Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways.
Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH2-terminal Jun kinase phosphorylates IRS-1 at Ser307, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser307 in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated alphapSer307. This antibody revealed that TNF-alpha, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser307 in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser307 on IRS-1 immunoprecipitated from muscle; moreover, Ser307 was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser307 was inhibited by LY294002 or wortmannin, whereas TNF-alpha-stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser307 to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.
Rui
L
L
Harvard Medical School, Joslin Diabetes Center, 1 Joslin Place, Boston, Massachusetts 02215, USA.
Aguirre
V
V
Kim
J K
JK
Shulman
G I
GI
Lee
A
A
Corbould
A
A
Dunaif
A
A
White
M F
MF
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D007328:Insulin / Q000494:pharmacology*; D007329:Insulin Antagonists / Q000493:pharmacokinetics; D007333:Insulin Resistance; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011972:Receptor, Insulin; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000737:chemistry
D000841:Anisomycin; D007328:Insulin; D007329:Insulin Antagonists; D007334:Insulin-Like Growth Factor I; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011972:Receptor, Insulin; D012694:Serine; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; C081827:insulin receptor serine kinase; C482125:MAP2K1 protein, human