Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 279, Issue 12, Pages 11320-11326Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M309262200
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Funding
- NCI NIH HHS [CA87584] Funding Source: Medline
- NHLBI NIH HHS [HL61378, HL072925, HL34303, HL10507] Funding Source: Medline
- NIGMS NIH HHS [GM08716, GM43825] Funding Source: Medline
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Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1beta and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206-38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A(2) (cPLA(2)) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA(2) is the key phospholipase A(2) downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 muM) induced the translocation of full-length cPLA(2) from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA(2) in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA(2) and with the CaLB domain in a calcium- and lipid-specific manner with a K-Ca of 1.54 muM. Furthermore, Cer-1-P induced a calcium- dependent increase in cPLA(2) enzymatic activity as well as lowering the EC50 of calcium for the enzyme from 191 to 31 nM. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA(2), demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.
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