TY - JOUR
T1 - Insulin-stimulated disassociation of the SOS-Grb2 complex
AU - Waters, Steven B.
AU - Yamauchi, Keishi
AU - Pessin, Jeffrey E.
PY - 1995/5
Y1 - 1995/5
N2 - 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 disassociation 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.
AB - 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 disassociation 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.
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U2 - 10.1128/MCB.15.5.2791
DO - 10.1128/MCB.15.5.2791
M3 - Article
C2 - 7739560
AN - SCOPUS:0028950637
SN - 0270-7306
VL - 15
SP - 2791
EP - 2799
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 5
ER -