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.
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Research Article|
January 01 1998
Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets
A. Katherine HINCHLIFFE;
A. Katherine HINCHLIFFE
1
*Cambridge University Department of Pharmacology, Tennis Court Rd, Cambridge CB2 1QJ, U.K.
1To whom correspondence should be addressed.
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F. Robin IRVINE;
F. Robin IRVINE
*Cambridge University Department of Pharmacology, Tennis Court Rd, Cambridge CB2 1QJ, U.K.
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Nullin DIVECHA
Nullin DIVECHA
2
†The Inositide Laboratory, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, U.K.
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Publisher: Portland Press Ltd
Received:
April 03 1997
Revision Received:
September 08 1997
Accepted:
September 10 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 329 (1): 115–119.
Article history
Received:
April 03 1997
Revision Received:
September 08 1997
Accepted:
September 10 1997
Citation
A. Katherine HINCHLIFFE, F. Robin IRVINE, Nullin DIVECHA; Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets. Biochem J 1 January 1998; 329 (1): 115–119. doi: https://doi.org/10.1042/bj3290115
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