Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action.
Okadaic acid has been described previously as being a negative regulator of insulin signaling, as it inhibits insulin stimulation of glucose transport. In addition, this drug induces on insulin receptor substrate-1 (IRS-1) a decrease in tyrosine phosphorylation, concomitantly with an increase in serine/threonine phosphorylation. The present work was aimed at the identification of the serine/threonine residues that, upon phosphorylation, might be involved in modulating insulin signaling. To this end, we studied double-point mutants of IRS-1, in which serines 612/632 and 662/731 were replaced with alanine. These are four plausible sites of phosphorylation by mitogen-activated protein kinases and are in the immediate proximity of tyrosine residues, which are potential sites of interaction with phosphatidylinositol 3-kinase Src homology 2 domains. Using transient expression in 293 EBNA cells, we demonstrate that serines 612, 632, 662, and 731 and mitogen-activated protein kinases are not involved in the okadaic acid effect on IRS-1. Rather, these serines appear to play a role in modulating basal and insulin-stimulated IRS-1 tyrosine phosphorylation, association of IRS-1, with p85, and phosphatidylinositol 3-kinase activity in the IRS-1.p85 immune complex, since mutation of these sites enhances these events. Our findings suggest the existence of an IRS-1 desensitization mechanism resulting from serine/threonine phosphorylation, occurring at least on serines 612, 632, 662, and 731.
Mothe
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INSERM Unité 145, Nice, France.
Van Obberghen
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D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013912:Threonine; D014162:Transfection
D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D005419:Flavonoids; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017871:Calcium-Calmodulin-Dependent Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one