Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity.
This laboratory first provided evidence for a potential signal transduction pathway involving sphingomyelin and its derivatives (Kolesnick, R.N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). Recently, this laboratory demonstrated the existence of the novel sphingolipid ceramide 1-phosphate in human leukemia (HL-60) cells. Ceramide 1-phosphate was synthesized from ceramide derived from sphingomyelin but not glycosphingolipids. This suggested that a specific pathway extended from sphingomyelin to ceramide 1-phosphate. The present studies provide additional support for this notion by demonstrating the existence of a ceramide kinase activity distinct from diacylglycerol (DG) kinase in HL-60 cells. Microsomal membranes contained a kinase activity that phosphorylated ceramide but not 1,2-DG in the presence of physiologic and higher Ca2+ concentrations (60 nM-3 mM). Kinetic analyses demonstrated an apparent Vmax for ceramide and ATP of 70 pmol.min-1.mg protein-1; apparent Km values were 45 and 25 microM, respectively. The pH optimum was within the physiologic range (pH 6-8). Magnesium but not other divalent cations (Mn2+, Ba2+, Cd2+, Zn2+) also stimulated ceramide phosphorylation. Magnesium also induced 1,2-DG phosphorylation. Since DG kinase is a Mg2(+)-stimulable enzyme that may utilize ceramide as substrate, additional studies separated calcium-dependent ceramide kinase from DG kinase activity. 1,2-DGs competitively inhibited magnesium- but not calcium-dependent ceramide phosphorylation. Hence, calcium-dependent ceramide kinase activity neither utilized DG as substrate nor was inhibited by DG. These activities were physically separable. Both activities were solubilized by n-octyl-beta-D-glucopyranoside and stabilized by glycerol. Ceramide kinase activity bound weakly to a DEAE-cellulose anion exchange column and eluted with 4-fold purification as a single peak of activity in the flow-through and 0.05 M NaCl elutions. In contrast, the majority of DG kinase activity bound more tightly and was recovered as a broad peak in the 0.2-0.35 M NaCl elutions. These studies demonstrate the existence of a ceramide kinase activity in HL-60 cells which is functionally and physically separable from DG kinase. These studies provide further support for the notion of a specific pathway from sphingomyelin to ceramide 1-phosphate.
Kolesnick
R N
RN
Department of Medicine, Memorial Sloan-Kettering Cancer Center, Cornell University Medical College, New York, New York 10021.
Hemer
M R
MR
D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008861:Microsomes / Q000201:enzymology; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor)
D002413:Cations, Divalent; D004075:Diglycerides; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; C066324:ceramide kinase; D002118:Calcium