Insulin-stimulated disassociation of the SOS-Grb2 complex.
Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.
Waters
S B
SB
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242-1109, USA.
Yamauchi
K
K
Pessin
J E
JE
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D005246:Feedback; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020664:Son of Sevenless Proteins; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007328:Insulin; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020664:Son of Sevenless Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins