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Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol
1Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Abstract:
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.
Insights
Platelet-activating factor (PAF) stimulates protein tyrosine phosphorylation and the translocation of pp60c-src tyrosine kinase from the cytosol to membranes in rabbit platelets.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Platelet activation involves complex signaling pathways.
- Protein tyrosine phosphorylation plays a critical role in cellular signaling.
- Platelet-activating factor (PAF) is a potent mediator of platelet activation.
Purpose of the Study:
- To investigate PAF-stimulated protein tyrosine phosphorylation in rabbit platelets.
- To determine the relationship between PAF signaling and pp60c-src activity.
- To elucidate the subcellular localization of pp60c-src in response to PAF.
Main Methods:
- 32P-labeled rabbit platelets were stimulated with PAF.
- Phosphotyrosine immunoprecipitation followed by SDS-PAGE and autoradiography.
- Immunoblotting with pp60v-src monoclonal antibody (mAb).
- Subcellular fractionation to isolate membrane and cytosol components.
Main Results:
- PAF increased radioactivity in approximately a dozen protein bands, notably at 50, 60, 71, 82, and 300 kDa.
- PAF treatment enhanced pp60c-src phosphorylation at 50 and 60 kDa.
- pp60c-src immunoreactivity significantly increased in the platelet membrane fraction and decreased in the cytosol fraction following PAF stimulation.
Conclusions:
- PAF stimulates the phosphorylation of pp60c-src tyrosine kinase.
- PAF induces the rapid translocation of pp60c-src from the cytosol to platelet membranes.
- These findings highlight a key mechanism in PAF-mediated platelet activation.