Journal of Lipid Research
Volume 47, Issue 2, February 2006, Pages 268-283
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Research Article
Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

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Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg2+ ions, much less on Ca2+ ions. A clear preference for the d-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but N-hexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and N-tetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg2+ dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain. No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.

N-acyl serinol
sphingosine
lipid kinase
ceramide phosphate
pleckstrin
testis
anandamide

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Abbreviations:

    CERK

    ceramide kinase

    Cer1P

    ceramide-1-phosphate

    CL

    cardiolipin

    CMC

    critical micellar concentration

    DMS

    N,N-dimethylsphingenine

    EST

    expressed sequence tag

    EtOH

    ethanol

    GFP

    green fluorescent protein

    HsCERK

    human ceramide kinase

    LDAO

    lauryldimethylammonium N-oxide

    MuLK

    multisubstrate lipid kinase

    C2-ceramide

    N-acetyl-sphingenine

    C6

    ceramide

    N

    hexanoyl-sphingenine

    OG

    octyl-β-d-glucopyranoside

    SeP

    sphingenine-1-phosphate

Published, JLR Papers in Press, November 3, 2005.

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Present address of S. Gijsbers: Katholieke Universiteit Leuven, Rega Instituut, Afdeling Virologie en Chemotherapie, Minderbroederstraat 10, B-3000 Leuven, Belgium.