Abstract
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.
Highlights
Insulin stimulation of Chinese hamster ovary (CHO)1 cells overexpressing insulin receptor (IR) at their surface leads to a rapid activation of the Ras signaling pathway [1, 2]
Insulin-induced Grb2/Sos Dissociation Requires Phosphorylation of Sos—It has been shown previously by several laboratories that insulin stimulation results in both the serine/ threonine phosphorylation of Sos and its dissociation from Grb2 [5,6,7,8]. These prior studies had not demonstrated whether Sos phosphorylation was related causally to its decreased affinity for Grb2. We approached this question by asking if treatment of cell lysates with alkaline phosphatase would prevent the destabilization of the Grb21⁄7Sos complex after insulin stimulation of CHO/IR cells
Sos.VII Is Still Phosphorylated Inducibly in CHO/IR Cells— Since we showed that the insulin-induced Grb2/Sos dissociation depends on Sos phosphorylation (Fig. 1), we were surprised by our finding that mutation of residues that eliminate the IR induced electrophoretic mobility shift, and the ability of extracellular signal-regulated kinase (ERK) to phosphorylate Sos in vitro did not affect the ability of insulin to alter the affinity of Sos for Grb2 (Fig. 5)
Summary
Insulin stimulation of Chinese hamster ovary (CHO)1 cells overexpressing insulin receptor (IR) at their surface leads to a rapid activation of the Ras signaling pathway [1, 2]. We have determined that Sos phosphorylation is directly responsible for the decreased binding affinity between Grb2 and Sos. ERK can phosphorylate Sos at specific carboxyl-terminal serine/threonine acceptor sites, those phosphorylation events do not affect the interaction between Grb2 and Sos. Together these data suggest that ERK may be a physiological kinase for Sos, its activity is not responsible for regulating the stability of the Grb21⁄7Sos complex following IR engagement on CHO cells.
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