Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets.
PtdIns(4,5)P2 production by the enzyme PtdIns4P 5-kinase C (PIPkin C) was examined in thrombin-stimulated human platelets. Thrombin caused a rapid, transient 2-3-fold increase in PIPkin activity and a transient net dephosphorylation of the enzyme. PIPkin C was phosphorylated on serine and threonine residues in unstimulated platelets; no evidence for tyrosine phosphorylation was found. The phosphatase inhibitor okadaic acid promoted PIPkin C hyperphosphorylation and a concomitant marked inhibition of its activity in immunoprecipitates. Activity was restored by treatment with alkaline phosphatase, suggesting the existence of an inhibitory phosphorylation site. In support of this idea, alkaline phosphatase treatment of PIPkin C immunoprecipitated from unstimulated platelets caused a modest (1.6-fold) but significant activation of the enzyme. However, alkaline phosphatase treatment of PIPkin C immunoprecipitated from thrombin-stimulated platelets caused a decrease in activity to approximately the same levels, suggesting that the phosphorylation of PIPkin C also contributes to the observed stimulation. Two-dimensional phosphopeptide mapping of immunoprecipitated PIPkin C revealed that the enzyme is multiply phosphorylated and that, whereas some phosphopeptides are indeed lost on stimulation, consistent with the net dephosphorylation of the enzyme, at least two novel sites become phosphorylated. This suggests that thrombin causes complex changes in the phosphorylation state of PIPkin C, one consequence of which is its activation.
Hinchliffe
K A
KA
Cambridge University Department of Pharmacology, Tennis Court Rd, Cambridge CB2 1QJ, U.K.
Irvine
R F
RF
Divecha
N
N
D000469:Alkaline Phosphatase / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology; D010449:Peptide Mapping; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011233:Precipitin Tests; D013917:Thrombin / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D000469:Alkaline Phosphatase; D014357:Trypsin; D013917:Thrombin