Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2.
Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.
Ichiba
T
T
Department of Pathology, National Institute of Health, Shinjuku-ku, Tokyo 162, Japan.
Kuraishi
Y
Y
Sakai
O
O
Nagata
S
S
Groffen
J
J
Kurata
T
T
Hattori
S
S
Matsuda
M
M
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D015800:Protozoan Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D020668:rap GTP-Binding Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D015852:Eukaryotic Initiation Factor-2; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D015800:Protozoan Proteins; D014157:Transcription Factors; D020663:ras Guanine Nucleotide Exchange Factors; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins