Identification and characterization of the phosphatidylinositol-(4, 5)-bisphosphate 5-phosphatase in human platelets

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)-P2) is the precursor of several second messenger molecules. In unstimulated cells PtdIns(4,5)P2 is hydrolyzed by a PtdIns(4,5)P2 5-phosphatase to form phosphatidylinositol 4-phosphate (PtdIns(4)P) which is subsequently recycled to phosphatidylinositol. PtdIns(4,5)P2 5-phosphatase activity was detected in platelet cytosolic and particulate fractions. The platelet PtdIns(4,5)P2 5-phosphatase activity was magnesium but not calcium dependent. The elution profile of platelet cytosolic PtdIns(4,5)P2 5-phosphatase from anion exchange resins, exactly matched that of the 75-kDa inositol-polyphosphate 5-phosphatase (Ins(1,4,5)P3 5-phosphatase). The latter is a signal terminating enzyme responsible for the hydrolysis of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) to inositol (1,4)-bisphosphate (Mitchell, C.A., Connolly, T.M., and Majerus, P.W. (1989) J. Biol. Chem. 264, 8873-8877). Polyclonal antibodies raised against recombinant 75-kDa Ins(1,4,5)P3 5-phosphatase specifically immunoprecipitated all PtdIns-(4,5)P2 5-phosphatase activity from both the platelet membrane and cytosolic fractions. Purified 75-kDa Ins(1,4,5)P3 5-phosphatase hydrolyzed PtdIns(4,5)P2 forming PtdIns(4)P (Km = 250 microM). By contrast, purified membrane-associated 43-kDa Ins(1,4,5)P3 5-phosphatase did not hydrolyze PtdIns(4,5)P2. In the unstimulated platelet, recycling of PtdIns-(4,5)P2 to PtdIns(4)P is mediated by the 75-kDa Ins-(1,4,5)P3 5-phosphatase.