Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: studies with genistein and monoclonal antibody to phosphotyrosine.
Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation. The inhibition of PLC by Genistein raised the possibility of involvement of tyrosine kinase in PAF receptor-coupled PLC activation. Western blot analysis using monoclonal antibody to phosphotyrosine demonstrated that PAF stimulated the tyrosine phosphorylation of two major proteins of 50,000 and 60,000 molecular weight. When platelets were challenged with PAF after treatment with either Genistein or CV-6209 (a PAF receptor antagonist), the reactivity of these proteins to monoclonal antibody was inhibited. Phosphoamino acid analysis of Mr 50,000 and 60,000 proteins confirmed that PAF increased the phosphorylation of tyrosine residues in both Mr 50,000 and 60,000 proteins and that this was inhibited by Genistein. Thus, PAF caused a receptor-dependent phosphorylation of tyrosine residues on Mr 50,000 and 60,000 proteins. Based on these observations, it is concluded that tyrosine kinase is involved in the PAF receptor-coupled PLC activation and signal transduction mechanism.
Dhar
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Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Paul
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Shukla
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SD
D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D015153:Blotting, Western; D005419:Flavonoids / Q000494:pharmacology*; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology*; D010766:Phosphorylation; D019000:Phosphotyrosine; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000187:drug effects*; D043562:Receptors, G-Protein-Coupled; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D005419:Flavonoids; D007529:Isoflavones; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D014316:Tritium; D019000:Phosphotyrosine; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases