PROTEIN-TYROSINE-PHOSPHATASE 1B COMPLEXES WITH THE INSULIN-RECEPTOR IN-VIVO AND IS TYROSINE-PHOSPHORYLATED IN THE PRESENCE OF INSULIN

In response to insulin, protein tyrosine phosphatase 1B (PTPase 1B) de phosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) protei ns (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysa tes from control and insulin-treated cells expressing catalytically in active PTPase 1B (CS) were immunoadsorbed and subsequently immunoblott ed using various combinations of phosphotyrosine, PTPase 1B, and insul in receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identi fied as the IR beta-subunit. Similarly, anti-IR antibodies coprecipita ted the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To ide ntify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in res ponse to insulin, three candidate sites (Tyr(66), Tyr(152), and Tyr(15 3)) were replaced with phenylalanine. Replacing Tyr(66) or Tyr(152) an d Tyr(153) significantly reduced insulin-stimulated PTPase 1B phosphot yrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B com plexes with the autophosphorylated insulin receptor in intact cells, e ither directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within bo th the receptor and PTPase 1B.