Functional identification of RASGEF1 family of exchange factors as activators of RAP2, and as interacting partners of CCDC124
Author
Yaman, Elif
Advisor
Tazebay, Uygar H.
Date
2009Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
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.