title: Protein kinase C-zeta phosphorylates insulin receptor substrate-1, -3, and -4 but not -2: isoform specific determinants of specificity in insulin signaling.
abstract: Protein kinase C-zeta, 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-zeta. In an in vitro kinase assay, purified recombinant PKC-zeta 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-zeta, phosphorylated IRS-1, -3, and -4 but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC-zeta in NIH-3T3(IR) cells cotransfected with epitope-tagged IRS proteins and either PKC-zeta or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC-zeta 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-zeta 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-zeta. We conclude that IRS-3 and -4 are novel substrates for PKC-zeta that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC-zeta to phosphorylate IRS-2 may help determine specific functional roles for IRS-2.
authors.affiliation: Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20892-0920, USA.
authors.forename: Sihoon
authors.initials: S
authors.lastname: Lee
authors.forename: Edward G
authors.initials: EG
authors.lastname: Lynn
authors.forename: Jeong-A
authors.initials: JA
authors.lastname: Kim
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Quon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation; D000099221:Protein Kinase C zeta
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D011493:Protein Kinase C; D014443:Tyrosine; D000099221:Protein Kinase C zeta; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; C108872:Irs3 protein, mouse; C491531:Irs4 protein, mouse