An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.
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.
Machicao
F
F
Institute for Diabetes Research, Munich, Federal Republic of Germany.
Häring
H
H
White
M F
MF
Carrascosa
J M
JM
Obermaier
B
B
Wieland
O H
OH
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
D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin