Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites.
A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.
Liu
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F
Department of Pharmacology, Stanford University School of Medicine, CA 94305.
Roth
R A
RA
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007328:Insulin; D009838:Oligodeoxyribonucleotides; D012694:Serine; D011972:Receptor, Insulin; D013755:Tetradecanoylphorbol Acetate