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Papers In Press, published online ahead of print June 1, 2007
J. Lipid Res., doi:10.1194/jlr.M700083-JLR200

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Journal of Lipid Research, Vol. 48, 1293-1304, June 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis

Nadia F. Lamour^*, Robert V. Stahelin, Dayanjan S. Wijesinghe^*, Michael Maceyka^*, Elaine Wang, Jeremy C. Allegood, Alfred H. Merrill, Jr., Wonhwa Cho^** and Charles E. Chalfant^1,*,

^* Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617
Department of Biology, Georgia Institute of Technology, Atlanta, GA 30332
^** Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607-7061
Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA 23249

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of four figures.

Published, JLR Papers in Press, March 27, 2007.

¹ To whom correspondence should be addressed. e-mail: cechalfant{at}vcu.edu

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C_16:0 C1P and C_18:0 C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A₂. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide "pool" transported in an active manner via CERT.

Supplementary key words ceramide-1-phosphate • prostaglandins • phospholipase A₂ • inflammation • arachidonic acid

Abbreviations: AA, arachidonic acid; C1P, ceramide-1-phosphate; CERK, ceramide kinase; CERT, ceramide transport protein; cPLA₂, cytosolic phospholipase A₂; COX, cyclooxygenase; EEA1, early endosome; EIA, enzyme immuno assay; IL, interleukin; PGE₂, prostaglandin E₂; siRNA, small interfering RNA; TGN, trans-Golgi network; TOM20, translocon of the outer membrane; SPR, surface plasmon resonance

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