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Journal of Lipid Research, Vol. 46, 2706-2716, December 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Substrate specificity of human ceramide kinase

Dayanjan S. Wijesinghe^*, Autumn Massiello^*, Preeti Subramanian^*, Zdzislaw Szulc, Alicja Bielawska and Charles E. Chalfant^1,*,

^* Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425
Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA 23249

Published, JLR Papers in Press, September 18, 2005. DOI 10.1194/jlr.M500313-JLR200

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

Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6xHis) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK.

Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.

Abbreviations: C1P, ceramide-1-phosphate; CERK, ceramide kinase; cPLA₂, cytosolic phospholipase A₂; K_m^b, intrafacial Michaelis-Menten constant; Ni-NTA, nickel-nitrilotriacetic acid agarose

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

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