Activation of protein kinase C stimulates the tyrosine phosphorylation and guanosine triphosphatase-activating protein association of p60 in rat hepatoma cells.
In the present studies, insulin was found to stimulate in a rat hepatoma cell line (called FAO cells) the tyrosine phosphorylation of the 60-kilodalton p21ras GTPase-activating protein (GAP)-associated protein called p60. Surprisingly, the tyrosine phosphorylation of this protein was also almost equally stimulated by an activator of protein kinase C (PKC), the phorbol ester phorbol 12-myristate 13-acetate (PMA). The tyrosine phosphorylation of p60 induced by either agent correlated with the formation of the GAP-p60 complex in situ and an increase in the ability of p60 to directly bind to the SH2 domain of GAP in vitro. Several lines of evidence indicated that the PMA-induced tyrosine phosphorylation of p60 occurred through a different mechanism than that induced by insulin. First, the stimulation of tyrosine phosphorylation of p60 by maximal concentrations of the two agents was almost additive. Second, down-regulation of PKC or pretreatment with a specific inhibitor of PKC abolished the ability of PMA to stimulate tyrosine phosphorylation of p60 but had no effect on the insulin stimulation. And third, long-term pretreatment with insulin abolished the insulin response but did not affect the response to PMA. The PMA effect did seem to be mediated via a tyrosine kinase, since it was blocked by quercetin, an inhibitor of tyrosine kinases. These results indicate that both PMA and insulin can equally stimulate in FAO cells the tyrosine phosphorylation of p60 and its association with GAP, although these two agents seem to act via different signaling systems.
Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305.
W
W
Ogawa
Y
Y
Hosomi
R A
RA
Roth
D000818:Animals; D015536:Down-Regulation; D020690:GTPase-Activating Proteins; D007328:Insulin / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D015290:Second Messenger Systems; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D007328:Insulin; D011506:Proteins; D014443:Tyrosine; D011972:Receptor, Insulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate