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 ac tivity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-leng th C3G cDNA isolated from human placenta markedly reduced the focus fo rming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transforma tion, 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 know n target of C3G. The inhibition of focus formation observed in the pre sence of C3G was not due to toxic effects on cell viability, since tra nsfected C3G cells exhibited the same survival and growth rates as unt ransfected NIH3T3 cells or cells transfected with plasmid vector alone . Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 onco gene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inh ibitory effect on Raf-induced transformation suggests that C3G has oth er functional targets in addition to Rap1. A C3G mutant (C3G Delta Cat ) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited sim ilar ability than full length C3G to inhibit focus formation. In contr ast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibit ory effect on transformation mediated by the oncogenes tested. The C3G -derived gene products overexpressed in our transfected cell lines loc alized to the cytoplasm and did not change the basal MAPK or JNK activ ity of those cell lines nor their ability to activate the kinases in r esponse to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibit ory effects observed.