Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments.
Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme responsible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) from phosphatidylinositol-5-phosphate (PI-5-P). In this study, we demonstrate the presence of PIPKII alpha in bovine photoreceptor rod outer segments (ROS) and the involvement of tyrosine phosphorylation in the regulation of its activity. PIPKII activity in bovine ROS was verified by the preferential conversion of synthetic dipalmitoyl PI-5-P to PI-4,5-P(2), lack of effect of phosphatidic acid, inhibition by heparin, immunoreaction with an anti-PIPKII alpha antibody on Western blots, and immunocytochemical localization in bovine and rat ROS by anti-PIPKII alpha. Immunoprecipitates of bovine ROS with the anti-PIPKII alpha antibody possessed PIPK enzymatic activity and preferentially used PI-5-P as substrate for PI-4,5-P(2) biosynthesis. The activity of PIPKII was greatly increased under conditions favoring tyrosine phosphorylation in ROS, and PIPKII activity was immunoprecipitated with anti-phosphotyrosine (anti-PY) antibodies from tyrosine phosphorylated ROS. Preincubation of ROS with tyrosine kinase inhibitors almost abolished the kinase activity in the anti-PY immunoprecipitates. Immunoblot analysis showed that PIPKII alpha was present in anti-PY immunoprecipitates from phosphorylated ROS but not from nonphosphorylated controls. We conclude that PIPKII alpha is present in ROS and that its activity is regulated by tyrosine phosphorylation.
Huang
Z
Z
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Guo
X X
XX
Chen
S X
SX
Alvarez
K M
KM
Bell
M W
MW
Anderson
R E
RE
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates; D020032:Tyrphostins; C055525:phosphatidylinositol 3-phosphate; C037178:phosphatidylinositol 4-phosphate; C113567:phosphatidylinositol 5-phosphate; C079836:tyrphostin 25; C086599:tyrphostin A46; D014443:Tyrosine; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D011505:Protein-Tyrosine Kinases