Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in a20 cells

The activation of phospholipase D in murine B cell lymphoma A20 cells treat ed with anti-Fas monoclonal antibody has been investigated. Fas cross-linki ng resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D act ivity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourf old increase within 3 h, Fas activation also caused an approximately twofol d increase of phosphatidylcholine-specific phospholipase C activity and 1,2 -diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 mu g/m l). Pretreatment of D609 also effectively inhibited the translocation of pr otein kinase C beta I and beta II from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1,2-diacylglycerol released from the cellular phosphatidylcholine pool thr ough phosphatidylcholine-specific phospholipase C plays a major role in pro tein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody fail ed to elicit phosphoinositide-specific phospholipase C activation and any c hanges in the intracellular Ca2+ level in A20 cells, indicating that the ph osphoinositide-mediated pathway is not involved in this Fas signaling. Ther efore, these results suggest that Fas-mediated phospholipase D activation m ay be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linki ng signaling downstream from phosphatidylcholine-specific phospholipase C. (C) 1999 Academic Press.