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 fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.
van den Berghe
N
N
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Cool
R H
RH
Horn
G
G
Wittinghofer
A
A
D018797:Cell Cycle Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020668:rap GTP-Binding Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D010446:Peptide Fragments; D011506:Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D010749:Phosphoprotein Phosphatases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins