Thrombin stimulates phosphoinositide 3-kinase-associated protein kinase activity in human platelets

A. Piaazn, S. Heydrick, F. Folli, J. Loscalzo

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Abstract

Thrombin activates platelets through a variety of intracellular signalling pathways, including the phosphomositide 3-kinase (PI 3-K) pathway. PI 3-Ks comprise a family of dual specificity enzymes that possess both protein and lipid kinase activities. The best studied PI 3-K is composed of a p85 regulatory subunit that binds to tyrosyl-phosphorylated proteins via its SH2 domain and a p110 catalytic subunit that possesses both lipid and serine kinase activities. While the lipid kinase activity of PI 3-K during platelet activation has been extensively studied, the serine kinase activity has yet to be characterized. To this end, we stimulated human platelets with 4 U/ml a-thrombin for 1.5 min, then lysed the cells for immunoprecipitation (IP) and protein kinase activity; results were compared with unactivated controls. IP with antiphosphotyrosine (anti-PY) antibodies from whole cell lysates of unactivated platelets and in vitro kinase assays revealed the phosphorylation of six bands: 143/115 KDa doublet, 87 kDa, 67/61 kDa doublet, and 46 kDa. Thrombin stimulated the phosphorylation of the 143/115 kDa and 67/61 kDa doublets. In subcellular localization studies, IPs with antibodies against the p85 subunit of PI 3-K and anti-PY antibodies revealed that the 143/115 kDa doublet was found almost exclusively in a 300,000 g membrane fraction, while the 67/61 kDa doublet appeared primarily in the cytosolic fraction. The 143/115 kDa doublet and the 87 kDa band comigrated with both the p110 catalytic subunit and the p85 regulatory subunit of PI 3-K, respectively, in Western analyses. The identities of the 67, 61, and 46 kDa phosphoproteins remain to be determined. These data show that thrombin stimulates the protein kinase activity of PI 3-K and/or other protein kinases associated with PI 3-K in the platelet, which leads to increased phosphorylation of the p110 subunit of the PI 3-K in the membrane fraction and of a 67/61 kDa doublet principally localized to the cytosolic fraction. The differential subcellular localization of these phosphorylated products may underlie their specific roles in the signal transduction accompanying platelet activation by thrombin.

Original languageEnglish (US)
Pages (from-to)214a
JournalJournal of Investigative Medicine
Volume44
Issue number3
Publication statusPublished - Jan 1 1996

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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