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

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.