Involvement of protein-tyrosine kinase p72syk in collagen-induced signal transduction in platelets.
Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including pp125FAK, through the interaction of collagen with integrin alpha 2 beta 1 (GP Ia-IIa). In this study, we report that p72syk is a potential candidate for the protein-tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p72syk in platelets also occurred in the same time course as the activation of p72syk. Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72syk induced by collagen. Furthermore, herbimycin A, a potent protein-tyrosine-kinase inhibitor, was capable of reducing collagen-evoked p72syk activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p72syk is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets.
Department of Biochemistry, Fukui Medical School, Japan.
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Fujii
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Yanagi
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Sada
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Nagai
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Taniguchi
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Yamamura
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013552:Swine; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D014443:Tyrosine; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D002118:Calcium