title: Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907.
abstract: The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser(675) and Ser(907) of mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters. Phosphorylation of both sites was a late and prolonged event during insulin treatment and was also detected in liver tissue of insulin-treated as well as refed mice. Inhibition and siRNA-mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser(907) was ERK dependent. Phosphorylation of Ser(907) did not prevent the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr(911) was proved to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser(675) was prevented by inhibition and siRNA-mediated knockdown of mTOR but not of p70(S6K1). Mutation of Ser(675) to Ala did not affect downstream insulin signaling but increased the half-life of the protein, suggesting an involvement of phospho-Ser(675) in an accelerated degradation of IRS-2. Moreover, the insulin-induced degradation of IRS-2 was blocked by inhibition of mTOR. We conclude that the two novel insulin-dependent serine phosphorylation sites of IRS-2 were not involved in the regulation of the adjacent PI 3-kinase and Grb2 binding domains but might be implicated in the ERK- and mTOR-mediated negative feedback control.
authors.affiliation: Dept. of Internal Medicine, Div. of Pathobiochemistry and Clinical Chemistry, Univ. of Tuebingen, Otfried-Mueller-Straße 10, 72076 Tuebingen, Germany.
authors.forename: Louise
authors.initials: L
authors.lastname: Fritsche
authors.forename: Sabine S
authors.initials: SS
authors.lastname: Neukamm
authors.forename: Rainer
authors.initials: R
authors.lastname: Lehmann
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.forename: Anita M
authors.initials: AM
authors.lastname: Hennige
authors.forename: Andrea
authors.initials: A
authors.lastname: Hunder-Gugel
authors.forename: Martin
authors.initials: M
authors.lastname: Schenk
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Häring
authors.forename: Erwin D
authors.initials: ED
authors.lastname: Schleicher
authors.forename: Cora
authors.initials: C
authors.lastname: Weigert
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D019295:Computational Biology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D047468:Immunoprecipitation; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects*; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering; D051381:Rats; D012694:Serine / Q000378:metabolism* / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D048049:Extracellular Signal-Regulated MAP Kinases; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D012313:RNA; D034741:RNA, Small Interfering; D012694:Serine; D058570:TOR Serine-Threonine Kinases; C526297:IRS2 protein, human; C546842:MTOR protein, human