Increased tyrosine kinase activity in pp60c-src immunoprecipitate from platelet activating factor stimulated human platelets: in vitro phosphorylation of a synthetic peptide.
The involvement of pp60c-src tyrosine kinase was studied in human platelets stimulated with platelet activating factor (PAF). Immunoprecipitation of pp60c-src from platelets followed by immunoblot with pp60v-src monoclonal antibody revealed four protein bands of 60, 56, 50 and 29 kDa as detected by enzymographic web. The phosphorylation of these bands was increased in the pp60c-src immunoprecipitate from PAF stimulated platelets. To assay the tyrosine kinase activity, we used a 13 amino acid synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg- Gly) which contains sequences similar to the phosphorylation site on pp60c-src. Incubation of the pp60c-src immunoprecipitate with the peptide and [32P]ATP caused phosphorylation of this peptide in vitro. This peptide phosphorylation was not observed when normal mouse IgG-bound protein(s) was used instead of pp60c-src immunoprecipitate. The peptide phosphorylation was markedly increased by pp60c-src immunoprecipitate obtained from PAF treated platelets. Lyso-PAF had no effect on the phosphorylation. PAF antagonists CV-6209 and WEB-2086 blocked PAF stimulated phosphorylation. This indicated structurally specific and PAF receptor dependency of this response. These results provide direct evidence that PAF stimulation of human platelets increased tyrosine kinase activity in pp60c-src immunoprecipitate.
Zhu
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CY
Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Shukla
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SD
D000328:Adult; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000502:physiology; D015539:Platelet Activation / Q000502:physiology; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000097:blood*; D012016:Reference Values
D010446:Peptide Fragments; D010972:Platelet Activating Factor; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)