Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not cytosolic phospholipase A2 which undergoes proteolytic inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A(2) (PLA(2)) activation. During apoptosis, type IV cytosolic PLA(2) (cPLA(2)), a PLA(2) isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA(2) correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA(2) protein in which Asp(522) was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD down arrow X), was resistant to apoptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA(2) truncated at Asp(522) comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA(2) cleavage eventually leads to destruction of a catalytic triad essential for cPLA, activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA(2) (iPLA(2)), a PLA(2) isozyme implicated in phospholipid remodeling, remained intact during apoptosis, Inhibitors of iPLA(2), but neither cPLA(2) nor secretory PLA(2) inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas, Therefore, we conclude that iPLA(2)-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.