Publications

• Differential role of human choline kinase alpha and beta enzymes in lipid metabolism: implications in cancer onset and treatment.

  Gallego-Ortega D., Ramirez de Molina A., Ramos M.A., Valdes-Mora F., Barderas M.G., Sarmentero-Estrada J., Lacal J.C.

  <h4>Background</h4>The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKal ... >> More

  <h4>Background</h4>The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKalpha and ChoKbeta isoforms, the first one with two different variants of splicing. Recently ChoKalpha has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKbeta in carcinogenesis has been reported.<h4>Methodology/principal findings</h4>Here we compare the in vitro and in vivo properties of ChoKalpha1 and ChoKbeta in lipid metabolism, and their potential role in carcinogenesis. Both ChoKalpha1 and ChoKbeta showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKbeta display an ethanolamine kinase role, ChoKalpha1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKalpha1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKbeta overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKalpha1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKbeta mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKalpha1 than ChoKbeta.<h4>Conclusion/significance</h4>This study represents the first evidence of the distinct metabolic role of ChoKalpha and ChoKbeta isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition. << Less

  PLoS ONE 4:E7819-E7819(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of critical residues of choline kinase A2 from Caenorhabditis elegans.

  Yuan C., Kent C.

  Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutag ... >> More

  Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase. << Less

  J. Biol. Chem. 279:17801-17809(2004) [PubMed] [EuropePMC]

• Purification and characterization of choline/ethanolamine kinase from rat liver.

  Porter T.J., Kent C.

  Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, w ... >> More

  Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/-0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa. << Less

  J Biol Chem 265:414-422(1990) [PubMed] [EuropePMC]

• Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization.

  Gee P., Kent C.

  Choline kinase is the first enzymatic step in the CDP-choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, a ... >> More

  Choline kinase is the first enzymatic step in the CDP-choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, and purified or partially purified three of the encoded enzymes. All expressed proteins had choline kinase activity; those that most closely resemble the mammalian choline kinases were the most active. CKA-2, a very active form, was purified to near homogeneity. The K(m) values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and k(cat) was 74 s(-1). CKA-2 was predominantly a homodimer as assessed by glycerol gradient sedimentation and dynamic light scattering. CKB-2, which was less similar to mammalian choline kinases, had K(m) values for choline and ATP of 13 and 0.7 mM, and k(cat) was 3.8 s(-1). Both of these highly purified enzymes required magnesium, had very alkaline pH optima, and were much more active with choline as substrate than with ethanolamine. These results provide a foundation for future studies on the structure and function of choline kinases, as well as studies on the genetic analysis of the function of the multiple isoforms in this organism. << Less

  Biochim. Biophys. Acta 1648:33-42(2003) [PubMed] [EuropePMC]

• Phosphatidylcholine synthesis in castor bean endosperm: characteristics and reversibility of the choline kinase reaction.

  Kinney A.J., Moore T.S. Jr.

  Choline kinase (EC 2.7.1.32) was measured in concentrated 100,000gav supernatants from castor bean endosperm (Ricinus communis L. var. Hale). Initial velocity analysis, along with competitive inhibitor (hemicholinium-3) and product inhibition (ADPMg2+) studies suggested that the forward reaction f ... >> More

  Choline kinase (EC 2.7.1.32) was measured in concentrated 100,000gav supernatants from castor bean endosperm (Ricinus communis L. var. Hale). Initial velocity analysis, along with competitive inhibitor (hemicholinium-3) and product inhibition (ADPMg2+) studies suggested that the forward reaction followed a sequentially ordered mechanism with ATPMg2+ binding to the enzyme first, followed by choline and then activation of the ternary complex by free Mg2+. The kinetic constants of the forward reaction are reported. A reverse reaction was measured which had a pH optimum of 6.5 and produced 1 mol of ATP for every mole of choline phosphate. The estimated maximum possible Keq at 7.25 was 5 X 10(-3) which suggested that this reaction is highly reversible in this tissue. The possible physiological significance of this is discussed. << Less

  Arch Biochem Biophys 260:102-108(1988) [PubMed] [EuropePMC]

• Choline kinase from brewers' yeast. Partial purification, properties, and kinetic mechanism.

  Brostrom M.A., Browning E.T.

  J Biol Chem 248:2364-2371(1973) [PubMed] [EuropePMC]

• Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase.

  Kim K., Kim K.-H., Storey M.K., Voelker D.R., Carman G.M.

  Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based o ... >> More

  Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p. << Less

  J. Biol. Chem. 274:14857-14866(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.