RAFTK/Pyk2 involvement in platelet activation is mediated by phosphoinositide 3-kinase.
Platelet activation by different agonists initiates a signalling cascade involving the phosphorylation of several protein kinases, which control key regulatory events. Previously, we demonstrated that the related adhesion focal tyrosine kinase (RAFTK, Pyk2) was involved in an early phase of platelet activation, independent of integrin and glycoprotein IIb-IIIa activation. In this study, we demonstrate that RAFTK is co-immunoprecipitated with phosphoinositide 3-kinase (PI3K) upon platelet activation, and that thrombin, ADP and collagen induced the phosphorylation of both PI3K and RAFTK. A low dose of thrombin (0.015 U/ml) induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, whereas a high dose of thrombin (0.1 U/ml) induced these events in a PI3K activity-independent manner. ADP and collagen also induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, similar to that of the low-dose thrombin. Furthermore, protein tyrosine phosphatase activity was associated with RAFTK in response to platelet activation, and was found to be that of protein tyrosine phosphatase-2 (SHP-2). The association of SHP-2 with RAFTK was PI3K-dependent and was increased upon RAFTK phosphorylation. Taken together, our results strongly suggest that the involvement of RAFTK in platelet activation is mediated via the PI3K pathway.
Koziak
K
K
Division of Experimental Medicine Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA.
Kaczmarek
E
E
Park
S Y
SY
Fu
Y
Y
Avraham
S
S
Avraham
H
H
D000244:Adenosine Diphosphate / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D015539:Platelet Activation / Q000502:physiology*; D011233:Precipitin Tests; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D000244:Adenosine Diphosphate; D002867:Chromones; D003094:Collagen; D004791:Enzyme Inhibitors; D005340:Fibrinogen; D051418:Focal Adhesion Kinase 2; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; D014638:Vanadates; C076715:pervanadate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human