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Journal of Lipid Research, Vol. 47, 268-283, February 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Helena Van Overloop, Sofie Gijsbers,¹ and Paul P. Van Veldhoven²

Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Afdeling Farmacologie, Leuven, Belgium

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

¹ Present address of S. Gijsbers: Katholieke Universiteit Leuven, Rega Instituut, Afdeling Virologie en Chemotherapie, Minderbroederstraat 10, B-3000 Leuven, Belgium.

³ Because of this fact, and by using another reading frame, this clone was predicted to code for KIAA1646 protein (AB051433).

⁴ Several lipophilic phosphorylatable compounds, including all four sphinganine and sphingenine stereoisomers, 4-hydroxysphinganine, (truncated) (dihydro)ceramides, (truncated) 1,2-sn-diacylglycerol, 1- or 2-monoacylglycerols, 1-monoalkylglycerols, and phosphatidylinositol, as well as less well-studied kinase substrates (tocopherol, cholesterol, farnesol) or putative substrates (galactosylceramide, anandamide), have been tested as substrates under different delivery modes (EtOH/BSA, octyl-ß-D-glucopyranoside/cardiolipin, CHAPS, DMSO) in the presence of Ca or Mg ions, with bacterially expressed kinase, lysates of CHO cells stably transfected with HsLK2-Myc-His, mitochondria isolated from such cells, or in vitro-transcribed/translated kinase. Compared with the appropriate controls, no remarkable differences were noticed. In addition, in lipid extracts from CHO cells cultured in the presence of labeled glucose (precursor of glycerolipids) or serine (precursor of sphingolipids) or uploaded with ³²P, no additional or enhanced radiolabeled products were revealed when analyzed by two-dimensional TLC/autoradiography and compared with cells stably transfected with the empty vector. On the other hand, these experiments showed that E. coli diacylglycerol kinase can phosphorylate anandamide and that coupled transcription/translation systems phoshorylate sphingoid bases, diacylglycerol, and ceramide (P. P. Van Veldhoven and K. De Greef, unpublished data).

² To whom correspondence should be addressed. e-mail: paul.vanveldhoven{at}med.kuleuven.be

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.

Supplementary key words N-acyl serinol • sphingosine • lipid kinase • ceramide phosphate • pleckstrin • testis • anandamide

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-ß-D-glucopyranoside; SeP, sphingenine-1-phosphate

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