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  • Title: Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.
    Author: Guo XX, Huang Z, Bell MW, Chen H, Anderson RE.
    Journal: Mol Vis; 2000 Nov 04; 6():216-21. PubMed ID: 11063755.
    Abstract:
    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 activation in these membranes. METHODS: Tyrosine-phosphorylated ROS (PY-ROS) were obtained by incubating ROS with ATP, MgCl2, and orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor. Non-phosphorylated ROS (N-ROS) were obtained by incubating ROS under the same conditions, but without ATP and orthovanadate. Both were subjected 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 assayed for PI 3-kinase activity. Enzyme assay products were separated by thin-layer chromatography (TLC), deacylated, and identified by high performance liquid chromatography (HPLC). RESULTS: PI 3-kinase activity in anti-p85 and p110alpha IPs was significantly higher in PY-ROS than in N-ROS. No enzyme activity was recovered in anti-p110beta IPs. PI 3-kinase activity in anti-PY IPs from PY-ROS was six-fold greater 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. However, tyrosine phosphorylation of p85 and p110alpha was not observed in anti-p85 and anti-p110alpha IPs that were probed with anti-PY. CONCLUSIONS: This study indicates that the p85/p110alpha complex of PI 3-kinase is present in ROS and tyrosine phosphorylation is involved in the regulation of its activity.
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