Regulation Of Membrane Binding By The C2-Domain Of Cytoplasmic Phospholipase A2 By Ceramide-1-Phosphate And Calcium

Cytoplasmic phospholipase A2α (cPLA2α) is a key generator of arachidonic acid that is used for downstream production of pro-inflammatory eicosanoids. The N-terminal C2-domain of cPLA2α helps drive calcium-dependent interaction with phosphatidylcholine (PC) membranes. The sphingolipid, ceramide-1-phosphate (C1P), also enhances cPLA2α C2‑domain partitioning to PC membranes. Here we show the usefulness of a new Forster resonance energy transfer (FRET) approach for gaining insights into the C1P and calcium interdependence of membrane partitioning by cPLA2α C2-domain. Our assay relies on energy transfer between intrinsic tryptophan (Trp71 in C2-domain) and anthryl­vinyl fluorophore that is omega-linked to the sn2 acyl chain of PC. A wide range of Ca++ concentration has been studied with PC membranes containing or lacking C1P. Enhancement of C2‑domain partitioning to the membrane by Ca++ and C1P is not simply additive because of their interaction with each other. Depending on conditions, binding of calcium by the C1P head group may mask the phosphate moiety and mitigate interaction with cationic residues that comprise the C1P head group binding site in the C2-domain. Thus, the C1P/Ca++ ratio appears to regulate membrane partitioning by the C2-domain of cPLA2α. Our findings shed light on the regulatory role played by bioactive sphingo­lipids, such as C1P, and changing calcium levels that occur during inflammatory events associated with diseases such as sepsis. [Support: NHLBI HL125353 u0026 NIGMS GM45928; Russian Foundation for Basic Research 012-04-00168; Hormel Foundation].