C2-di-ethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A2
Ceramide-1-phosphate (C1P) has been shown to bind with C2 domain in group IVA cytosolic phospholipase A(2) (cPLA(2)α, PLA2G4A) and activate the enzyme activity directly. In cells, C1P causes translocation of cPLA(2)α to perinuclear regions including the Golgi complex by interacting with C2 domain in the enzyme, and then cPLA(2)α releases arachidonic acid from substrate phospholipids in the regions. In this study, we synthesized new di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and examined their effects on cPLA(2)α. A DE-C1P analog with a C2-N-acyl chain (C2-DE-C1P), but not DE-C1P analogs with longer N-acyl chain, such as C6- and C16-DE-C1P, inhibited release of arachidonic acid via cPLA(2)α activation in CHO-W11A cells expressing platelet-activating factor (PAF) receptors without changing secretory phospholipase A(2)-induced release. Treatment with C2-DE-C1P did not modify phosphorylation of extracellular signal-regulated kinase 1/2 and cPLA(2)α and increase of intracellular Ca(2+) level induced by PAF, but inhibited Ca(2+)- and PAF-induced accumulation of cPLA(2)α in the Golgi complex. Phosphatidylcholine vesicles containing C2-DE-C1P reduced cPLA(2)α activity in vitro. C2-DE-C1P disturbed the binding of the enzyme to glycerophospholipids in the lipid-protein overlay assay, and the reagent alone did not bind to the enzyme. Interestingly, C2-DE-C1P inhibited neither Ca(2+)- and PAF-induced accumulation of C2 domain of cPLA(2)α in the Golgi complex nor binding of cPLA(2)α to C16-C1P. These results suggest that C2-DE-C1P appeared to inhibit cPLA(2)α, probably by interaction with a site in the catalytic domain of the enzyme, not with the site in C2 domain responsible for native C1P.