Abstract
Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.
Highlights
From the Department of Medicine, Division of Endocrinology and Metabolism, University of California, Sun Diego, La Jolla, California 92093,the Veterans Administration Medical Center, Medical Research Service, Sun Diego, California 92161, and the $Medical Research Service and the Department of Medicine, Veterans AffairsMedical Center and the University of Colorado Health Sciences Center, Denver, Colorado 80220
The proline rich region of Sos binds to current results indicate that Shc plays a more impothretanStH3domain of Grb2, whichis an adapter proteicnomposed role thanIRS-1 in insulin stimulation ofGNRF activity of one Srchomology 2 (SH2) domain andtwo SH3 domains[21], and preformed and subsequentp21"GTP formation
Through the Grb2 SH2 domain, Grb2.Sos complexes can bind to phosphorylated epidermal growthfactor (EGF)and Insulin binding to the extracellular a-subunits activates thpelatelet-derived growth factorreceptors,providing a mechaintrinsic tyrosine kinase activityof the cytoplasmic portion of nism whereby these growth factors can stimulate p2lm8-GTP the insulin receptor P-subunit [1].One early molecular event formation [16, 22,23,24,25]
Summary
Materials-Rat cells expressing wild type insulin receptors (HI&) were maintained as previously described [29]. The cell lysates were centrifuged, and supernatants were used for immunoprecipitation with the indicated antibodies. Cells were lysed in a buffer containing 50 m Hepes, 150 m NaCl, 5 m MgCl,, 1m PMSF, 1 m Na,VO,, 1%Triton X-100, 0.05% SDS, 10 pg/ml aprotinin, 10 pg/ml leupeptin, 500 w GTP, and 500 w GDP, pH. Measurements of GTP- and GDP-bound p21"Serum-starved cells were incubated for 1h in phosphate-free serum-free Dulbecco's modified Eagle's medium and labeled with [32Plorthophosphatefor 4 h. Insulin (17 m)was added a t 37 "C.f i r the indicated time, cells were lysed in a buffer containing 50 m Hepes, 150 mM NaCl, 5 m MgCI,, 1 m PMSF, 1m dithiothreitol, 1m sodium vanadate, 1m Na,PO,,1% Triton X-100, 0.05% SDS, 10 pg/ml aprotinin, 10 pg/ml leupeptin, 500 w GTP, 500 GDP,pH 7.5. Separation of GTP from GDP was performed by thin layer chromatography [17]
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