BIOCHEMICAL-CHARACTERIZATION OF C3G - AN EXCHANGE FACTOR THAT DISCRIMINATES BETWEEN RAP1 AND RAP2 AND IS NOT INHIBITED BY RAP1A(S17N)

A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluoresce nce spectroscopy, C3G stimulates GDP dissociation from Rap1, but not f rom Rap2, neither from Bud1, which is believed to be the yeast homolog ue of Rap1 nor from all other proteins of the human Ras-subfamily, Lik e the corresponding fragment from CDC25(Mm), the increase in the GDP d issociation rate is linear with increasing concentration of Rap1A.GDP up to 100 mu M, indicating an apparent K-M higher than 100 mu M. Unlik e the Ras-CDC25(Mm) system, the Rap1A(S17N) mutant does not inhibit th e C3G-activated guanine nucleotide dissociation from wild-type Rap1A i n vitro. These data suggest that Rap1A(S17N) is unlikely to titrate aw ay C3G in vivo, the proposed mechanism by which S17N-mutants exert the ir dominant negative effects.