Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling.
Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.
Giraud
Jodel
J
Howard Hughes Medical Institute, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Leshan
Rebecca
R
Lee
Yong-Hee
YH
White
Morris F
MF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D016466:CHO Cells; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011233:Precipitin Tests; D051381:Rats; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors; D000077191:Wortmannin
D000596:Amino Acids; D000730:Androstadienes; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D012694:Serine; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; D001973:Bromodeoxyuridine; D005947:Glucose; D020123:Sirolimus; D000077191:Wortmannin