Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.
We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.
Dhar
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A
Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Shukla
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SD
D000818:Animals; D000911:Antibodies, Monoclonal; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015688:Oncogene Protein pp60(v-src) / Q000276:immunology; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000502:physiology*; D015539:Platelet Activation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits; D015398:Signal Transduction
D000911:Antibodies, Monoclonal; D010972:Platelet Activating Factor; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)