INCREASED TYROSINE KINASE-ACTIVITY IN PP60C-SRC IMMUNOPRECIPITATE FROM PLATELET-ACTIVATING-FACTOR STIMULATED HUMAN PLATELETS - INVITRO PHOSPHORYLATION OF A SYNTHETIC PEPTIDE

The involvement of pp60c-src tyrosine kinase was studied in human plat elets stimulated with platelet activating factor (PAF). Immunoprecipit ation of pp60c-src from platelets followed by immunoblot with pp60v-sr c monoclonal antibody revealed four protein bands of 60, 56, 50 and 29 kDa as detected by enzymographic web. The phosphorylation of these ba nds was increased in the pp60c-src immunoprecipitate from PAF stimulat ed platelets. To assay the tyrosine kinase activity, we used a 13 amin o acid synthetic peptide g-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 pept ide and [P-32]ATP caused phosphorylation of this peptide in vitro. Thi s peptide phosphorylation was not observed when normal mouse IgG-bound protein(s) was used instead of pp60c-src immunoprecipitate. The pepti de phosphorylation was markedly increased by pp60c-src immunoprecipita te obtained from PAF treated platelets. Lyso-PAF had no effect on the phosphorylation. PAF antagonists CV-6209 and WEB-2086 blocked PAF stim ulated phosphorylation. This indicated structurally specific and PAF r eceptor dependency of this response. These results provide direct evid ence that PAF stimulation of human platelets increased tyrosine kinase activity in pp60c-src immunoprecipitate.