title: Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation.
abstract: Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Greene
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakaue
authors.forename: Lihong
authors.initials: L
authors.lastname: Wang
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Richard A
authors.initials: RA
authors.lastname: Roth
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014407:Tumor Cells, Cultured
chemical_list: D000873:Anthracenes; D017931:DNA Primers; D004791:Enzyme Inhibitors; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun; D012694:Serine; D058570:TOR Serine-Threonine Kinases; C526292:IRS1 protein, human; C526296:Irs1 protein, rat; C432165:pyrazolanthrone; C546842:MTOR protein, human; C546845:mTOR protein, rat