-49536. Balendran, A., A. Casamayor, et al. (1999). "PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2." Current Biology 9(8): 393-404. Ballestrem, C., B. Wehrle-Haller, et al. (2000). "Actin-dependent Lamellipodia Formation and Microtubule-dependent Tail Retraction Control-directed Cell Migration." Mol. Biol. Cell 11(9): 2999-3012. Bass, M. D., K. A. Roach, et al. (2007). "Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix." J. Cell Biol. 177(3): 527-538. Bellacosa, A., T. O. Chan, et al. (1998). "Akt activation by growth factors is a multiple-step process: the role of the PH domain." Oncogene 17(3): 313-325. Bellacosa, A., J. R. Testa, et al. (1991). "A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region." Science 254(5029): 274-277. Benard, V., B. P. Bohl, et al. (1999). "Characterization of Rac and Cdc42 Activation in Chemoattractant- stimulated Human Neutrophils Using a Novel Assay for Active GTPases." Journal of Biological Chemistry 274(19): 13198-13204. Beningo, K. A., M. Dembo, et al. (2001). "Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts." J. Cell Biol. 153(4): 881-888. Bennett, R. G., F. G. Hamel, et al. (2000). "Insulin Inhibits the Ubiquitin-Dependent Degrading Activity of the 26S Proteasome." Endocrinology 141(7): 2508-2517. Benzinger, A., N. Muster, et al. (2005). "Targeted Proteomic Analysis of 14-3-3, a p53 Effector Commonly Silenced in Cancer." Mol Cell Proteomics 4(6): 785-795. 143 Berman, H. M., J. Westbrook, et al. (2000). "The Protein Data Bank." Nucleic Acids Research 28(1): 235-242. Berwick, D. C., G. C. Dell, et al. (2004). "Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles." J Cell Sci 117(Pt 25): 5985-5993. Bhaskar, P. T. and N. Hay (2007). "The Two TORCs and Akt." Developmental Cell 12(4): 487-502. Blommaart, E. F. C., J. J. F. P. Luiken, et al. (1995). "Phosphorylation of Ribosomal Protein S6 Is Inhibitory for Autophagy in Isolated Rat Hepatocytes." Journal of Biological Chemistry 270(5): 2320-2326. Boston, P. F., P. Jackson, et al. (1982). "Human 14-3-3 protein: radioimmunoassay, tissue distribution, and cerebrospinal fluid levels in patients with neurological disorders." J Neurochem 38(5): 1475- 1482. Boureux, A., E. Vignal, et al. (2007). "Evolution of the Rho Family of Ras-Like GTPases in Eukaryotes." Mol Biol Evol 24(1): 203-216. Braselmann, S. and F. McCormick (1995). "Bcr and Raf form a complex in vivo via 14-3-3 proteins." EMBO J 14(19): 4839-4848. Bridges, D. and G. B. G. Moorhead (2005). "14-3-3 Proteins: A Number of Functions for a Numbered Protein." Sci. STKE 2005(296): re10-. Brodbeck, D., P. Cron, et al. (1999). "A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain." J Biol Chem 274(14): 9133-9136. Brognard, J., E. Sierecki, et al. (2007). "PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms." Mol Cell 25(6): 917-931. Browne, T. R., A. Van Langenhove, et al. (1981). "Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin." Clin Pharmacol Ther 29(4): 511-515. Brummer, T., M. Larance, et al. (2008). "Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein." EMBO J 27(17): 2305-2316. Brunet, A., A. Bonni, et al. (1999). "Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor." Cell 96(6): 857-868. Bryant, N. J., R. Govers, et al. (2002). "Regulated transport of the glucose transporter GLUT4." Nat Rev Mol Cell Biol 3(4): 267-277. Burgess, R. W., K. A. Peterson, et al. (2004). "Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice." Mol Cell Biol 24(3): 1096-1105. Bustos, D. M. and A. A. Iglesias (2006). "Intrinsic disorder is a key characteristic in partners that bind 14- 3-3 proteins." Proteins: Structure, Function, and Bioinformatics 63(1): 35-42. Calleja, V. r., D. Alcor, et al. (2007). "Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo." PLoS Biol 5(4): e95. Campbell, J. K., R. Gurung, et al. (1997). "Activation of the 43 kDa Inositol Polyphosphate 5- Phosphatase by 14-3-3 " Biochemistry 36(49): 15363-15370. Cantley, L. C. (2002). "The phosphoinositide 3-kinase pathway." Science 296(5573): 1655-1657. Cardone, M. H., N. Roy, et al. (1998). "Regulation of Cell Death Protease Caspase-9 by Phosphorylation." Science 282(5392): 1318-1321. Celis, J. E., B. Gesser, et al. (1990). "Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data." Electrophoresis 11(12): 989-1071. Chahdi, A. and A. Sorokin (2008). "Protein Kinase A-Dependent Phosphorylation Modulates 1Pix Guanine Nucleotide Exchange Factor Activity through 14-3-3 Binding." Mol. Cell. Biol. 28(5): 1679-1687. Chaudhri, M., M. Scarabel, et al. (2003). "Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo." Biochemical and Biophysical Research Communications 300(3): 679-685. Chavez, J. A., W. G. Roach, et al. (2008). "Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase- activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation." J Biol Chem 283(14): 9187-9195. Chen, W. S., P. Z. Xu, et al. (2001). "Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene." Genes Dev 15(17): 2203-2208. Chen, W. T. (1981). "Mechanism of retraction of the trailing edge during fibroblast movement." Journal of Cell Biology 90(1): 187-200. Cheresh, D. A., J. Leng, et al. (1999). "Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells." J Cell Biol 146(5): 1107-1116.

144 Chiang, C.-W., G. Harris, et al. (2001). "Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation." Blood 97(5): 1289-1297. Cho, H., J. L. Thorvaldsen, et al. (2001). "Akt1/PKB is required for normal growth but dispensable for maintenance of glucose homeostasis in mice." J Biol Chem 276(42): 38349-38352. Coblitz, B., S. Shikano, et al. (2005). "C-terminal Recognition by 14-3-3 Proteins for Surface Expression of Membrane Receptors." Journal of Biological Chemistry 280(43): 36263-36272. Coffer, P. J. and J. R. Woodgett (1991). "Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families." Eur J Biochem 201(2): 475-481. Cohen, P. (2001). "The role of protein phosphorylation in human health and disease." European Journal of Biochemistry 268(19): 5001-5010. Collins, B. J., M. Deak, et al. (2003). "In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation." EMBO J 22(16): 4202-4211. Collins, B. J., M. Deak, et al. (2005). "In vivo role of the phosphate groove of PDK1 defined by knockin mutation." J Cell Sci 118(Pt 21): 5023-5034. Cox, J. and M. Mann (2008). "MaxQuant enables high peptide identification rates, individualized p.p.b.- range mass accuracies and proteome-wide protein quantification." Nat Biotechnol. 26(12): 1367- 1372. Cross, D. A., D. R. Alessi, et al. (1995). "Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B." Nature 378(6559): 785-789. Cross, D. A. E., D. R. Alessi, et al. (1995). "Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B." Nature 378(6559): 785-789. Currie, R. A., K. S. Walker, et al. (1999). "Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1." Biochem J 337 ( Pt 3): 575-583. Datta, S. R., H. Dudek, et al. (1997). "Akt Phosphorylation of BAD Couples Survival Signals to the Cell- Intrinsic Death Machinery." Cell 91(2): 231-241. Datta, S. R., A. Katsov, et al. (2000). "14-3-3 Proteins and Survival Kinases Cooperate to Inactivate BAD by BH3 Domain Phosphorylation." Molecular Cell 6(1): 41-51. Dawe, H. R., L. S. Minamide, et al. (2003). "ADF/Cofilin Controls Cell Polarity during Fibroblast Migration." Current Biology 13(3): 252-257. Didsbury, J., R. F. Weber, et al. (1989). "rac, a novel ras-related family of proteins that are botulinum toxin substrates." J Biol Chem 264(28): 16378-16382. Diehl, J. A., M. Cheng, et al. (1998). "Glycogen synthase kinase-3 regulates cyclin D1 proteolysis and subcellular localization." Genes & Development 12(22): 3499-3511. Diekmann, D., A. Abo, et al. (1994). "Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity." Science 265(5171): 531-533. Diekmann, D., S. Brill, et al. (1991). "Bcr encodes a GTPase-activating protein for p21." Nature 351(6325): 400-402. Dijkers, P. F., K. U. Birkenkamp, et al. (2002). "FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity." J. Cell Biol. 156(3): 531-542. Dong, L. Q. and F. Liu (2005). "PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle." Am J Physiol Endocrinol Metab 289(2): E187-196. Dovas, A. and J. R. Couchman (2005). "RhoGDI: multiple functions in the regulation of Rho family GTPase activities." Biochem. J. 390(1): 1-9. Dubois, F., F. Vandermoere, et al. (2009). "Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling." Mol Cell Proteomics 8(11): 2487-2499. Dubois, T., C. Rommel, et al. (1997). "14-3-3 Is Phosphorylated by Casein Kinase I on Residue 233." Journal of Biological Chemistry 272(46): 28882-28888. Dunaway, S., H.-Y. Liu, et al. (2005). "Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function." J Cell Sci 118(1): 39-50. Easton, R. M., H. Cho, et al. (2005). "Role for Akt3/Protein Kinase B in Attainment of Normal Brain Size." Mol. Cell. Biol. 25(5): 1869-1878. Eckes, B., E. Colucci-Guyon, et al. (2000). "Impaired wound healing in embryonic and adult mice lacking vimentin." J Cell Sci 113(13): 2455-2462. Eckes, B., D. Dogic, et al. (1998). "Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts." J Cell Sci 111(13): 1897-1907. 145 Eden, S., R. Rohatgi, et al. (2002). "Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck." Nature 418(6899): 790-793. Edwards, D. C., L. C. Sanders, et al. (1999). "Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics." Nature Cell Biology 1(5): 253-259. Etienne-Manneville, S. (2004). "Cdc42 - the centre of polarity." J Cell Sci 117(8): 1291-1300. Etienne-Manneville, S. and A. Hall (2001). "Integrin-Mediated Activation of Cdc42 Controls Cell Polarity in Migrating Astrocytes through PKC[zeta]." Cell 106(4): 489-498. Etienne-Manneville, S. and A. Hall (2003). "Cdc42 regulates GSK-3 and adenomatous polyposis coli to control cell polarity." Nature 421(6924): 753-756. Eulalio, A., J. Rehwinkel, et al. (2007). "Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing." Genes & Development 21(20): 2558-2570. Euteneuer, U. and M. Schliwa (1984). "Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules." Nature 310(5972): 58-61. Franke, T. F., C. P. Hornik, et al. "PI3K//Akt and apoptosis: size matters." Oncogene 22(56): 8983-8998. Franke, T. F., C. P. Hornik, et al. (2003). "PI3K//Akt and apoptosis: size matters." Oncogene 22(56): 8983-8998. Franke, T. F., S. I. Yang, et al. (1995). "The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase." Cell 81(5): 727-736. Frodin, M., T. L. Antal, et al. (2002). "A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation." EMBO J 21(20): 5396-5407. Frost, J. A., A. Khokhlatchev, et al. (1998). "Differential Effects of PAK1-activating Mutations Reveal Activity-dependent and -independent Effects on Cytoskeletal Regulation." Journal of Biological Chemistry 273(43): 28191-28198. Fuglsang, A. T., S. Visconti, et al. (1999). "Binding of 14-3-3 Protein to the Plasma Membrane H+- ATPase AHA2 Involves the Three C-terminal Residues Tyr946-Thr-Val and Requires Phosphorylation of Thr947." Journal of Biological Chemistry 274(51): 36774-36780. Fukuhara, N., J. Ebert, et al. (2005). "SMG7 Is a 14-3-3-like Adaptor in the Nonsense-Mediated mRNA Decay Pathway." Molecular Cell 17(4): 537-547. Gail, M. (1973). Locomotion of Tissue Cells. Amsterdam, Elsevier. Gammeltoft, S. and E. Van Obberghen (1986). "Protein kinase activity of the insulin receptor." Biochem. J. 235(1): 1-11. Gao, T., F. Furnari, et al. (2005). "PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth." Mol Cell 18(1): 13-24. Garcia, Z., V. Silio, et al. (2006). "A PI3K activity-independent function of p85 regulatory subunit in control of mammalian cytokinesis." EMBO J 25(20): 4740-4751. Gardiner, E. M., K. N. Pestonjamasp, et al. (2002). "Spatial and Temporal Analysis of Rac Activation during Live Neutrophil Chemotaxis." Current Biology 12(23): 2029-2034. Gavin, A.-C., M. Bosche, et al. (2002). "Functional organization of the yeast proteome by systematic analysis of protein complexes." Nature 415(6868): 141-147. Ghosh, M., X. Song, et al. (2004). "Cofilin Promotes Actin Polymerization and Defines the Direction of Cell Motility." Science 304(5671): 743-746. Glading, A., D. A. Lauffenburger, et al. (2002). "Cutting to the chase: calpain proteases in cell motility." Trends in Cell Biology 12(1): 46-54. Gotlieb, A. I., L. Subrahmanyan, et al. (1983). "Microtuble-organizing Microtubule-organizing and cell migration: Effect of inhibition of migration and microtubule disruption in endothelial cells." Journal of Cell Biology 96(5): 1266-1272. Gu, H. and B. G. Neel (2003). "The 'Gab' in signal transduction." Trends in Cell Biology 13(3): 122-130. Gygi, S. P., B. Rist, et al. (1999). "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags." Nat Biotechnol 17(10): 994-999. Haas, W., B. K. Faherty, et al. (2006). "Optimization and use of peptide mass measurement accuracy in shotgun proteomics." Mol. Cell. Proteomics. 5(7): 1326-1337. Haataja, L., J. Groffen, et al. (1997). "Characterization of RAC3, a novel member of the Rho family." J Biol Chem 272(33): 20384-20388. Hall, A. (1998). "Rho GTPases and the Actin Cytoskeleton." Science 279(5350): 509-514. Heasman, S. J. and A. J. Ridley (2008). "Mammalian Rho GTPases: new insights into their functions from in vivo studies." Nat Rev Mol Cell Biol 9(9): 690-701. Henriksson, M. L., M. S. Francis, et al. (2002). "A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo." European Journal of Biochemistry 269(20): 4921-4929. Hermeking, H., C. Lengauer, et al. (1997). "14-3-3 Is a p53-Regulated Inhibitor of G2/M Progression." Molecular Cell 1(1): 3-11. 146 Higuchi, M., K. Onishi, et al. (2008). "Scaffolding function of PAK in the PDK1-Akt pathway." Nat Cell Biol 10(11): 1356-1364. Hill, C. S., J. Wynne, et al. (1995). "The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF." Cell 81(7): 1159-1170. Hornbeck, P. V., I. Chabra, et al. (2004). "PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation." Proteomics 4(6): 1551-1561. Hresko, R. C. and M. Mueckler (2005). "mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes." J Biol Chem 280(49): 40406-40416. Hynes, R. O. (1987). "Integrins: A family of cell surface receptors." Cell 48(4): 549-554. Hynes, R. O. (2002). "Integrins: Bidirectional, Allosteric Signaling Machines." Cell 110(6): 673-687. Ichimura, T., T. Isobe, et al. (1988). "Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases." Proceedings of the National Academy of Sciences of the United States of America 85(19): 7084- 7088. Ichimura, T., T. Isobe, et al. (1987). "Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+,calmodulin- dependent protein kinase II." FEBS Letters 219(1): 79-82. Ichimura, T., M. Ito, et al. (1997). "The 14-3-3 protein binds its target proteins with a common site located towards the C-terminus." FEBS Letters 413(2): 273-276. Ichimura, T., J. Uchiyama, et al. (1995). "Identification of the Site of Interaction of the 14-3-3 Protein with Phosphorylated Tryptophan Hydroxylase." Journal of Biological Chemistry 270(48): 28515-28518. Iizuka, K., A. Yoshii, et al. (1999). "A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea." Br J Pharmacol 128(4): 925-933. Ikemoto, S., K. S. Thompson, et al. (1995). "Expression of an insulin-responsive glucose transporter (GLUT4) minigene in transgenic mice: effect of exercise and role in glucose homeostasis." Proc Natl Acad Sci U S A 92(3): 865-869. Inoki, K., Y. Li, et al. (2002). "TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling." Nat Cell Biol 4(9): 648-657. Irie, H. Y., R. V. Pearline, et al. (2005). "Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition." J. Cell Biol. 171(6): 1023-1034. Ishizaki, T., Y. Morishima, et al. (2001). "Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia1." Nature Cell Biology 3(1): 8-14. Jacinto, E., V. Facchinetti, et al. (2006). "SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity." Cell 127(1): 125-137. James, D. E., R. Brown, et al. (1988). "Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein." Nature 333(6169): 183-185. Jin, J., F. D. Smith, et al. (2004). "Proteomic, Functional, and Domain-Based Analysis of In Vivo 14-3-3 Binding Proteins Involved in Cytoskeletal Regulation and Cellular Organization." Current Biology 14(16): 1436-1450. Jones, D. H., S. Ley, et al. (1995). "Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins." FEBS Letters 368(1): 55-58. Jones, P. F., T. Jakubowicz, et al. (1991). "Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily." Proceedings of the National Academy of Sciences of the United States of America 88(10): 4171-4175. Joost, H. G., G. I. Bell, et al. (2002). "Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators." Am J Physiol Endocrinol Metab 282(4): E974-976. Kandel, E. S. and N. Hay (1999). "The Regulation and Activities of the Multifunctional Serine/Threonine Kinase Akt/PKB." Experimental Cell Research 253(1): 210-229. Kane, S., H. Sano, et al. (2002). "A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain." J Biol Chem 277(25): 22115-22118. Kanzaki, M. (2006). "Insulin Receptor Signals Regulating GLUT4 Translocation and Actin Dynamics." Endocrine Journal 53(3): 267-293. Kater, S. B. and V. Rehder (1995). "The sensory-motor role of growth cone filopodia." Curr Opin Neurobiol 5(1): 68-74. Katoh, M. (2004). "Characterization of human ARHGAP10 gene in silico." Int J Oncol 25(4): 1201-1206. Katoh, M. (2004). "Identification and characterization of ARHGAP24 and ARHGAP25 genes in silico." Int J Mol Med 14(2): 333-338. 147 Kaverina, I., O. Krylyshkina, et al. (1999). "Microtubule Targeting of Substrate Contacts Promotes Their Relaxation and Dissociation." J. Cell Biol. 146(5): 1033-1044. Kemphues, K. J., J. R. Priess, et al. (1988). "Identification of genes required for cytoplasmic localization in early C. elegans embryos." Cell 52(3): 311-320. King, F. W., J. Skeen, et al. (2004). "Inhibition of Chk1 by activated PKB/Akt." Cell Cycle 3(5): 634- 637. Knighton, D. R., J. H. Zheng, et al. (1991). "Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase." Science 253(5018): 407-414. Kohn, A. D., S. A. Summers, et al. (1996). "Expression of a Constitutively Active Akt Ser/Thr Kinase in 3T3-L1 Adipocytes Stimulates Glucose Uptake and Glucose Transporter 4 Translocation." Journal of Biological Chemistry 271(49): 31372-31378. Kolsch, V., P. G. Charest, et al. (2008). "The regulation of cell motility and chemotaxis by phospholipid signaling." J Cell Sci 121(5): 551-559. Kostelecky, B., A. T. Saurin, et al. (2009). "Recognition of an intra-chain tandem 14-3-3 binding site within PKC ." EMBO Rep 10(9): 983-989. Kovacina, K. S., G. Y. Park, et al. (2003). "Identification of a proline-rich Akt substrate as a 14-3-3 binding partner." J Biol Chem 278(12): 10189-10194. Krebs, E. G., D. J. Graves, et al. (1959). "Factors Affecting the Activity of Muscle Phosphorylase b Kinase." Journal of Biological Chemistry 234(11): 2867-2873. Krüger, M., M. Moser, et al. (2008). "SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function." 134(2): 353-364. Ku, N.-O., J. Liao, et al. (1998). "Phosphorylation of human keratin 18 serine 33 regulates binding to 14- 3-3 proteins." EMBO J 17(7): 1892-1906. Kulik, G., A. Klippel, et al. (1997). "Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt." Molecular and Cellular Biology 17(3): 1595-1606. Laemilli, U. (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature 227(5259): 680-685. Lai, W. S., E. A. Kennington, et al. (2003). "Tristetraprolin and its family members can promote the cell- free deadenylation of AU-rich element-containing mRNAs by poly(A) ribonuclease." Mol Cell Biol 23(11): 3798-3812. Larance, M., G. Ramm, et al. (2005). "Characterization of the Role of the Rab GTPase-activating Protein AS160 in Insulin-regulated GLUT4 Trafficking." Journal of Biological Chemistry 280(45): 37803-37813. Larkin, M. A., G. Blackshields, et al. (2007). "Clustal W and Clustal X version 2.0." Bioinformatics 23(21): 2947-2948. Lavelin, I. and B. Geiger (2005). "Characterization of a Novel GTPase-activating Protein Associated with Focal Adhesions and the Actin Cytoskeleton." Journal of Biological Chemistry 280(8): 7178- 7185. Lee, J. W., H. S. Choi, et al. (1995). "Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor." Mol Endocrinol 9(2): 243-254. Leffers, H., P. Madsen, et al. (1993). "Molecular Cloning and Expression of the Transformation Sensitive Epithelial Marker Stratifin : A Member of a Protein Family that has been Involved in the Protein Kinase C Signalling Pathway." Journal of Molecular Biology 231(4): 982-998. Letunic, I., T. Doerks, et al. (2009). "SMART 6: recent updates and new developments." Nucl. Acids Res. 37(suppl_1): D229-232. Leung, T., X. Q. Chen, et al. (1996). "The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton." Mol Cell Biol 16(10): 5313- 5327. Li, Y., K. Inoki, et al. (2003). "The p38 and MK2 Kinase Cascade Phosphorylates Tuberin, the Tuberous Sclerosis 2 Gene Product, and Enhances Its Interaction with 14-3-3." Journal of Biological Chemistry 278(16): 13663-13671. Liang, J., J. Zubovitz, et al. (2002). "PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest." Nat Med 8(10): 1153-1160. Liang, X. H., S. Jackson, et al. (1999). "Induction of autophagy and inhibition of tumorigenesis by beclin 1." Nature 402(6762): 672-676. Liu, B. P., M. Chrzanowska-Wodnicka, et al. (1998). "Microtubule depolymerization induces stress fibers, focal adhesions, and DNA synthesis via the GTP-binding protein Rho." Cell Adhesion and Communication 5(4): 249-255.

148 Liu, D., J. Bienkowska, et al. (1995). "Crystal structure of the zeta isoform of the 14-3-3 protein." Nature 376(6536): 191-194. Liu, D., J. Bienkowska, et al. (1995). "Crystal structure of the zeta isoform of the 14-3-3 protein." Nature 376(6536): 191-194. Liu, M. L., E. M. Gibbs, et al. (1993). "Transgenic mice expressing the human GLUT4/muscle-fat facilitative glucose transporter protein exhibit efficient glycemic control." Proc Natl Acad Sci U S A 90(23): 11346-11350. Liu, X., Y. Shi, et al. (2006). "Quantitative analysis of anti-apoptotic function of Akt in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells under normal and stressed conditions." J Biol Chem 281(42): 31380-31388. Liu, Y.-C., Y. Liu, et al. (1997). "Serine Phosphorylation of Cbl Induced by Phorbol Ester Enhances Its Association with 14-3-3 Proteins in T Cells via a Novel Serine-rich 14-3-3-binding Motif." Journal of Biological Chemistry 272(15): 9979-9985. Luk, S. C. W., S.-m. Ngai, et al. (1998). "Developmental regulation of 14-3-3 isoform in rat heart." Journal of Cellular Biochemistry 68(2): 195-199. Ma, S. and R. L. Chisholm (2002). "Cytoplasmic dynein-associated structures move bidirectionally in vivo." Journal of Cell Science 115(7): 1453-1460. Machacek, M., L. Hodgson, et al. (2009). "Coordination of Rho GTPase activities during cell protrusion." Nature 461(7260): 99-103. Machesky, L. M., R. D. Mullins, et al. (1999). "Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex." Proc Natl Acad Sci U S A 96(7): 3739-3744. Maekawa, M., T. Ishizaki, et al. (1999). "Signaling from Rho to the Actin Cytoskeleton Through Protein Kinases ROCK and LIM-kinase." Science 285(5429): 895-898. Mammucari, C., G. Milan, et al. (2007). "FoxO3 Controls Autophagy in Skeletal Muscle In Vivo." Cell Metabolism 6(6): 458-471. Manning, B. D., A. R. Tee, et al. (2002). "Identification of the Tuberous Sclerosis Complex-2 Tumor Suppressor Gene Product Tuberin as a Target of the Phosphoinositide 3-Kinase/Akt Pathway." 10(1): 151-162. Margolis, S. S., S. Walsh, et al. (2003). "PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation." EMBO J 22(21): 5734-5745. Martinez-Quiles, N., R. Rohatgi, et al. (2001). "WIP regulates N-WASP-mediated actin polymerization and filopodium formation." Nat Cell Biol 3(5): 484-491. McGough, A., B. Pope, et al. (1997). "Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function." J. Cell Biol. 138(4): 771-781. Medema, R. H., G. J. Kops, et al. (2000). "AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1." Nature 404(6779): 782-787. Meek, S. E. M., W. S. Lane, et al. (2004). "Comprehensive Proteomic Analysis of Interphase and Mitotic 14-3-3-binding Proteins." Journal of Biological Chemistry 279(31): 32046-32054. Meier, R., M. Thelen, et al. (1998). "Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress." EMBO J 17(24): 7294-7303. Meriane, M., S. Mary, et al. (2000). "Cdc42Hs and Rac1 GTPases Induce the Collapse of the Vimentin Intermediate Filament Network." Journal of Biological Chemistry 275(42): 33046-33052. Middleton, C. A., A. F. Brown, et al. (1989). "The polarization of fibroblasts in early primary cultures is independent of microtubule integrity." Journal of Cell Science 94(1): 25-32. Miki, H., K. Miura, et al. (1996). "N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases." EMBO J 15(19): 5326-5335. Miki, H., S. Suetsugu, et al. (1998). "WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac." EMBO J 17(23): 6932-6941. Milburn, C. C., M. Deak, et al. (2003). "Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change." Biochem J 375(Pt 3): 531-538. Minoshima, Y., T. Kawashima, et al. (2003). "Phosphorylation by Aurora B Converts MgcRacGAP to a RhoGAP during Cytokinesis." 4(4): 549-560. Mitchison, T. J. and L. P. Cramer (1996). "Actin-based cell motility and cell locomotion." Cell 84(3): 371-379. Moore, B. and V. J. Perez (1967). Physiological and Biochemical Aspects of Nervous Integration. F. D. Carlson. Englewood Cliffs, NJ, Prentice-Hall: 343–359. Moreland, J., A. Gramada, et al. (2005). "The Molecular Biology Toolkit (MBT): a modular platform for developing molecular visualization applications." BMC Bioinformatics 6(1): 21. 149 Morita, S., T. Kojima, et al. (2000). "Plat-E: an efficient and stable system for transient packaging of retroviruses." Gene Therapy 7(12): 1063-1069. Mullins, R. D., J. A. Heuser, et al. (1998). "The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments." Proc Natl Acad Sci U S A 95(11): 6181-6186. Muslin, A. J., J. W. Tanner, et al. (1996). "Interaction of 14-3-3 with Signaling Proteins Is Mediated by the Recognition of Phosphoserine." Cell 84(6): 889-897. Nakamura, F., O. Heikkinen, et al. (2009). "Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations." PLoS ONE 4(3): e4928. Nakashima, N., P. M. Sharma, et al. (2000). "The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes." J Biol Chem 275(17): 12889-12895. Nakayama, M., T. M. Goto, et al. (2008). "Rho-Kinase Phosphorylates PAR-3 and Disrupts PAR Complex Formation." Developmental Cell 14(2): 205-215. Nishimura, T., T. Yamaguchi, et al. (2005). "PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1." Nat Cell Biol 7(3): 270-277. Nobes, C. D., P. Hawkins, et al. (1995). "Activation of the small GTP-binding proteins rho and rac by growth factor receptors." J Cell Sci 108 ( Pt 1): 225-233. Norman, B. H., C. Shih, et al. (1996). "Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs." J Med Chem 39(5): 1106-1111. Norman, J. C., L. S. Price, et al. (1996). "The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways." Mol Biol Cell 7(9): 1429-1442. Obata, T., M. B. Yaffe, et al. (2000). "Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKB." Journal of Biological Chemistry 275(46): 36108-36115. Obenauer, J. C., L. C. Cantley, et al. (2003). "Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs." Nucleic Acids Res. 31(13): 3635-3641. Obsil, T., R. Ghirlando, et al. (2003). "Two 14-3-3 Binding Motifs Are Required for Stable Association of Forkhead Transcription Factor FOXO4 with 14-3-3 Proteins and Inhibition of DNA Binding." Biochemistry 42(51): 15264-15272. Obsil, T., R. Ghirlando, et al. (2001). "Crystal Structure of the 14-3-3:Serotonin N-Acetyltransferase Complex: A Role for Scaffolding in Enzyme Regulation." Cell 105(2): 257-267. Obsilova, V., P. Herman, et al. (2004). "14-3-3 C-terminal Stretch Changes Its Conformation upon Ligand Binding and Phosphorylation at Thr232." Journal of Biological Chemistry 279(6): 4531- 4540. Oda, Y., K. Huang, et al. (1999). "Accurate quantitation of protein expression and site-specific phosphorylation." Proceedings of the National Academy of Sciences of the United States of America 96(12): 6591-6596. Ohta, Y., J. H. Hartwig, et al. (2006). "FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling." Nat Cell Biol 8(8): 803-814. Olsen, J. V., B. Blagoev, et al. (2006). "Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks." Cell 127(3): 635-648. Olsen, J. V., M. Vermeulen, et al. "Quantitative Phosphoproteomics Reveals Widespread Full Phosphorylation Site Occupancy During Mitosis." Sci. Signal. 3(104): ra3-. Olson, M. F., A. Ashworth, et al. (1995). "An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1." Science 269(5228): 1270-1272. Ong, S.-E., B. Blagoev, et al. (2002). "Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics." Molecular & Cellular Proteomics 1(5): 376-386. Ottmann, C., S. Marco, et al. (2007). "Structure of a 14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H+-ATPase by Combining X-Ray Crystallography and Electron Cryomicroscopy." Molecular Cell 25(3): 427-440. Palazzo, A. F., T. A. Cook, et al. (2001). "mDia mediates Rho-regulated formation and orientation of stable microtubules." Nature Cell Biology 3(8): 723-729. Pallas, D. C., H. Fu, et al. (1994). "Association of polyomavirus middle tumor antigen with 14-3-3 proteins." Science 265(5171): 535-537. Pankov, R., Y. Endo, et al. (2005). "A Rac switch regulates random versus directionally persistent cell migration." J. Cell Biol. 170(5): 793-802. Pantaloni, D., R. Boujemaa, et al. (2000). "The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins." Nat Cell Biol 2(7): 385-391. 150 Parker, R. and U. Sheth (2007). "P Bodies and the Control of mRNA Translation and Degradation." Molecular Cell 25(5): 635-646. Paul, A.-L., P. C. Sehnke, et al. (2005). "Isoform-specific Subcellular Localization among 14-3-3 Proteins in Arabidopsis Seems to be Driven by Client Interactions." Mol. Biol. Cell 16(4): 1735-1743. Peck, J., G. Douglas, et al. (2002). "Human RhoGAP domain-containing proteins: structure, function and evolutionary relationships." FEBS Letters 528(1-3): 27-34. Peng, J., B. J. Wallar, et al. (2003). "Disruption of the Diaphanous-Related Formin Drf1 Gene Encoding mDia1 Reveals a Role for Drf3 as an Effector for Cdc42." Current Biology 13(7): 534-545. Peng, X. D., P. Z. Xu, et al. (2003). "Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2." Genes Dev 17(11): 1352-1365. Perona, R., S. Montaner, et al. (1997). "Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins." Genes Dev 11(4): 463-475. Petosa, C., S. C. Masters, et al. (1998). "14-3-3 Binds a Phosphorylated Raf Peptide and an Unphosphorylated Peptide via Its Conserved Amphipathic Groove." Journal of Biological Chemistry 273(26): 16305-16310. Powell, D. W., M. J. Rane, et al. (2002). "Identification of 14-3-3 as a Protein Kinase B/Akt Substrate." Journal of Biological Chemistry 277(24): 21639-21642. Pozuelo Rubio, M., K. M. Geraghty, et al. (2004). "14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking." Biochem. J. 379(2): 395-408. Primo, L., L. di Blasio, et al. (2007). "Essential role of PDK1 in regulating endothelial cell migration." J. Cell Biol. 176(7): 1035-1047. Puc, J., M. Keniry, et al. (2005). "Lack of PTEN sequesters CHK1 and initiates genetic instability." Cancer Cell 7(2): 193-204. Puigserver, P., J. Rhee, et al. (2003). "Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC- 1[alpha] interaction." Nature 423(6939): 550-555. Puntervoll, P., R. Linding, et al. (2003). "ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins." Nucleic Acids Res. 31(13): 3625-3630. Raftopoulou, M. and A. Hall (2004). "Cell migration: Rho GTPases lead the way." Developmental Biology 265(1): 23-32. Ramm, G., M. Larance, et al. (2006). "A Role for 14-3-3 in Insulin-stimulated GLUT4 Translocation through Its Interaction with the RabGAP AS160." Journal of Biological Chemistry 281(39): 29174-29180. Rea, S., L. B. Martin, et al. (1998). "Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface." J Biol Chem 273(30): 18784-18792. Regen, C. and A. Horwitz (1992). "Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts." J. Cell Biol. 119(5): 1347-1359. Reuther, G. W., H. Fu, et al. (1994). "Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family." Science 266(5182): 129-133. Rich, A. M. and S. T. Hoffstein (1981). "Inverse correlation between neutrophil microtubule numbers and enhanced random migration." Journal of Cell Science Vol. 48: 181-191. Ridley, A. J. and A. Hall (1992). "The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors." Cell 70(3): 389-399. Ridley, A. J. and A. Hall (1992). "The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors." 70(3): 389-399. Ridley, A. J., H. F. Paterson, et al. (1992). "The small GTP-binding protein rac regulates growth factor- induced membrane ruffling." Cell 70(3): 401-410. Ridley, A. J., A. J. Self, et al. (1993). "rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo." EMBO Journal 12(13): 5151-5160. Rittinger, K., J. Budman, et al. (1999). "Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding." Mol Cell 4(2): 153-166. Roach, W. G., J. A. Chavez, et al. (2007). "Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1." Biochem J 403(2): 353-358. Rohatgi, R., L. Ma, et al. (1999). "The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly." Cell 97(2): 221-231. Rosenquist, M., P. Sehnke, et al. (2000). "Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity?" Journal of Molecular Evolution 51(5): 446-458. 151 Ross, P. L., Y. N. Huang, et al. (2004). "Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents." Molecular & Cellular Proteomics 3(12): 1154-1169. Rossetti, L., A. E. Stenbit, et al. (1997). "Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene." J Clin Invest 100(7): 1831- 1839. Rossman, K. L., C. J. Der, et al. (2005). "GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors." Nat Rev Mol Cell Biol 6(2): 167-180. Rossman, K. L., D. K. Worthylake, et al. (2002). "A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange." EMBO J 21(6): 1315-1326. Rottner, K., A. Hall, et al. (1999). "Interplay between Rac and Rho in the control of substrate contact dynamics." Current Biology 9(12): 640-648, S641. Sancak, Y., C. C. Thoreen, et al. (2007). "PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase." Mol Cell 25(6): 903-915. Sander, E. E., J. P. ten Klooster, et al. (1999). "Rac Downregulates Rho Activity: Reciprocal Balance between Both Gtpases Determines Cellular Morphology and Migratory Behavior." J. Cell Biol. 147(5): 1009-1022. Sanders, L. C., F. Matsumura, et al. (1999). "Inhibition of Myosin Light Chain Kinase by p21-Activated Kinase." Science 283(5410): 2083-2085. Sano, H., S. Kane, et al. (2003). "Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation." J Biol Chem 278(17): 14599-14602. Sanz-Moreno, V., G. Gadea, et al. (2008). "Rac Activation and Inactivation Control Plasticity of Tumor Cell Movement." Cell 135(3): 510-523. Saoncella, S., F. Echtermeyer, et al. (1999). "Syndecan-4 signals cooperatively with integrins in a Rhodependent manner in the assembly of focal adhesions and actin stress fibers." Proceedings of the National Academy of Sciences of the United States of America 96(6): 2805-2810. Sarbassov, D. D., D. A. Guertin, et al. (2005). "Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex." Science 307(5712): 1098-1101. Scheffzek, K., M. R. Ahmadian, et al. (1997). "The Ras-RasGAP Complex: Structural Basis for GTPase Activation and Its Loss in Oncogenic Ras Mutants." Science 277(5324): 333-338. Schmelzle, T. and M. N. Hall (2000). "TOR, a central controller of cell growth." Cell 103(2): 253-262. Schmidlin, M., M. Lu, et al. (2004). "The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B." EMBO J 23(24): 4760-4769. Scholz, R.-P., J. Regner, et al. (2009). "DLC1 interacts with 14-3-3 proteins to inhibit RhoGAP activity and block nucleocytoplasmic shuttling." J Cell Sci 122(1): 92-102. Scott, P. H., G. J. Brunn, et al. (1998). "Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway." Proceedings of the National Academy of Sciences of the United States of America 95(13): 7772- 7777. Sehnke, P. C., R. Henry, et al. (2000). "Interaction of a Plant 14-3-3 Protein with the Signal Peptide of a Thylakoid-Targeted Chloroplast Precursor Protein and the Presence of 14-3-3 Isoforms in the Chloroplast Stroma." Plant Physiol. 122(1): 235-242. Sekimoto, T., M. Fukumoto, et al. (2004). "14-3-3 suppresses the nuclear localization of threonine 157- phosphorylated p27Kip1." EMBO J 23(9): 1934-1942. Sekuli, A., C. C. Hudson, et al. (2000). "A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells." Cancer Research 60(13): 3504-3513. Settleman J, Albright CF, et al. (1992). "Association between GTPase activators for Rho and Ras families." Nature 359(6391): 153-154. Shepherd, P. R. and B. B. Kahn (1999). "Glucose Transporters and Insulin Action -- Implications for Insulin Resistance and Diabetes Mellitus." N Engl J Med 341(4): 248-257. Shin, I., F. M. Yakes, et al. (2002). "PKB/Akt mediates cell-cycle progression by phosphorylation of p27Kip1 at threonine 157 and modulation of its cellular localization." Nat Med 8(10): 1145- 1152. Shirsat, N. V., R. J. Pignolo, et al. (1990). "A member of the ras gene superfamily is expressed specifically in T, B and myeloid hemopoietic cells." Oncogene 5(5): 769-772. Shulman, G. I., D. L. Rothman, et al. (1990). "Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by ¹³C nuclear magnetic resonance spectroscopy." New England Journal of Medicine 322(4): 223-228.

152 Silhan, J., V. Obsilova, et al. (2004). "14-3-3 Protein C-terminal Stretch Occupies Ligand Binding Groove and Is Displaced by Phosphopeptide Binding." Journal of Biological Chemistry 279(47): 49113-49119. Sin, W.-C., X.-Q. Chen, et al. (1998). "RhoA-Binding Kinase alpha Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network." Mol. Cell. Biol. 18(11): 6325-6339. Slot, J. W., H. J. Geuze, et al. (1991). "Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat." Proc Natl Acad Sci U S A 88(17): 7815-7819. Somlyo, A. V., D. Bradshaw, et al. (2000). "Rho-Kinase Inhibitor Retards Migration and in Vivo Dissemination of Human Prostate Cancer Cells." Biochemical and Biophysical Research Communications 269(3): 652-659. Staal, S. P. (1987). "Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma." Proceedings of the National Academy of Sciences of the United States of America 84(14): 5034-5037. Stenbit, A. E., T. S. Tsao, et al. (1997). "GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes." Nat Med 3(10): 1096-1101. Stockli, J., J. R. Davey, et al. (2008). "Regulation of glucose transporter 4 translocation by the Rab guanosine triphosphatase-activating protein AS160/TBC1D4: role of phosphorylation and membrane association." Mol Endocrinol 22(12): 2703-2715. Stokoe, D., L. R. Stephens, et al. (1997). "Dual Role of Phosphatidylinositol-3,4,5-trisphosphate in the Activation of Protein Kinase B." Science 277(5325): 567-570. Su, Z.-J., C. N. Hahn, et al. (2004). "A vascular cell-restricted RhoGAP, p73RhoGAP, is a key regulator of angiogenesis." Proceedings of the National Academy of Sciences of the United States of America 101(33): 12212-12217. Summers, S. A., A. W. Kao, et al. (1999). "The Role of Glycogen Synthase Kinase 3 in Insulin- stimulated Glucose Metabolism." Journal of Biological Chemistry 274(25): 17934-17940. Tee, A. R., D. C. Fingar, et al. (2002). "Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling." Proc Natl Acad Sci U S A 99(21): 13571-13576. The UniProt Consortium (2009). "The Universal Protein Resource (UniProt) 2009." Nucl. Acids Res. 37(suppl_1): D169-174. Thomas, C. C., M. Deak, et al. (2002). "High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate." Curr Biol 12(14): 1256-1262. Toker, A., C. A. Ellis, et al. (1990). "Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein." European Journal of Biochemistry 191(2): 421-429. Toker, A. and A. C. Newton (2000). "Akt/Protein Kinase B Is Regulated by Autophosphorylation at the Hypothetical PDK-2 Site." Journal of Biological Chemistry 275(12): 8271-8274. Tong, A. H. Y., G. Lesage, et al. (2004). "Global Mapping of the Yeast Genetic Interaction Network." Science 303(5659): 808-813. Truong, A. B., S. C. Masters, et al. (2002). "Role of the 14-3-3 C-terminal loop in ligand interaction." Proteins: Structure, Function, and Bioinformatics 49(3): 321-325. Tschopp, O., Z.-Z. Yang, et al. (2005). "Essential role of protein kinase B (PKB/Akt3) in postnatal brain development but not in glucose homeostasis." Development 132(13): 2943-2954. Tsuruta, F., J. Sunayama, et al. (2004). "JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins." EMBO J 23(8): 1889-1899. Tu, H. and M. Wigler (1999). "Genetic evidence for Pak1 autoinhibition and its release by Cdc42." Mol Cell Biol 19(1): 602-611. Ueda, K., Y. Ohta, et al. (2003). "The carboxy-terminal pleckstrin homology domain of ROCK interacts with filamin-A." Biochemical and Biophysical Research Communications 301(4): 886-890. Vadlamudi, R. K., F. Li, et al. (2002). "Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1." Nat Cell Biol 4(9): 681-690. Vander Haar, E., S. I. Lee, et al. (2007). "Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40." Nat Cell Biol 9(3): 316-323. Velasco, G., C. Armstrong, et al. (2002). "Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin." FEBS Lett 527(1-3): 101-104. Verkhovsky, A. B., T. M. Svitkina, et al. (1999). "Self-polarization and directional motility of cytoplasm." Curr Biol 9(1): 11-20.

153 Viglietto, G., M. L. Motti, et al. (2002). "Cytoplasmic relocalization and inhibition of the cyclin- dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer." Nat Med 8(10): 1136-1144. Vincent, S., P. Jeanteur, et al. (1992). "Growth-regulated expression of rhoG, a new member of the ras homolog gene family." Mol Cell Biol 12(7): 3138-3148. Vincenz, C. and V. M. Dixit (1996). "14-3-3 Proteins Associate with A20 in an Isoform-specific Manner and Function Both as Chaperone and Adapter Molecules." Journal of Biological Chemistry 271(33): 20029-20034. Wang, B., H. Yang, et al. (1999). "Isolation of High-Affinity Peptide Antagonists of 14-3-3 Proteins by Phage Display." Biochemistry 38(38): 12499-12504. Waterman, M. J. F., E. S. Stavridi, et al. (1998). "ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins." Nat Genet 19(2): 175-178. Webb, D. J., J. T. Parsons, et al. (2002). "Adhesion assembly, disassembly and turnover in migrating cells - over and over and over again." Nat Cell Biol 4(4): E97-E100. Wei, W., J. Jin, et al. (2005). "The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase." Cancer Cell 8(1): 25-33. Welcker, M., J. Singer, et al. (2003). "Multisite Phosphorylation by Cdk2 and GSK3 Controls Cyclin E Degradation." Molecular Cell 12(2): 381-392. Wherlock, M. and H. Mellor (2002). "The Rho GTPase family: a Racs to Wrchs story." J Cell Sci 115(2): 239-240. Whiteman, E. L., H. Cho, et al. (2002). "Role of Akt/protein kinase B in metabolism." Trends Endocrinol Metab 13(10): 444-451. Wilker, E. W., R. A. Grant, et al. (2005). "A Structural Basis for 14-3-3 Functional Specificity." Journal of Biological Chemistry 280(19): 18891-18898. Woodcock, J. M., J. Murphy, et al. (2003). "The Dimeric Versus Monomeric Status of 14-3-3 Is Controlled by Phosphorylation of Ser58 at the Dimer Interface." Journal of Biological Chemistry 278(38): 36323-36327. Worthylake, R. A., S. Lemoine, et al. (2001). "RhoA is required for monocyte tail retraction during transendothelial migration." J. Cell Biol. 154(1): 147-160. Wu, Y. I., D. Frey, et al. (2009). "A genetically encoded photoactivatable Rac controls the motility of living cells." Nature 461(7260): 104-108. Xiao, B., S. J. Smerdon, et al. (1995). "Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways." Nature 376(6536): 188-191. Xiao, B., S. J. Smerdon, et al. (1995). "Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways." Nature 376(6536): 188-191. Yaffe, M. B. (2002). "How do 14-3-3 proteins work? - Gatekeeper phosphorylation and the molecular anvil hypothesis." FEBS Letters 513(1): 53-57. Yaffe, M. B., K. Rittinger, et al. (1997). "The Structural Basis for 14-3-3:Phosphopeptide Binding Specificity." Cell 91(7): 961-971. Yamada, E., S. Okada, et al. (2005). "Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles." J Cell Biol 168(6): 921-928. Yang, J., P. Cron, et al. (2002). "Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP." Nat Struct Biol 9(12): 940-944. Yang, Z. Z., O. Tschopp, et al. (2005). "Dosage-dependent effects of Akt1/protein kinase Balpha (PKBalpha) and Akt3/PKBgamma on thymus, skin, and cardiovascular and nervous system development in mice." Mol Cell Biol 25(23): 10407-10418. Yeh, E., M. Cunningham, et al. (2004). "A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells." Nat Cell Biol 6(4): 308-318. Yoeli-Lerner, M., G. K. Yiu, et al. (2005). "Akt Blocks Breast Cancer Cell Motility and Invasion through the Transcription Factor NFAT." Molecular Cell 20(4): 539-550. Yonemoto, W., M. McGlone, et al. (1997). "Autophosphorylation of the catalytic subunit of cAMP- dependent protein kinase in Escherichia coli." Protein Eng. 10(8): 915-925. Yoshida, K., T. Yamaguchi, et al. (2005). "JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage." Nat Cell Biol 7(3): 278-285. Yuan, Z., A. Agarwal-Mawal, et al. (2004). "14-3-3 Binds to and Mediates Phosphorylation of Microtubule-associated Tau Protein by Ser9-phosphorylated Glycogen Synthase Kinase 3 in the Brain." Journal of Biological Chemistry 279(25): 26105-26114. Zamir, E. and B. Geiger (2001). "Components of cell-matrix adhesions." J Cell Sci 114(20): 3577-3579.

154 Zhao, X., L. Gan, et al. (2004). "Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms." Biochem. J. 378(3): 839-849. Zheng, Y., S. Bagrodia, et al. (1994). "Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85." Journal of Biological Chemistry 269(29): 18727-18730. Zheng, Y., M. J. Hart, et al. (1993). "Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain." Journal of Biological Chemistry 268(33): 24629-24634. Zhou, B. P., Y. Liao, et al. (2001). "Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells." Nat Cell Biol 3(3): 245-252. Zhou, G.-L., D. F. Tucker, et al. (2006). "Opposing Roles for Akt1 and Akt2 in Rac/Pak Signaling and Cell Migration." Journal of Biological Chemistry 281(47): 36443-36453. Zisman, A., O. D. Peroni, et al. (2000). "Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance." Nat Med 6(8): 924-928.

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