Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments

PURPOSE: We have previously shown that phosphatidylinositol 3-kinase (PI 3- kinase) activity is present in bovine rod outer segments (ROS). The present study was undertaken to investigate the mechanism of PI 3-kinase activatio n in these membranes. METHODS: Tyrosine-phosphorylated ROS (PY-ROS) were obtained by incubating R OS with ATP, MgCl2, and orthovanadate (Na3VO4), a tyrosine phosphatase inhi bitor. Non-phosphorylated ROS (N-ROS) were obtained by incubating ROS under the same conditions, but without ATP and orthovanadate. Both were subjecte d to immunoprecipitation using antibodies against the regulatory p85 (anti- p85) subunit of PI 3-kinase, the catalytic p110 (anti-p110) subunit of PI 3 -kinase, or phosphotyrosine (anti-PY). The immunoprecipitates (IPs) were as sayed for PI 3-kinase activity. Enzyme assay products were separated by thi n-layer chromatography (TLC), deacylated, and identified by high performanc e liquid chromatography (HPLC). RESULTS: PI 3-kinase activity in anti-p85 and p110a IPs was significantly h igher in PY-ROS than in N-ROS. No enzyme activity was recovered in anti-p11 0b IPs. PI 3-kinase activity in anti-PY IPs from PY-ROS was six-fold greate r than those from N-ROS. Immunoblot analysis showed that the amount of p85 in PY IPs from PY-ROS was significantly higher than those from N-ROS. Howev er, tyrosine phosphorylation of p85 and p110a was not observed in anti-p85 and anti-p110a IPs that were probed with anti-PY. CONCLUSIONS: This study indicates that the p85/p110a complex of PI 3-kinase is present in ROS and tyrosine phosphorylation is involved in the regulati on of its activity.