Interaction of the insulin receptor beta-subunit with phosphatidylinositol3-kinase in bovine ROS

PURPOSE. To identify the tyrosine-phosphorylated protein(s) in bovine rod o uter segments (ROS) that are associated with phosphatidylinositol 3-kinase (PI3K). METHODS. Glutathione-S-transferase (GST) fusion proteins containing two SH2 domains of the p85 regulatory subunit of PI3K-GST-p85 (N-SH2), GST-p85 (C- SH2), and respective SH2 mutants (N-SH2, R358A, and C-SH2, R649A)-were prep ared and used to pull down tyrosine-phosphorylated proteins in bovine ROS. Protein identity was established by Western blot analysis. PI3K activity wa s determined in the pull-down mixtures and in immunoprecipitates by incubat ion with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P-2) and [gamma P-32 ]adenosine triphosphate (ATP). RESULTS. The GST pull-down assays indicated the binding of a 97-kDa protein by GST-p85 (N-SH2) in tyrosine-phosphorylated (PY)-ROS that was not presen t in nonphosphorylated (N)-ROS. Binding was completely abolished when the A rg 358 in the N-SH2 domain was mutated to Ala. Increased binding of the p11 0 alpha catalytic subunit to GST-p85 (N-SH2) fusion protein was also observ ed in the presence of the 97-kDa phosphorylated protein. Biochemical eviden ce indicated that the 97-kDa protein was the beta -subunit of the insulin r eceptor beta -subunit (IR beta). Immunoprecipitates of PY-ROS and N-ROS wit h anti-PY antibodies, probed with anti-IR beta, indicated the presence of I R beta only in PY-ROS. Immunoprecipitates of PY-ROS and N-ROS with anti-IR beta antibodies, probed with anti-p85 and anti-p110 alpha antibodies, indic ated increased amounts of both p85 and p110 alpha in PY-ROS compared to N-R OS. Treatment of ROS with insulin, followed by immunoprecipitation with eit her anti-IR beta or anti-PY, resulted in increased PI3K activity. Expressio n and phosphorylation of the cytoplasmic tail of retina insulin receptor sh owed direct involvement with the p85 subunit of P13K in vitro. CONCLUSIONS. Tyrosine phosphorylation of the beta -subunit of the insulin r eceptor is involved in the regulation of P13K activity in ROS.