Transformation suppressor activity of C3G is independent of its CDC25-homology domain

The guanine nucleotide releasing protein C3G was initially identified as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5-10-fold. The observed inhibitory effect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic effects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory effect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G deltaCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory effect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory effects observed.