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Protein Kinase C- Phosphorylates Insulin Receptor Substrate-1, -3, and -4 But Not -2: Isoform Specific Determinants of Specificity in Insulin Signaling

Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Chief, Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, 9 Memorial Drive, Building 9, Room 1N-105 MSC 0920, Bethesda, Maryland 20892-0920. E-mail: quonm{at}nih.gov.

Protein kinase C-, a downstream effector of phosphatidylinositol 3-kinase (PI3K), phosphorylates insulin receptor substrate (IRS)-1 on serine residues impairing activation of PI3K in response to insulin. Because IRS-1 is upstream from PI3K, this represents a negative feedback mechanism that may contribute to signal specificity in insulin action. To determine whether similar feedback pathways exist for other IRS isoforms, we evaluated IRS-2, -3, and -4 as substrates for PKC-. In an in vitro kinase assay, purified recombinant PKC- phosphorylated IRS-1, -3 and -4 but not IRS-2. Similar results were obtained with an immune-complex kinase assay demonstrating that wild-type, but not kinase-deficient mutant PKC-, phosphorylated IRS-1, -3, and -4 but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC- in NIH-3T3^IR cells cotransfected with epitope-tagged IRS proteins and either PKC- or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC- for IRS-1, -3, and -4 but not IRS-2. Significant insulin-stimulated increases in PI3K activity was coimmunoprecipitated with all IRS isoforms. In cells overexpressing PKC- there was marked inhibition of insulin-stimulated PI3K activity associated with IRS-1, -3 and -4 but not IRS-2. That is, PI3K activity associated with IRS-2 in response to insulin was similar in control cells and cells overexpressing PKC-. We conclude that IRS-3 and -4 are novel substrates for PKC- that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC- to phosphorylate IRS-2 may help determine specific functional roles for IRS-2.