Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.
OBJECTIVE
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.
Guo
X X
XX
Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Huang
Z
Z
Bell
M W
MW
Chen
H
H
Anderson
R E
RE
D000255:Adenosine Triphosphate; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases