Ethanolamine Kinase Activity Research Articles

Mutant IDH1 gliomas downregulate phosphocholine and phosphoethanolamine synthesis in a 2-hydroxyglutarate-dependent manner

BackgroundMagnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas.MethodsSteady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies.ResultsDe novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic.ConclusionsThis study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.

Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.

Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells.

BackgroundEthanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.Methodology/Principal FindingsIn this report, we mapped the important regulatory regions in the human ek1 promoter. 5’ deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.Conclusion/SignificanceIn conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.

Abstract 1195: Ethanolamine kinase-1 and phosphoethanolamine are potential diagnostic markers and therapeutic targets in breast cancer

Abstract Increased phosphoethanolamine (PE) has been observed in tumors almost as consistently as increased phosphocholine (PC), but the role of PE in cancer is relatively unexplored. Our ongoing studies have shown that choline kinase (ChK)-α, the enzyme that forms PC from choline, has a dual choline/ethanolamine kinase activity but ChK-β has no significant role in maintaining PE levels in vivo. We observed that ethanolamine kinase (EtnK)-1siRNA significantly reduces PE levels in triple negative MDA-MB-231 human breast cancer cells. We further investigated the role of ethanolamine kinase-2 (EtnK-2) in contributing to the increased PE observed in cancers, and the effect of various small interfering RNA (siRNA) combinations downregulating ChK and EtnK on cell viability, as potential therapeutic strategies in these triple negative breast cancer cells. Cells were cultured in RPMI-1640 medium supplemented with 20μM choline and 50μM ethanolamine. While 50nM siRNA was used in all individual siRNA treatment, for combination siRNA treatment 50nM of each specific siRNA was used. Silencing of 80-90% message was confirmed using qRT-PCR for the siRNAs used. Approximately 40 million cells were harvested 48h post transfection and cell extracts were prepared using a dual-phase extraction method based on methanol/chloroform/water (1/1/1; v/v/v). 31P MR spectra were acquired on water soluble fraction and metabolites were quantitated. For assaying cell viability, 4000 cells per well were plated in a 96 well plate and transfected with 50nM siRNA and viability assessed 96h post transfection. PC decreased consistently when cells were treated with Chk-α siRNA or in the presence of high levels of ethanolamine in the medium showing dual choline/ethanolamine kinase activity of ChK-α. In cells treated with EtnK-1 siRNA, a significant reduction in PE levels was observed. ChK-β or EtnK-2 siRNA treatment did not affect PC or PE levels. These results indicate that ChK-β and EtnK-2 do not have significant choline or ethanolamine kinase activity in these breast cancer cells. Etnk-1 is the major contributor to PE levels in these breast cancer cells with some contribution from Chk-α. ChK-β siRNA did not have a significant effect on cell viability whereas ChK-α and EtnK1 siRNA resulted in ∼ 60% and 50% reduction of cell viability respectively. Combined treatment with ChK-α+ EtnK-1 siRNA was not more effective than treatment with the individual siRNA alone, suggesting that both siRNA act through the same path in decreasing cell viability. The reduction of cell viability caused by treatment with EtnK-1 siRNA and its major contribution to elevated PE levels in tumors indicates its therapeutic potential that warrants further investigation. Additionally, like radiolabeled choline, radiolabeled ethanolamine may be useful in detecting breast and other cancers with PET imaging. Acknowledgements: Supported by P50 CA103175. Citation Format: Tariq Shah, Balaji Krishnamachary, Flonne Wildes, Jannie Wijnen, Kristine Glunde, Zaver M. Bhujwalla. Ethanolamine kinase-1 and phosphoethanolamine are potential diagnostic markers and therapeutic targets in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2015-1195

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

BackgroundThe 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 ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα 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 ChoKβ in carcinogenesis has been reported.Methodology/Principal FindingsHere we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ 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 ChoKβ display an ethanolamine kinase role, ChoKα1 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 ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ 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 ChoKα1 than ChoKβ.Conclusion/SignificanceThis study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ 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.

Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion

Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The beta-galactosidase activity driven by the P(EKI1)-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of P(EKI1)-lacZ reporter gene activity in the zrt1deltazrt2delta mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1delta mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p.

Ethanolamine kinase controls neuroblast divisions in Drosophila mushroom bodies

The Drosophila mushroom bodies (MBs), paired brain structures composed of vertical and medial lobes, achieve their final organization at metamorphosis. The alpha lobe absent ( ala) mutant randomly lacks either the vertical lobes or two of the median lobes. We characterize the ala axonal phenotype at the single-cell level, and show that the ala mutation affects Drosophila ethanolamine (Etn) kinase activity and induces Etn accumulation. Etn kinase is overexpressed in almost all cancer cells. We demonstrate that this enzymatic activity is required in MB neuroblasts to allow a rapid rate of cell division at metamorphosis, linking Etn kinase activity with mitotic progression. Tight control of the pace of neuroblast division is therefore crucial for completion of the developmental program in the adult brain.

Regulation of the Yeast EKI1-encoded Ethanolamine Kinase by Inositol and Choline

Regulation of the EKI1-encoded ethanolamine kinase by inositol and choline was examined in Saccharomyces cerevisiae. Transcription of the EKI1 gene was monitored by following the expression of beta-galactosidase activity driven by a P(EKI1)-lacZ reporter gene. The addition of inositol to the growth medium resulted in a dose-dependent decrease in EKI1 expression. Supplementation of choline to inositol-containing growth medium brought about a further decrease in expression, whereas choline supplementation alone had no effect. Analysis of EKI1 expression in ino2Delta, ino4Delta, and opi1Delta mutants indicated that the transcription factors Ino2p, Ino4p, and Opi1p played a role in this regulation. Moreover, mutational analysis showed that the UAS(INO) element in the EKI1 promoter was required for the inositol-mediated regulation. The regulation of EKI1 expression by inositol and choline was confirmed by corresponding changes in ethanolamine kinase mRNA, protein, and activity levels. The repression of ethanolamine kinase by inositol supplementation correlated with a decrease in the incorporation of ethanolamine into CDP-ethanolamine pathway intermediates and into phosphatidylethanolamine and phosphatidylcholine.

Tissue-specific and developmental effects of the easily shocked mutation on ethanolamine kinase activity and phospholipid composition in Drosophila melanogaster.

The easily shocked (eas) gene of Drosophila melanogaster encodes ethanolamine kinase (EK), the first step in phosphatidylethanolamine (PE) synthesis via the CDP-ethanolamine pathway Flies mutant for eas display a complex neurological phenotype. In this paper we look at the contribution of EK to lipid metabolism during Drosophila development with the goal of linking the eas biochemical defect with the organismal phenotype. Using a chromatography-based assay, EK activity was detected in wild-type flies throughout development. Most of the activity in the adult was present in heads, which is primarily tissue of neural origin. Flies mutant for eas showed severely reduced levels of activity at each stage assayed. Using standard extraction methods and thin layer chromatography, phospholipid composition was assayed in wholeflies and in heads. While PE levels were decreased significantly in both tissues, heads also had significantly less phosphatidylserine (PS). Therefore, decreases in both phospholipids may play a role in producing the aberrant phenotype in eas flies.

Abnormal activity of membrane phospholipid synthetic enzymes in the brain of patients with Friedreich's ataxia and spinocerebellar atrophy type-1.

Much evidence, derived from biochemical studies of both blood and autopsied brain, has suggested that phospholipid metabolism is abnormal in patients with Friedreich's ataxia (FA), a disorder characterized by severe neuronal loss in the spinal cord and lower brain stem with no, or only modest, damage in other brain regions. To establish the cause of our recent finding of reduced brain levels of phospholipids in FA, we assayed activities of 10 phospholipid-metabolizing enzymes in the autopsied cerebellar cortex of patients with the disorder and, for comparison, in a group of patients with spinocerebellar ataxia type 1 (SCA-1), a disease characterized, unlike FA, by marked neuronal loss in the cerebellar cortex. Enzyme activities were also measured in four brain areas which are relatively unaffected morphologically in both FA and SCA-1. We found that ethanolamine kinase activity was increased in multiple brain regions of patients with FA (increased 31%-137%) and, more modestly, in SCA-1 (increased 39%-60%), suggesting a nonspecific enhancement of phosphoethanolamine production in both disorders. In contrast, the activity of phosphoethanolamine cytidylyltransferase (PECT), the rate-limiting enzyme of phosphatidylethanolamine synthesis, was significantly and markedly decreased by 35%-78% in the cerebellar, frontal, and occipital cortices of patients with FA but was normal in SCA-1. Reduced PECT activity in FA may explain the lower brain levels of phosphatidylethanolamine in the disorder. Moreover, because decreased PECT activity in FA occurs in brain regions having no, or only modest, morphologic damage, this may represent a systemic change consequent to the frataxin gene defect. Our data also suggest that therapeutic intervention in FA designed to increase synthesis of membrane phospholipids may warrant further investigation.

Isolation and Characterization of the Saccharomyces cerevisiae EKI1 Gene Encoding Ethanolamine Kinase

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.

Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase).

Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1.82) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycerol:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycerol:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (Suc) density gradient centrifugation and broken by osmotic shock and homogenization. The membrane-bound ECT was solubilized with 1.5% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate and purified approximately 118-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then Suc gradient centrifugation. The continuous presence of both salt (0.5 M NaCl) and detergent (1% [w/v] 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both Suc gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.

A novel high-molecular mass mammalian ethanolamine kinase

The present report describes, for the first time, a mammalian kinase highly specific for ethanolamine ( K m=41 μM). Ethanolamine kinase catalyzes the initial step in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. Although plant and protozoan kinases are known to exhibit remarkable specificity for ethanolamine, no equivalent mammalian kinase has been characterized previously. The easily shocked ( eas) mutant of Drosophila has been characterized and found to lack ethanolamine kinase activity while still possessing normal choline kinase activity (Pavlidis, P., Ramaswami, M., and Tanouye, M.A. (1994) Cell 79, 23–33). The gene compensating this mutation encodes a sequence resembling that of choline kinase. In the present report, the eas gene product, expressed in Escherichia coli as fusion protein, is found to have highly specific ethanolamine kinase activity. Anti- eas antibody revealed a protein with a molecular mass of approximately 86 kDa in rat tissues and human HeLa cells by Western blotting, but did not bind rat choline kinase isozymes. Rat liver kinase activity specific for ethanolamine was separated chromatographically and by an immunological procedure using anti-choline kinase antibody, associated with the 86 kDa protein, and immunoprecipitated by anti- eas antibody. This 86 kDa protein is characterized as ethanolamine kinase. Relations with previously reported kinases are discussed.

Characterization of soybean choline kinase cDNAs and their expression in yeast and Escherichia coli.

An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase. A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length. Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity. In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity. Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME. DME effectively inhibited only the GmCK2-encoded choline kinase activity. Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme. These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.

Rat lens choline and ethanolamine kinases: Independent kinetics in intact tissue-competition in homogenates

In the committed step of phosphatidylcholine and phosphatidylethanolamine synthesis, choline and ethanolamine are phosphorylated to form phosphocholine and phosphoethanolamine. Studies of rat tissues in several laboratories attribute these two kinase activities to a single enzyme for which choline and ethanolamine are competing alternative substrates. However, in this study of intact cultured lenses, choline and ethanolamine were phosphorylated independently, and neither compound inhibited phosphorylation of the other, even at high lenticular concentrations. In contrast, choline kinase in lens homogenates was competitively inhibited by ethanolamine ( K i = 2.3 mM), and choline strongly inhibited ethanolamine kinase activity. The results suggest a fragile metabolic compartmentation or organization of kinase enzyme(s) or substrates within the intact, physiologically integrated lens which results in striking changes in kinetic characteristics when the biological organization of the lens is disrupted.

Serine regulates phosphatidylethanolamine biosynthesis in the hamster heart.

The role of serine as a precursor and metabolic regulator for phosphatidylethanolamine biosynthesis in the hamster heart was investigated. Hearts were perfused with 50 microM [1-3H]ethanolamine in the presence or absence of serine for up to 60 min. Ethanolamine uptake was attenuated by 0.05-10 mM serine in a noncompetitive manner, and the incorporation of labeled ethanolamine into phosphatidylethanolamine was also inhibited by serine. Analysis of the ethanolamine-containing metabolites in the CDP-ethanolamine pathway revealed that the conversion of ethanolamine to phosphoethanolamine was reduced. The reduction was a result of an inhibition of ethanolamine kinase activity by an elevated pool of intracellular serine. Perfusion of the heart with 1 mM serine caused a 5-fold increase in intracellular serine pool. In order to examine the action of serine on other phosphatidylethanolamine metabolic pathways, hearts were perfused with [1-3H]glycerol in the presence and absence of serine. Serine did not cause any enhancement of phosphatidylethanolamine hydrolysis. The base-exchange reaction for phosphatidylserine formation or the decarboxylation of phosphatidylserine was not affected by serine perfusion. We conclude that circulating serine plays an important role in the modulation of phosphatidylethanolamine biosynthesis via the CDP-ethanolamine pathway in the hamster heart but does not affect the contribution of the decarboxylase pathway for phosphatidylethanolamine formation.

15] Choline/ethanolamine kinase from rat liver

Choline kinase is the initial enzyme in the CDPcholine pathway for phosphatidylcholine biosynthesis in mammalian cells. It catalyzes the Mg 2+ -ATP-dependent phosphorylation of choline. Choline kinase is rate limiting for phosphatidylcholine formation in several cells and tissues. The enzyme exists as at least two isoelectric forms in all tissues examined; a third form is inducible with polycyclic aromatic hydrocarbons and hepatoxic agents in liver. Both constitutive isoforms in liver have ethanolamine kinase activity, and kinetic and immunological data show that both kinase activities are catalyzed by the same enzymes. These results on the regulation and enzymatic activities of choline kinase indicate that the enzyme plays an important role in the long-term regulation of the CDPcholine pathway and may be involved in the coordinate regulation of phosphatidylethanolamine biosynthesis. To study choline and ethanolamine phosphorylation with a purified kinase preparation and to investigate the nature of the isoforms, a purification protocol for rat liver choline-ethanolamine kinase is developed. With the use of size-exclusion chromatography as the final step, a preparation of very high specific activity can be obtained. To obtain homogeneous, though less active, enzyme, chromatofocusing is used as the final step.

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

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.

Cloning and Characterization of the Yeast CKI Gene Encoding Choline Kinase and Its Expression in Escherichia coli

Using a mutant defective in choline kinase (Hosaka, K., and Yamashita, S. (1980) J. Bacteriol. 143, 176-181; Hosaka, K., and Yamashita, S. (1987) Eur. J. Biochem. 162, 7-13), the structural gene (CKI) for choline kinase of the yeast, Saccharomyces cerevisiae, was isolated by means of genetic complementation. Within its sequence there was an open reading frame capable of encoding 582 amino acids with a calculated molecular weight of 66,316. The primary translation product contained a segment closely related to the phosphotransferase consensus sequence (Brenner, S. (1987) Nature 329, 21). A yeast transformant carrying CKI in multiple copies exhibited very high choline kinase activity as well as ethanolamine kinase activity. In-frame insertion of the CKI coding frame into lacZ' on the pUC19 vector led to efficient expression of choline kinase in Escherichia coli cells in the presence of a lac inducer, isopropylthiogalactoside, proving that CKI is the structural gene for choline kinase. Concomitantly, ethanolamine kinase activity was also expressed. When the CKI locus in the wild-type yeast genome was inactivated by its replacement with the in vitro disrupted cki gene, the yeast cells lost virtually all of the choline kinase activity and most of the ethanolamine kinase activity. Thus, it is concluded that choline kinase is mono-cistronic and that the ethanolamine kinase activity is a second activity of choline kinase in the yeast.

Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 ± 1.4 nmol phosphethanolamine/10 7 infected cells per h) was higher than choline kinase specific activity (1.9 ± 0.2 nmol phosphocholine/10 7 infected cells per h). The K m of choline kinase for choline was 79 ± 20 μM, and ethanolamine was a weak competitive inhibitor of the reaction ( K i = 92 mM). Ethanolamine kinase had a K m for ethanolamine of 188 ± 19 μM, and choline was a competitive inhibitor of ethanolamine kinase with a very high K m of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50°C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.