Tyrosine phosphorylation of Phosphoinositide-dependent kinase-1 by the insulin receptor is necessary for insulin metabolic signaling.

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IRƒ´43) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IRƒ´43 (L6IRƒ´43) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr308. Insulin induced the phosphatidylinositol 3 kinase-dependent copptn. of PDK-1 with wild-type IR (IRWT), but not IRƒ´43. Based on overlay blotting, PDK-1 directly bound IRWT, but not IRƒ´43. Insulin-activated IRWT, and not IRƒ´43, phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr„_Phe substitution prevented this inhibitory action. In the L6hIR cells, the C-terminal peptide copptd. with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 copptn. with IRWT, on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr308, and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6IRƒ´43 compared to control cells. In L6 cells expressing IRWT, the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.