Platelet-activating factor induces the tyrosine phosphorylation and activation of phospholipase C-gamma 1, Fyn and Lyn kinases, and phosphatidylinositol 3-kinase in a human B cell line.
Platelet-activating factor (PAF) is a versatile lipid mediator of inflammation in a variety of biologic systems. We have previously reported that one of the earliest events in the signal transduction pathway of PAF in a human B lymphoblastoid cell line was the induction of tyrosine kinase activity concomitant with the activation of phospholipase C (PLC). We now demonstrate the occurrence of multiple tyrosine phosphorylation-dependent events which follow the interaction of PAF with its receptor on B cells. Anti-phosphotyrosine immunoprecipitates from lysates of PAF-stimulated cells, when fractionated by SDS-PAGE and analyzed by Western blotting with anti-PLC-gamma 1, showed that maximal tyrosine phosphorylation of this enzyme occurred within 2 min of stimulation. This phenomenon was verified by immunoprecipitating with anti-PLC-gamma 1 and subsequently probing with anti-phosphotyrosine. Immunoprecipitation of the tyrosine kinases, Fyn and Lyn, from PAF-stimulated cells, and use of these immunoprecipitates in kinase assays established that the activation of both kinases also occurred within the first 2 min of stimulation with phosphorylation occurring on their tyrosine residues. Additionally, we also provide evidence for the tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PtdIns 3-kinase) and activation of this kinase by PAF in a dose-dependent manner, maximal activation occurring within 10 min post-stimulation. We have thus demonstrated that the activation of tyrosine kinases is an important proximate step in PAF-mediated signal transduction in B cells, leading to tyrosine phosphorylation and activation of PLC-gamma 1, Fyn and Lyn kinases, and PtdIns 3-kinase.
Kuruvilla
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Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Pielop
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C
Shearer
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WT
D000936:Antigen-Antibody Complex / Q000276:immunology; D001402:B-Lymphocytes / Q000201:enzymology*; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000502:physiology*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D010738:Type C Phospholipases / Q000378:metabolism*; D019061:src-Family Kinases
D000936:Antigen-Antibody Complex; D007527:Isoenzymes; D010972:Platelet Activating Factor; D011518:Proto-Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma