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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

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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 .............................................................................................................
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