Insulin receptor-mediated dissociation of Grb2 from Sos involves phosphorylation of Sos by kinase(s) other than extracellular signal-regulated kinase.
The Ras signaling pathway is rapidly activated and then down-regulated following stimulation of multiple cell-surface receptors including the insulin receptor (IR). Much recent attention has focused on elucidating the mechanism of Ras inactivation following IR engagement. Previous data suggest that IR-mediated serine/threonine phosphorylation of the Ras guanine nucleotide exchange factor Sos correlates with its decreased affinity for the adapter protein Grb2. This phosphorylation-induced disassembly of the Grb2.Sos complex is thought to be responsible, at least in part, for diminishing Ras activity in Chinese hamster ovary cells. In this report, we confirm the causal relationship between Sos phosphorylation and Grb2/Sos dissociation. We then examine several putative phosphorylation sites of Sos that could potentially regulate this event. Since a number of reports suggest that extracellular signal-regulated kinase (ERK) phosphorylates Sos, we generated a Sos mutant lacking all seven canonical phosphorylation sites for ERK. This mutant is a poor substrate of activated ERK in vitro and fails to undergo a change in its electrophoretic mobility following IR stimulation. It is, however, phosphorylated after IR stimulation when expressed in Chinese hamster ovary cells. Interestingly, the mutant protein still dissociates from Grb2 following insulin stimulation, suggesting that ERK is not the kinase responsible for regulating the stability of the Grb2.Sos complex.
Zhao
H
H
Molecular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA.
Okada
S
S
Pessin
J E
JE
Koretzky
G A
GA
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D020664:Son of Sevenless Proteins
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008565:Membrane Proteins; D011506:Proteins; D020664:Son of Sevenless Proteins; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases