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.     

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.     

Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues

Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate, K., Nakagomi, K. and Nakazawa, Y. (1984) J. Biol. Chem. 259, 14706-14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3-0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.     

Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Multiple isoforms of Arabidopsis casein kinase I combine conserved catalytic domains with variable carboxyl-terminal extensions.

Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana. One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7. In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation. These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms. Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others. One of them also shows a carboxyl-terminal extension, but of only 80 amino acids. Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein. This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor. Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers.     

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.     

Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae

The wild-type yeast nuclear gene, PIS, encodes phosphatidylinositol synthase (CDPdiacylglycerol-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (Nikawa, J., and Yamashita, S. (1984) Eur. J. Biochem. 143, 251-256). We now report the sequence of the cloned 2, 129-base pair DNA and the location of the PIS coding region within the sequence. The PIS coding frame is capable of encoding 220 amino acid residues with a calculated molecular weight of 24,823. On Northern blot analysis, an RNA species that hybridized with the coding region was detected in the total poly(A)+ RNA of the wild-type yeast. The primary translation product contains a region showing local sequence homology with yeast phosphatidylserine synthase (EC 2.7.8.8) and Escherichia coli 3-phosphatidyl-1'-glycerol-3'-phosphate synthase (EC 2.7.8.5), suggesting that these three enzymes are evolutionarily related. The PIS gene was disrupted in vitro through insertion of the yeast HIS3 gene into the coding region. A heterozygous diploid, PIS/pis::HIS3, constructed from a PIS/PIS his3/his3 diploid by replacing one of the wild-type PIS genes with the disrupted PIS gene, showed no segregation of viable His+ spores on tetrad analysis, indicating that disruption of the PIS gene is lethal. The nonviable spores were in an arrested state with a characteristic terminal phenotype, suggesting that the function of the PIS gene is essential for progression of the yeast cell cycle.     

Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase.

We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

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.     

Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (phosphatidylethanolamine) [corrected] and GPCho (phosphatidylcholine) [corrected] . Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine. The K(m) values for choline, ethanolamine and ATP were 31.4+/-2.6 microM, 2.56+/-0.31 mM and 20.6+/-1.96 microM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.     

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.     

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 phosphoethanolamine/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 Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki 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 degrees 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.     

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.