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 vit ro 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 C3 G-Rap1 pathway, Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cel ls significantly increased the ratio of GTP/GDP bound to Rap1, Both th e 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 al losteric mechanism. The requirement of the SH2 domain of Crk for the e nhancement of guanine nucleotide exchange activity for Rap1 could be c ompensated for by the addition of a farnesylation signal to Crk, indic ating that Crk enhanced the guanine nucleotide exchange activity of C3 G by membrane recruitment of C3G, These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by r ecruiting C3G to the cell membrane.