Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant two-dimensional gel electrophoresis.
We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.
Levenson
R M
RM
Howard Hughes Medical Institute Laboratory, Durham, North Carolina 27710.
Blackshear
P J
PJ
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014443:Tyrosine / Q000032:analysis
D007328:Insulin; D010710:Phosphates; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin