Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.

PURPOSE To identify the tyrosine-phosphorylated protein(s) in bovine rod outer 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 prepared and used to pull down tyrosine-phosphorylated proteins in bovine ROS. Protein identity was established by Western blot analysis. PI3K activity was determined in the pull-down mixtures and in immunoprecipitates by incubation with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) and [gamma(32)P]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 present in nonphosphorylated (N)-ROS. Binding was completely abolished when the Arg 358 in the N-SH2 domain was mutated to Ala. Increased binding of the p110alpha catalytic subunit to GST-p85 (N-SH2) fusion protein was also observed in the presence of the 97-kDa phosphorylated protein. Biochemical evidence indicated that the 97-kDa protein was the beta-subunit of the insulin receptor beta-subunit (IRbeta). Immunoprecipitates of PY-ROS and N-ROS with anti-PY antibodies, probed with anti-IRbeta, indicated the presence of IRbeta only in PY-ROS. Immunoprecipitates of PY-ROS and N-ROS with anti-IRbeta antibodies, probed with anti-p85 and anti-p110alpha antibodies, indicated increased amounts of both p85 and p110alpha in PY-ROS compared to N-ROS. Treatment of ROS with insulin, followed by immunoprecipitation with either anti-IRbeta or anti-PY, resulted in increased PI3K activity. Expression and phosphorylation of the cytoplasmic tail of retina insulin receptor showed direct involvement with the p85 subunit of PI3K in vitro. CONCLUSIONS Tyrosine phosphorylation of the beta-subunit of the insulin receptor is involved in the regulation of PI3K activity in ROS.