MODULATION OF INSULIN-RECEPTOR SUBSTRATE-1 TYROSINE PHOSPHORYLATION AND FUNCTION BY MITOGEN-ACTIVATED PROTEIN-KINASE

Increased serine phosphorylation of insulin receptor substrate-1 (IRS- 1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogen ous substrate and to inhibit its subsequent association with phosphati dylinositol 3-kinase. In the present studies we have examined the pote ntial role of the mitogen-activated protein (MAP) kinase in the increa sed serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myris tate 13-acetate. First, recombinantly produced kinase was shown to pho sphorylate intact IRS-1 in a way that decreased the ability of isolate d insulin receptor to phosphorylate the tyrosines recognized by the SH 2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor o f MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13- acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP ki nase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increas e in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinas e in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of t he kinases capable of phosphorylating and regulating IRS-1 tyrosine ph osphorylation.