Functional identification of RASGEF1 family of exchange factors as activators of RAP2, and as interacting partners of CCDC124
Tazebay, Uygar H.
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Coiled coil domain-124 gene is highly conserved among eukaryotes and the human counterpart encodes a protein with no domain similarities with any previously characterized eukaryotic proteins. In this study, we aimed to identify biological functions and interaction partners of human Ccdc-124. A yeast-two-hybrid analysis carried in this study has revealed that Ccdc-124 interacts with RasGEF1B which was predicted to be a member of Ras guanine exchange factors. The highly conserved RasGEF1 family of proteins contain C-terminal CDC25-homology domain (CDC25- HD) and an N-terminal RasGEF-N domain (Ras Exchange Motif, REM), and is of unknown function and specificity. In this thesis, the interaction of Ccdc-124 and RasGEF1 family of proteins was also established with co-immunoprecipitation and GST pull down assays. On the other hand, by using purified RasGEF1A and RasGEF1B proteins, as well as a large number of Ras family of G-proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. On the other hand, Ccdc-124 protein did not change the stimulatory effect of RasGEF1 family of proteins on any of the tested G proteins in vitro. Furthermore, using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B. This study describes for the first time GEFs that are uniquely specific for Rap2 among Rap family of G-proteins.