Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.
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 microm) 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 microm. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA(2) enzymatic activity as well as lowering the EC(50) 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.
Pettus
Benjamin J
BJ
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Bielawska
Alicja
A
Subramanian
Preeti
P
Wijesinghe
Dayanjan S
DS
Maceyka
Michael
M
Leslie
Christina C
CC
Evans
John H
JH
Freiberg
Jessica
J
Roddy
Patrick
P
Hannun
Yusuf A
YA
Chalfant
Charles E
CE
D001483:Base Sequence; D045744:Cell Line, Tumor; D002518:Ceramides / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology*; D020536:Enzyme Activators / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D034622:RNA Interference
D002518:Ceramides; D020536:Enzyme Activators; C065576:ceramide 1-phosphate; D010741:Phospholipases A; D054467:Phospholipases A2