Ceramide 1-Phosphate Is a Direct Activator of Cytosolic Phospholipase A2

Abstract

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-1β 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 A2 (cPLA2) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA2 is the key phospholipase A2 downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 μM) induced the translocation of full-length cPLA2 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 cPLA2 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 cPLA2 and with the CaLB domain in a calcium- and lipid-specific manner with a KCa of 1.54 μM. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA2 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 cPLA2, demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.