SHC IS THE PREDOMINANT SIGNALING MOLECULE COUPLING INSULIN-RECEPTORS TO ACTIVATION OF GUANINE-NUCLEOTIDE RELEASING-FACTOR AND P21(RAS)-GTP FORMATION

Insulin stimulates tyrosine phosphorylation of insulin receptor substr ate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type ins ulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p 21(ras)-GTP formation. The time course of insulin stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas She phosphorylation was re latively slow. Growth factor receptor bound protein-2 (Grb2) associate d with IRS-1 rapidly and gradually dissociated after 5 min, whereas Gr b2 association with She was slower and reached a maximum at 10 min aft er insulin stimulation. Thus, the kinetics of Grb2 association with IR S-1 and She corresponded closely to the time course of tyrosine phosph orylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetic s of insulin-stimulated GNRF activity and p21(ras)-GTP formation corre sponded more closely to the time course of She phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitati on of She proteins from cell lysates of insulin-stimulated cells remov ed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negl igible effect on GNRF activity. Thus, although both IRS-1 and She asso ciate with Grb2, the current results indicate that She plays a more im portant role than IRS-1 in insulin stimulation of GNRF activity and su bsequent p21(ras)-GTP formation.