Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosp horylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for t he biological effects of insulin, and understanding regulation of insulin r eceptor phosphorylation and kinase activity is essential to understanding i nsulin action. Receptor tyrosine kinase activity may be altered by direct c hanges in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin s timulation, the insulin receptor was tyrosine phosphorylated 8-fold more an d Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and in sulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin re ceptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin re ceptors and a form of IRS-1 that cannot be phosphorylated on tyrosine resid ues (32D/IR+IRS-1(F18)). Therefore, IRS-1 and IRS-2 appeared to have differ ent effects an insulin receptor phosphorylation and downstream signaling. P reincubation of cells with pervanadate greatly decreased protein-tyrosine p hosphatase activity in all four cell lines. After pervanadate treatment, ty rosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1(F18) cells was markedly increased, but pervanada te had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 a ppear to function differently in their effects on signaling downstream of t he insulin receptor. IRS-1 may play a major role in regulating insulin rece ptor phosphorylation and enhancing downstream signaling after insulin stimu lation.