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Tyrosine phosphorylation and activation of PYK2 in platelets: involvement in PI 3-kinase activation

University of British Columbia

Platelets play a central role in the hemostatic response. They also contribute significantly to the initiation of responses by other cells which occur concomitantly with, or as a necessary sequel to, the initial events that prevent loss of blood, and are directly involved in wide range of diseases. Platelets activate various intracellular signaling pathways in thrombus formation, upon blood vessel injury. These intracellular signaling events involve activation of protein-tyrosine kinases which play key regulatory roles in controlling platelet aggregation and in regulating the activity of p85/p110 form of PI 3-kinase. The exact mechanism leading to the activation of p85/PI 3-kinase through the G-protein-coupled receptor is not understood. We show, for the first time, a novel association of PYK2 with p85/PI 3-kinase. The activation of PYK2 and its association with PI 3-kinase were shown to be increased in a time dependent manner and uninhibited by RGDS peptide which blocks the interaction of ligands such as fibrin/fibrinogen with the platelet integrin GPIIb/IIIa. This was concomitant with increased PI 3-kinase activity and association in PYK2 immunoprecipitates. We also have shown that the PI 3-kinase inhibitor LY-294002 had no effect on the activity and association of PYK2 with PI 3-kinase. We suggest that a physical or functional association of PYK2 with PI 3-kinase may occur in vivo that is responsible for the initial activation of PI 3-kinase in thrombinstimulated human platelets independendy of integrin ligation. Furthermore, we observed major tyrosine phosphorylated proteins p115 and p102 were inducibly associated with p85/PI 3-kinase in PAF-stimulated rabbit and thrombin-stimulated human platelets respectively. These proteins were not immunoreactive with several antibodies in this molecular weight range known to be tyrosine phosphorylated and associate with PI 3- kinase on cell activation. We attempted to purify p115 which was shown to be composed of more than one polypeptide or different phosphorylation states of. the same polypeptide, p102 was shown to be inducibly associated with PI 3-kinase, but was dependent upon fibrinogen binding to the integrin. While there is some evidence of aggregation-dependent degradation of proteins in platelets involved a protein at 123 kD that immunoreacted with PYK2 antibody and could be a splice variant of PYK2, the exact mechanisms have not been clearly demonstrated. These events may influence the regulation of PYK2 and PI 3- kinase.

Thesis/Dissertation

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2009-05-26

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http://hdl.handle.net/2429/8240

Experimental Medicine

University of British Columbia

UBCV

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