H2O2 potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells.
Western blotting with anti-phosphotyrosine antibodies was employed in order to study insulin-dependent protein tyrosine phosphorylation in intact Fao cells. In insulin-treated cells, a prominent 180-kDa protein underwent tyrosine phosphorylation, which peaked at 45 s and then rapidly declined. Pretreatment of the cells with 1 mM Bt2cAMP or 0.16 microM 12-O-tetradecanoylphorbol-13-acetate inhibited the insulin-dependent phosphorylation of pp 180, while 1 mM vanadate or 3 mM H2O2 markedly potentiated it. These results indicate that phosphorylation of pp 180 is respectively regulated by agents that are known to synergize with or antagonize the action of the insulin receptor kinase. pp 180 is therefore likely to mediate physiological functions of this receptor kinase. Incubation of Fao cells with 3 mM H2O2 for 30 min prior to their treatment with insulin for 45 s allowed the detection of additional, previously undescribed, proteins pp 150, 114, 100, 85, 68, and 56 kDa that underwent insulin-dependent tyrosine phosphorylation. The potentiating effects of H2O2 were time- and dose-dependent and could be reversed by 2 mM dithiothreitol. Proteins phosphorylated in response to H2O2 plus insulin maintained their fully phosphorylated state for at least 20 min. We suggest that these phosphoproteins are potential physiological substrates for the insulin receptor kinase.
Heffetz
D
D
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Zick
Y
Y
D003994:Bucladesine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007328:Insulin; D003994:Bucladesine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D013755:Tetradecanoylphorbol Acetate