The insulin-elicited 60-kDa phosphotyrosine protein in rat adipocytes is associated with phosphatidylinositol 3-kinase

Abstract

Insulin stimulates the tyrosine phosphorylation of a 60-kDa protein (pp60) in rat adipocytes. After insulin treatment of these cells, pp60, as well as the 160-kDa insulin receptor substrate-1 (IRS-1), were found to be associated with the enzyme phosphatidylinositol 3-kinase (PtdIns-3-kinase) in separate complexes. By contrast, pp60 was not detected in insulin-treated mouse 3T3-L1 adipocytes, which contain abundant IRS-1. PtdIns-3-kinase complex. The pp60.PtdIns 3-kinase complex was located in both the soluble and membrane fractions of the rat adipocytes. Fusion proteins containing the isolated src homology 2 domains from the 85-kDa subunit of PtdIns-3-kinase bound to pp60 in lysates of insulin-treated rat adipocytes. This finding indicates that the most likely mode of association of pp60 with PtdIns-3-kinase is through binding of phosphotyrosine residues in pp60 to these domains. By immunoaffinity chromatography on a monoclonal antibody against phosphotyrosine, pp60 was purified in high percentage yield from insulin-stimulated rat adipocytes, but the low amount of the protein obtained (about 3 ng from the adipocytes of one rat) precluded sequence analysis.