Protein kinase C-ζ phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin

Protein kinase C-zeta (PKC-zeta is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta Under in vitro conditions, wild-type PKC-C (but not kinase-deficient mutant :PKC-zeta significantly phosphorylated IRS-1, This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A, In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [P-32]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-5, and this association was increased S-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-l-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-5. Because PKC-5 is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-C may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.