Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40‐transformed 3T3 T cells by insulin or vanadate

Abstract

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen-transformed 3T3 T cells (CSV3-1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3-1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin- and vanadate-stimulated CSV3-1 cells. The insulin receptor beta-subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3-1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate-1 and complex formation between the phosphorylated insulin receptor substrate-1 and the 85 kDa subunit of phosphatidylinositol 3'-kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate-1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate-treated CSV3-1 cells. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorylation of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor beta-subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40-transformed and nontransformed 3T3 T cells.