Insulin regulation of protein phosphorylation in H4 hepatoma cells. Examination of rapid effects using two-dimensional electrophoresis.
Activation of the insulin receptor tyrosine kinase is thought to initiate a chain of phosphorylation changes which regulate various metabolic and growth processes. Insulin has been shown to regulate some kinases by affecting their phosphorylation states; however, direct connections between activation of the receptor tyrosine kinase and subsequent altered phosphorylations of these kinases have yet to be established. To define the constituents of these regulated phosphorylation cascade systems, the time course of insulin-induced phosphorylation changes has been examined. 32P-Labeled H4IIE hepatoma cells were treated with insulin for 5-300 s, and then cell extracts were subjected to two-dimensional gel electrophoresis. Computer analysis of autoradiograms was employed to quantify insulin-induced phosphorylation changes. Phosphotyrosine was assessed by KOH digestion, by anti-phosphotyrosine immunoprecipitation, and by phenylarsine oxide treatment. Insulin induced a hierarchy of phosphorylation changes over the 300-s time course, with most increases being detectable within 10 s. Of these, only four did not undergo enhanced tyrosine phosphorylation. Eight proteins were found to undergo transient phosphorylation changes. Phenylarsine oxide elicited the appearance of novel low molecular weight phosphoproteins. These results support the concept of insulin-directed phosphorylation cascades, but indicate the magnitude of potential targets of the insulin receptor has been underestimated.
Schwartz
C F
CF
Department of Surgery, University of Virginia, Charlottesville 22908.
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D001152:Arsenicals; D007328:Insulin; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011480:Protease Inhibitors; C029341:oxophenylarsine; D019000:Phosphotyrosine; D014443:Tyrosine