Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets

J. Zhang, J. R. Falck, K. K. Reddy, C. S. Abrams, W. Zhao, S. E. Rittenhouse

Research output: Contribution to journalArticle

62 Scopus citations

Abstract

We have reported that platelets exposed to thrombin or thrombin receptor- directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein- dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN- stimulated 3-phosphorylated phosphoinositide accumulation is 10 mM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)- tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating β-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C → diacylglycerol → protein kinase C.

Original languageEnglish (US)
Pages (from-to)22807-22810
Number of pages4
JournalJournal of Biological Chemistry
Volume270
Issue number39
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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