A DOMINANT-NEGATIVE MUTANT OF MSOS1 INHIBITS INSULIN-INDUCED RAS ACTIVATION AND REVEALS RAS-DEPENDENT AND RAS-INDEPENDENT INSULIN SIGNALINGPATHWAYS

The role of the Grb2-SOS complex in insulin signal transduction was in vestigated with a deletion mutant of mSOS1 that lacks the guanine nucl eotide exchange domain of the wild-type protein. Cells overexpressing either wild-type (CHO IR/SOS cells) or mutant (CHO-IR/Delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropri ate expression plasmid. The mutant mSOS1 protein did not contain the g uanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insuli n rapidly stimulated the formation of GTP bound pas and the phosphoryl ation of mitogen-activated protein (MAP) kinase;,both these effects of insulin were markedly inhibited in CHO-IR/Delta SOS cells. Insulin-in duced glycogen synthase and 70-kDa S6 kinase activities were not affec ted by expression of either wild-type or mutant mSOS1. These results s how that the mutant mSOS1 acts in a dominant-negative manner and sugge st that the Grb2-SOS complex mediates, at least in part, insulin-induc ed activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.