Nutrient-dependent and Insulin-stimulated Phosphorylation of Insulin Receptor Substrate-1 on Serine 302 Correlates with Increased Insulin Signaling

• Published in: The Journal of biological chemistry Vol. 279; no. 5; pp. 3447 - 3454
• Main Authors: Giraud, Jodel, Leshan, Rebecca, Lee, Yong-Hee, White, Morris F
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30.01.2004
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Androstadienes - pharmacology; Animals; Blotting, Western; Bromodeoxyuridine - pharmacology; Cell Division; Cell Line; CHO Cells; Cricetinae; Culture Media, Serum-Free - pharmacology; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Glucose - metabolism; Glycogen Synthase Kinase 3 - metabolism; Glycogen Synthase Kinase 3 beta; Insulin - metabolism; Insulin Receptor Substrate Proteins; insulin receptor substrate-1 protein; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases - chemistry; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Phosphoproteins - metabolism; Phosphorylation; Point Mutation; Precipitin Tests; Rats; Serine - chemistry; Signal Transduction; Sirolimus - pharmacology; Time Factors; Wortmannin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M308631200

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 3β 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.