Regulation of type II PIP kinase by PKD phosphorylation.
The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.
Faculty of Life Sciences, The University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, United Kingdom. Katherine.a.hinchliffe@cam.ac.uk
Katherine A
KA
Hinchliffe
Robin F
RF
Irvine
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013552:Swine; D014162:Transfection
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C113567:phosphatidylinositol 5-phosphate; D019870:1-Phosphatidylinositol 4-Kinase; C089212:protein kinase D; D011493:Protein Kinase C