A dominant-negative mutant of mSOS1 inhibits insulin-induced Ras activation and reveals Ras-dependent and -independent insulin signaling pathways.
The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing 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 appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced 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.
Sakaue
M
M
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Bowtell
D
D
Kasuga
M
M
D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D005799:Genes, Dominant; D006006:Glycogen Synthase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011972:Receptor, Insulin / Q000502:physiology*; D019893:Ribosomal Protein S6 Kinases; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020663:ras Guanine Nucleotide Exchange Factors
D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D011506:Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D004247:DNA; D006006:Glycogen Synthase; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)