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The E3 Ligase TRIM32 Is an Effector of the RAS Family Gtpase RAP2

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The E3 Ligase TRIM32 Is an Effector of the RAS Family Gtpase RAP2 The E3 Ligase TRIM32 is an effector of the RAS family GTPase RAP2 Berna Demiray A thesis submitted towards the degree of Doctor of Philosophy Cancer Institute University College London 2014 Declaration I, Berna Demiray, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated. London, 2014 The E3 Ligase TRIM32 is an Effector of the RAS family GTPase RAP2 Classical RAS oncogenes are mutated in approximately 30% of human tumours and RAP proteins are closely related to classical RAS proteins. RAP1 has an identical effector domain to RAS whereas RAP2 differs by one amino acid. RAP2 not only shares effectors with other classical RAS family members, but it also has its own specific effectors that do not bind to RAP1 or classical RAS family proteins. Thus, although closely related, RAP2 performs distinct functions, although these have been poorly characterised. Using RAP2 as bait in Tandem Affinity Purifications, we have identified several RAP2 interacting proteins including TRIM32; a protein implicated in diverse pathological processes such as Limb-Girdle Muscular Dystrophy (LGMD2H), and Bardet-Biedl syndrome (BBS). TRIM32 was shown to interact specifically with RAP2 in an activation- and effector domain-dependent manner; demonstrating stronger interaction with the RAP2 V12 mutant than the wild-type RAP2 and defective binding to the effector mutant RAP2 V12A38. The interaction was mapped to the C-terminus of TRIM32 (containing the NHL domains) while mutations found in LGMD2H (R394H, D487N, ∆588) were found to disrupt binding to RAP2. The TRIM32 P130S mutant linked to BBS did not affect binding to RAP2, suggesting that the RAP2-TRIM32 interaction may be functionally involved in LGMD2H. Because TRIM32 is an E3 ubiquitin ligase, the possible ubiquitination of interacting proteins by TRIM32 was assessed along with the potential for modulation by RAP2. RAP2 stimulates the ubiquitin ligase activity of TRIM32 against some substrates but not others. We propose that RAP2 uses TRIM32 to regulate the signalling properties of other RAP2 effectors. Furthermore, our data also shows that the overexpression of TRIM32 may increase C2C12 mouse myoblast cell differentiation whereas the inhibition of RAP2 expression decreases differentiation in C2C12 cells. Further study could lead to a potential link to Limb-Girdle Muscular Dystrophy that remains to be elucidated. Acknowledgements I would like to thank all those who have supported me throughout this PhD, both in my work and outside. At times when I didn’t believe I could make it, or did think I could carry on, my family and close friends, you were there to help motivate me, to console me, to feed me wine and dance around like a fool until I could smile again and keep going. I couldn’t have done it without you all. Contents Declaration ................................................................................................................... 2 The E3 Ligase TRIM32 is an Effector of the RAS family GTPase RAP2 ......................... 3 Acknowledgements ...................................................................................................... 5 Contents ....................................................................................................................... 6 List of Figures ............................................................................................................. 13 List of Tables ............................................................................................................... 16 List of Abbreviations .................................................................................................. 17 CHAPTER 1: RAS Proteins form Interesting Targets for Research ............................. 19 RAS proteins ........................................................................................................... 19 Significance of RAS proteins for study ................................................................ 19 RAS superfamily biochemistry and regulation ................................................... 21 Lipid modification and membrane targeting ...................................................... 23 Prenylation ...................................................................................................... 25 Palmitoylation ................................................................................................. 26 Other common posttranslational modifications ............................................ 26 Myristoylation ............................................................................................. 26 Glycosylation ............................................................................................... 27 Phosphorylation .......................................................................................... 27 S-nitrosylation ............................................................................................. 27 Others .......................................................................................................... 28 RAP proteins ........................................................................................................... 29 Localisation ......................................................................................................... 29 Activation ............................................................................................................ 30 RAP1 .................................................................................................................... 31 6 RAP2 .................................................................................................................... 31 Structure.......................................................................................................... 31 Biological functions of RAP2: Neural function ................................................ 32 Biological functions of RAP2: Receptor trafficking ......................................... 33 RAP effectors ...................................................................................................... 33 Ste20 kinases – TNIK, MINK and MAP4K4/NIK ............................................... 33 Background .................................................................................................. 34 Phylogenic grouping .................................................................................... 34 Structure ...................................................................................................... 35 Expression .................................................................................................... 36 Function ....................................................................................................... 36 TNIK ............................................................................................................. 36 MINK ............................................................................................................ 38 MAP4K4/NIK ................................................................................................ 38 RIN ................................................................................................................... 39 RalGEF family................................................................................................... 41 RGL/Rlf ......................................................................................................... 41 AF6/Afadin ...................................................................................................... 41 Potential RAP2 Interactors ................................................................................. 42 VPS35/VPS29 ................................................................................................... 42 TBC1D10B ........................................................................................................ 43 TRIM32 ............................................................................................................ 44 TRIM ............................................................................................................ 44 TRIM32 ........................................................................................................ 45 Role in Cancer .......................................................................................... 46 Role in Bardet-Biedl Syndrome (BBS) ...................................................... 47 TRIM32 is required for neuronal differentiation ..................................... 48 7 Role in Muscular Dystrophy ..................................................................... 49 Limb-Gridle Muscular Dystrophy ............................................................. 51 LGMD2H ............................................................................................... 53 NOEY2/DIRAS/ARHI ............................................................................................ 56 The DIRAS family ............................................................................................. 56 NOEY2/DIRAS3/ARHI ....................................................................................... 56 NOEY2 is an imprinted tumour suppressor .................................................... 58 NOEY2 mouse model ...................................................................................... 60 DIRAS1 ............................................................................................................
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