Insulin rapidly stimulates tyrosine phosphorylation of a M_r-185,000 protein in intact cells

Abstract

Phosphotyrosine-containing proteins are minor components of normal cells^1,2 which appear to be associated primarily with the regulation of cellular metabolism and growth^3,4. The insulin receptor is a tyrosine-specific protein kinase^5,6, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its β-subunit^7–9. Tyrosine autophosphorylation activates the phosphotransferase in the β-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates^10,11. When incubated in vitro with [γ-³²P]APT and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues^12–16. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (M_r) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, ppl85 does not adhere to wheat-germ agglutinin–agarose or bind to anti-insulin receptor antibodies. Phosphorylation of ppl85 is maximal within seconds after exposure of the cells to insulin and exhibits a dose–response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.