Demiray, B; (2014) The E3 Ligase TRIM32 is a Novel Effector of the RAS Family GTPase Rap2. Doctoral thesis , UCL (University College London).

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Abstract

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, Δ588) 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.

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