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Ceramide kinase (CerK) and ceramide-1-phosphate (C1P) are involved in various cellular functions, while regulation of the enzyme activity has not been well elucidated. We herein investigated the effects of several glycerophospholipids on human recombinant CerK activity with CaCl 2 and MgCl 2 by measuring the formation of fluorescent labeled C1P in vitro. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl 2 , and 144±32 with 2 mM MgCl 2 in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P 2 ), and phosphatidic acid had no effect on CerK activity with CaCl 2 , although PI(4,5)P 2 and phosphatidic acid bound to CerK in the lipid-protein overlay assay. The addition of cardiolipin (diphosphatidylglycerol) at concentrations up to 0.1 µM increased, whereas those more than 1 µM decreased CerK activity with CaCl 2 /MgCl 2 . In the lipid-protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl 2 -independent manner. Cardiolipin also bound to CerK in the multilamellar vesicle binding assay. A deviation from the normal range of cellular cardiolipin, both the decrease by phospholipase D6 expression and increase by an exogenous addition of the lipid, negatively regulated C1P formation in intact HepG2 cells. Our results revealed that cardiolipin bound to CerK and regulated the formation of C1P in vitro and in cells.

Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca(2+) suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca(2+)-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca(2+)-independent manner., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chimeric enzymes between a cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia maris, (CmICL) and a mesophilic ICL of a nitrogen-fixing bacterium, Azotobacter vinelandii, (AvICL) were constructed by dividing the ICL genes into four regions of almost equal length and exchanging regions in various combinations. The chimeric ICL, which was replaced C-terminal region 4 of AvICL by the corresponding region of CmICL, showed much lower specific activity and lower optimum temperature and thermostability for activity than wild-type AvICL, indicating that region 4 is involved in its thermal properties. Furthermore, mutual substitution between the Met501 residue in region 4 of CmICL and the corresponding Ile504 residue of AvICL influenced the temperature dependence of their activities, suggesting that these amino acid residues are important to the respective mesophilic and cold-adapted properties of AvICL and CmICL.