title: An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.
abstract: The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.
authors.affiliation: Institute for Diabetes Research, Munich, Federal Republic of Germany.
authors.forename: F
authors.initials: F
authors.lastname: Machicao
authors.forename: H
authors.initials: H
authors.lastname: Häring
authors.forename: M F
authors.initials: MF
authors.lastname: White
authors.forename: J M
authors.initials: JM
authors.lastname: Carrascosa
authors.forename: B
authors.initials: B
authors.lastname: Obermaier
authors.forename: O H
authors.initials: OH
authors.lastname: Wieland
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin
chemical_list: D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin