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Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis.

ABSTRACT: Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), the initial rate-limiting step. The current view is that cPLA(2)alpha associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA(2)alpha in vitro via a novel site in the cationic beta-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467-204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA(2)alpha to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA(2)alpha interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA(2)alpha(-/-)) coupled to "rescue" studies. Therefore, this study provides a paradigm shift in how cPLA(2)alpha is activated during inflammation.

SUBMITTER: Lamour NF

PROVIDER: S-EPMC2785377 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

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Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis.

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