title: 14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain.
abstract: Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with 32P-labeled recombinant IRS-1 and obtained two isoforms (epsilon and zeta) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.
authors.affiliation: Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ogihara
authors.forename: T
authors.initials: T
authors.lastname: Isobe
authors.forename: T
authors.initials: T
authors.lastname: Ichimura
authors.forename: M
authors.initials: M
authors.lastname: Taoka
authors.forename: M
authors.initials: M
authors.lastname: Funaki
authors.forename: H
authors.initials: H
authors.lastname: Sakoda
authors.forename: Y
authors.initials: Y
authors.lastname: Onishi
authors.forename: K
authors.initials: K
authors.lastname: Inukai
authors.forename: M
authors.initials: M
authors.lastname: Anai
authors.forename: Y
authors.initials: Y
authors.lastname: Fukushima
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Y
authors.initials: Y
authors.lastname: Yazaki
authors.forename: Y
authors.initials: Y
authors.lastname: Oka
authors.forename: T
authors.initials: T
authors.lastname: Asano
mesh_terms: D048948:14-3-3 Proteins; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D015723:Gene Library; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; C526292:IRS1 protein, human; C526297:IRS2 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C526298:Irs2 protein, mouse; C526299:Irs2 protein, rat; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011994:Recombinant Proteins; D019319:Okadaic Acid; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D014446:Tyrosine 3-Monooxygenase; D011972:Receptor, Insulin