FAS-INDUCED ARACHIDONIC-ACID RELEASE IS MEDIATED BY CA2-INDEPENDENT PHOSPHOLIPASE A(2) BUT NOT CYTOSOLIC PHOSPHOLIPASE A(2) WHICH UNDERGOESPROTEOLYTIC 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)) activatio n. During apoptosis, type IV cytosolic PLA(2) (cPLA(2)), a PLA(2) isoz yme with an apparent molecular mass of 110 kDa critical for stimulus-c oupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA(2) correlated with incre ased caspase-3-like protease activity in apoptotic cells and was abrog ated by a caspase-3 inhibitor. A mutant cPLA(2) protein in which Asp(5 22) was replaced by Asn, which aligns with the consensus sequence of t he caspase-3 cleavage site (DXXD down arrow X), was resistant to apopt osis-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 essentia l for cPLA, activity, thereby terminating its AA-releasing function. I n contrast, the activity of type VI Ca2+-independent PLA(2) (iPLA(2)), a PLA(2) isozyme implicated in phospholipid remodeling, remained inta ct during apoptosis, Inhibitors of iPLA(2), but neither cPLA(2) nor se cretory PLA(2) inhibitors, suppressed AA release markedly and, importa ntly, delayed cell death induced by Fas, Therefore, we conclude that i PLA(2)-mediated fatty acid release is facilitated in Fas-stimulated ce lls and plays a modifying although not essential role in the apoptotic cell death process.