Stimulation of platelets with platelet-activating factor induces changes in the subcellular distribution and activity of the tyrosine kinase pp60c-src.

Activation of platelets by different agonists induces rapid tyrosine phosphorylation of a number of proteins by stimulating one or more protein tyrosine kinase (PTKase) [l]. The presence of several members of the src family of PTKases, namely pp60""', pp6OfY", pp61 hc*, ~ ~ 6 2 ~ " and pp54/56Iv", has now been identified in platelets 121. The major PTKase expressed in platelets is pp60"'" which comprises 0.2-0.4% of total cell protein [31. The abundance of pp60""' in platelets together with the increase in tyrosine phosphorylation of proteins upon platelet activation implicates pp60""' as having a role in the signal transduction mechanism(s) operating within these cells. The effect of PAF-stimulation on the number o f proteins phosphorylated on tyrosine residues, together with the subcellular distribution of these phosphoproteins has been determined. Moreover, both the subcellular distribution and the in vifro kinase activity of the major PTKase, pp6Oc"'", has been monitored in platelets treated with PAF. The association of pp60""' and tyrosine phosphorylated protein(s) with phosphatidylinositol (PI)kinase activity was also investigated. Washed rabbit platelets were prepared as previously described [4]. lmmunoblotting with a specific monoclonal anti-phosphotyrosine antibody (PY20) detected four tyrosine phosphorylated proteins (52 62 kDa) in unstimulated platelets. Within 5s of stimulation with 300 nM PAF, phosphorylation of two groups of proteins in the molecular mass groups of 35 45 kDa and 66 90 kDa was detected. At 30s post-PAF a third group of proteins (90 150 kDa) was detected. These findings are in agreement with the temporal waves of phosphorylation previously detected in thrombinstimulated platelets [ l ] . Cytoskeletal fractions, detergent soluble fractions and membrane skeletal fractions were prepared from equal numbers of platelets as previously described 151. PAFstimulation induced a rapid increase in the number of tyrosine phosphorylated proteins in both the cytoskeletal and the detergent soluble fraction. lmmunoblotting was also used to determine the subcellular distribution of pp60"-*". In resting platelets pp60""' was located in the detergent soluble fraction and the membrane skeletal fraction, however, upon cell stimulation pp60""' became associated with the cytoskeletal fraction (Fig 1). In order to investigate the difference in activity of pp60"-"" before and after stimulation with PAF, platelets were lysed and extracted in RlPA buffer, pp60""' was immunoprecipitated and its in vifro kinase activity was determined by autophosphorylation. There was a rapid decrease in the in vifro kinase activity of pp60""' immunoprecipitated from platelets which had been stimulated with PAF for 5 s compared to basal levels. After a 5 min stimulation with PAF the in vifro kinase activity of pp60"-"' was back to basal levels.