title: Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.
abstract: IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb(Irs1)). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)(Irs1)) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302(Irs1), Ser(P)-307(Irs1), and four others) correlated significantly with impaired insulin-stimulated Tyr(P)(Irs1). Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)(Irs1) in CHO(IR)/IRS1 cells.
authors.affiliation: From the Division of Endocrinology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Hançer
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Christine
authors.initials: C
authors.lastname: Cherella
authors.forename: Yedan
authors.initials: Y
authors.lastname: Li
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Copps
authors.forename: Morris F
authors.initials: MF
authors.lastname: White
mesh_terms: D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000841:Anisomycin; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D012694:Serine; D058570:TOR Serine-Threonine Kinases; D019284:Thapsigargin; D013912:Threonine; D014415:Tunicamycin; D014443:Tyrosine; C526296:Irs1 protein, rat; C546842:MTOR protein, human; C435941:INSR protein, human
keywords: CHO Cells; Cell Signaling; IRS1; Insulin Resistance; Insulin Signaling; Metabolic Stress; Monoclonal Antibodies; Phosphotyrosine Signaling; Protein Phosphorylation; Signal Transduction