Abstract
Cytosolic phospholipase A(2)alpha (cPLA(2)alpha; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp(522) during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA(2)(1-522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A(2)s (PLA(2)s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA(2)s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA(2)(1-522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca(2+)-independent PLA(2) (iPLA(2); type VI), a phospholipid remodeling PLA(2), contributes to the cell death-associated increase in fatty acid release. iPLA(2) was cleaved at Asp(183) by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA(2) functions. iPLA(2) had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA(2)alpha behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA(2) accelerates phospholipid turnover that may lead to apoptotic membranous changes.
Highlights
Cytosolic phospholipase A2␣, an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp522 during apoptosis
Pharmacological and transfection studies revealed that Ca2؉-independent phospholipase A2s (PLA2s), a phospholipid remodeling PLA2, contributes to the cell death-associated increase in fatty acid release. iPLA2 was cleaved at Asp183 by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA2 functions. iPLA2 had a significant influence on cell growth or death, according to cell type
In order to investigate whether the cleaved cytosolic PLA2 (cPLA2)␣ plays some roles in cellular AA metabolism, death, survival, or proliferation, we prepared cDNA for the mutant cPLA2␣ truncated at Asp522 (cPLA2(1–522)) and transfected it into HEK293 cells
Summary
Cytosolic phospholipase A2␣ (cPLA2␣; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp522 during apoptosis. To verify whether iPLA2 is a substrate for caspase-3 and whether the resultant truncated iPLA2 retains its activity to facilitate cell death-associated fatty acid release, we constructed an iPLA2 mutant, iPLA2(184-C), that would correspond to the truncated enzyme after cleavage at Asp183 and transfected it into HEK293 cells.
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