Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments

Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme respons ible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI- 4,5-P- 2) from phosphatidylinositol-5-phosphate (PI-5-P). In this study, we demons trate the presence of PIPKII alpha in bovine photoreceptor rod outer segmen ts (ROS) and the involvement of tyrosine phosphorylation in the regulation of its activity. PIPKII activity in bovine ROS was verified by the preferen tial conversion of synthetic dipalmitoyl PI-5-P to PI-4,5-P-2, lack of effe ct of phosphatidic acid, inhibition by heparin, immunoreaction with an anti -PIPKII alpha antibody on Western blots. and immunocytochemical localizatio n in bovine and rat ROS by anti-PIPKII alpha. Immunoprecipitates of bovine ROS with the anti-PIPKII alpha antibody possessed PIPK enzymatic activity a nd preferentially used PI-5-P as substrate for PI-4,5-P2 biosynthesis. The activity of PIPKII was greatly increased under conditions favoring tyrosine phosphorylation in ROS, and PIPKII activity was immunoprecipitated with an ti-phosphotyrosine (anti-PY) antibodies from tyrosine phosphorylated ROS, P reincubation of BOS with tyrosine kinase inhibitors almost abolished the ki nase activity in the anti-PY immunoprecipitates. Immunoblot analysis showed that PIPKII alpha was present in anti-PY immunoprecipitates from phosphory lated ROS but not from nonphosphorylated controls, We conclude that PIPKII alpha is present in ROS and that its activity is regulated by tyrosine phos phorylation.