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

Abstract

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 Mg 21 ions, much less on Ca 21 ions. A clear preference for the D-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but Nhexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and Ntetradecanoyl-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 Mg 21 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.

Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

“…In the cardiolipin/Triton X-100 buffer, the addition of 1 mM MgCl 2 alone increased CerK activity by 8-fold, which was much greater than that by 1 mM CaCl 2 , and the co-addition of CaCl 2 -MgCl 2 did not show additive and/or synergic effects. The results were consistent with those in a previous report by Van Overloop et al 19) In kinase reactions, divalent cations such as Mg 2+ and Ca 2+ interact and reduce negative charges in the phosphate groups of ATP, and may generally enhance the transfer of phosphate groups to substrates and the association between enzymes and ATP. Previous findings and the present results suggest that Ca 2+ is not a direct activator of CerK, while divalent cations such as Mg 2+ and Ca 2+ appear to be essential factors for a kinase reaction of CerK.…”

“…[28][29][30] Also, activity of CerK was measured in cardiolipin-containing buffers in many previous reports. 1,19,20,22,23) However, direct evidence to show the physical association between cardiolipin and CerK as well as activation of this enzyme by the lipid does not currently exist. In the present study, we showed for the first time that cardiolipin directly bound to recombinant CerK in vitro using two lipid-protein binding assays, an overlay assay and large multilamellar vesicle binding assay (Fig.…”

“…CerK was mainly detected in the plasma membranes, Golgi complex, and endosomes in cells, 1,4,19,21) although the localization of CerK in the cytosol was reported in rat basophilic leukemia (RBL-CK3) cells. 10) CerK appeared to be attached to the cytosolic face of the cellular organelles including the plasma membranes.…”

“…Cardiolipin (diphosphatidylglycerol), is a unique phospholipid with four acyl chains and two negative charges, and this lipid is generally used to measure CerK activity in vitro. 1,[19][20][21][22][23] However, it currently remains unknown whether cardiolipin directly interacts with and activates CerK. Thus, we investigated the effects of glycerophospholipids including cardiolipin on recombinant human CerK activity in the glycerol/albumin buffer.…”

Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

“…Thus, the method may not be suitable for determination of ceramide metabolites triggered by the stimuli-induced breakdown of SM. The substrate specificity for enzymes including CERK (12,38) and ceramidases (32,39) is dependent on the length of fatty acyl chains. In addition, the NBD-headgroup changed the reactivity of the attached ceramides to the enzymes involved in ceramide metabolism (40).…”