Protein kinase C-zeta 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 fro m phosphatidylinositol 3-kinase in insulin signaling pathways. However, spe cific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insuli n receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta Under in v itro conditions, wild-type PKC-C (but not kinase-deficient mutant :PKC-zeta significantly phosphorylated IRS-1, This phosphorylation was reversed by t reatment with the serine-specific phosphatase, protein phosphatase 2A, In a ddition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused signifi cant phosphorylation of cotransfected IRS-1 as demonstrated by [P-32]orthop hosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimm unoprecipitated 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 ph osphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-l-associated phosphatidylinositol 3-kinase activity. Taken tog ether, our results raise the possibility that IRS-1 is a novel physiologica l substrate for PKC-5. Because PKC-5 is located downstream from IRS-1 and p hosphatidylinositol 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.