Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization.
Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.
Sugiura
Masako
M
Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan.
Kono
Keita
K
Liu
Hong
H
Shimizugawa
Tetsuya
T
Minekura
Hiroyuki
H
Spiegel
Sarah
S
Kohama
Takafumi
T
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase