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A more recent version of this article appeared on December 1, 2005

Papers In Press, published online ahead of print September 18, 2005
J. Lipid Res., doi:10.1194/jlr.M500313-JLR200
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Submitted on July 21, 2005
Revised on September 12, 2005
Accepted on September 18, 2005

Substrate specificity of human ceramide kinase

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

Department of Biochemistry, Virginia Commonwealth University, Richmond, VA 23298-0614

Corresponding Author: cechalfant{at}vcu.edu

Previous studies by 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 hCERK (6XHis) and a newly designed assay based on mixed-micelles of Triton X-100. Results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific as CERK only phosphorylated D-e-C16:0 ceramide and not the ceramide stereoisomers, L-e-C16:0 ceramides, D-t-C16:0 ceramides, and L-t-C16:0 ceramides. A minimum of a 12 carbon acyl chain was required for normal CERK activity as shortening of the acyl chain below 12 carbons dramatically reduced the substrate specificity. The 4-5 trans double bond was important for substrate recognition as D-e-4,5-Cis, D-e-dihydro, D-e-dehydro, and D-e-phyto ceramides demonstrated a marked decrease in CERK activity when compared to natural ceramide. These results did not show any significant discrimination by CERK between ceramides with long saturated and long unsaturated fatty acyl chains as substrates. Methylation of the primary hydroxyl group resulted in a loss of activity confirming that CERK produces C1P versus C3P. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation of the substrate by the enzyme suggesting the 3´ OH group is required for substrate recognition. These results also indicated that the free hydrogen of the secondary amide group to be critical for substrate recognition as N-methyl ceramides failed to become phosphorylated by CERK. 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 nor diacylglycerol as substrates.


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