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Kozo Ishidate - One of the best experts on this subject based on the ideXlab platform.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.
The Biochemical journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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expression and characterization of the active molecular forms of choline Ethanolamine Kinase α and β in mouse tissues including carbon tetrachloride induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α/β hetero-oligomers, with relatively small parts of activity expressed by α/α and β/β homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2–4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was α, not β. The level of α mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the α isoform. Consequently, the composition of α/α homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.
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structure and characterization of the genes for murine choline Ethanolamine Kinase isozymes α and β
Journal of Lipid Research, 2000Co-Authors: Chieko Aoyama, Naoshi Yamazaki, Hiroshi Terada, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (CK/EK) is the first enzyme in phosphatidylcholine/phosphatidylethanol- amine biosynthesis in all animal cells. The highly purified CKs from mammalian sources and their recombinant gene products so far were all shown to have EK activity also, in- dicating that both activities reside on the same protein. CK/EK in most animal cells exists as several isoforms, for two of which ( a and b ) their cDNAs have been cloned from both the rat and mouse, and they are found to be separate gene products. The physiological significance for the existence of more than one CK/EK enzyme, however, remains to be clarified. In this study, we isolated mouse genes encoding both types of CK/EK isozyme and deter- mined their entire structure. The 5 9 -flanking promoter re- gions were found to have quite different features from each other, indicating that their expression could be un- der distinct control. Comparison of the nucleotide se- quence between the corresponding coding exons showed the best homology (75%) residing on exon VIII. A search of the database resulted in the possible existence of 17 different origins of eukaryotic CK and/or EK, each of which presumably contained the entire amino acid se- quence. Multialignment of their putative amino acid sequences led to an identification of the novel consensus sequence possibly required for the expression of either CK or EK activity, which corresponded to the sequence within exons VII and VIII of CK/EK- a and - b genes from the mouse. This sequence was localized in close proximity to the C-terminal region of the general (Brenner's) phos- photransferase concensus sequence which was also com- pletely conserved in all of the putative eukaryotic CK/EK proteins. The results demonstrated that, while both CK/EK- a and - b genes were composed of 11 major exons, the size of their genes was quite different: 40 kb for CK/EK- a , whereas it was only 3.5 kb for CK/EK- b . —Aoyama, C., N. Yamazaki, H. Terada, and K. Ishidate. Structure and char- acterization of the genes for murine choline/ethanol- amine Kinase isozymes a and b . J. Lipid Res. 2000. 41: 452- 464.
George M. Carman - One of the best experts on this subject based on the ideXlab platform.
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Isolation and Characterization of the Saccharomyces cerevisiae
2015Co-Authors: Eki Gene, Encoding Ethanolamine Kinase, George M. CarmanAbstract:transferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylEthanolamine synthesis via the CDP-etha-nolamine pathway. The gene encoding Ethanolamine ki-nase (EKI1) was identified from the Saccharomyces Ge-nome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline Kinase, which also exhibits Ethanolamine Kinase activ-ity. The EKI1 gene was isolated and used to construct eki1D and eki1D cki1D mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of Ethanolamine Kinase activity in wild-type, eki1D mu-tant, cki1D mutant, and eki1D cki1D double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold over-expression of Ethanolamine Kinase activity relative t
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Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion*
2014Co-Authors: Michael C. Kersting, George M. CarmanAbstract:Ethanolamine Kinase catalyzes the committed step in the synthe-sis of phosphatidylEthanolamine via the CDP-Ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanol-amine Kinase by the essential nutrient zinc was examined in Saccha-romyces cerevisiae. The level of Ethanolamine Kinase activity increased whenzincwasdepleted fromthegrowthmedium.This regulation cor-related with increases in the CDP-Ethanolamine pathway intermedi-atesphosphoEthanolamineandCDP-Ethanolamine, andan increase in the methylated derivative of phosphatidylEthanolamine, phosphati-dylcholine. The -galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in Ethanolamine Kinase activity was attributed to a tran-scriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1zrt2 mutant (defective in plasma mem-brane zinc transport) cells grown with zinc was similar to the activ
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Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion
The Journal of biological chemistry, 2006Co-Authors: Michael C. Kersting, George M. CarmanAbstract:Abstract 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 β-galactosidase activity driven by the PEKI1-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 PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ 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 zap1Δ 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.
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Regulation of the Yeast EKI1-encoded Ethanolamine Kinase by Inositol and Choline
The Journal of biological chemistry, 2004Co-Authors: Michael C. Kersting, Hyeon-son Choi, George M. CarmanAbstract:Abstract 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 β-galactosidase activity driven by a PEKI1-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 ino2Δ, ino4Δ, and opi1Δ mutants indicated that the transcription factors Ino2p, Ino4p, and Opi1p played a role in this regulation. Moreover, mutational analysis showed that the UASINO 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.
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Regulation of Phospholipid Synthesis in the Yeastcki1Δeki1Δ Mutant Defective in the Kennedy Pathway: THECHO1-ENCODED PHOSPHATIDYLSERINE SYNTHASE IS REGULATED BY mRNA STABILITY
The Journal of biological chemistry, 2004Co-Authors: Hyeon-son Choi, Avula Sreenivas, Gil-soo Han, George M. CarmanAbstract:Abstract In the yeast Saccharomyces cerevisiae, the most abundant phospholipid phosphatidylcholine is synthesized by the complementary CDP-diacylglycerol and Kennedy pathways. Using a cki1Δ eki1Δ mutant defective in choline Kinase and Ethanolamine Kinase, we examined the consequences of a block in the Kennedy pathway on the regulation of phosphatidylcholine synthesis by the CDP-diacylglycerol pathway. The cki1Δ eki1Δ mutant exhibited increases in the synthesis of phosphatidylserine, phosphatidylEthanolamine, and phosphatidylcholine via the CDP-diacylglycerol pathway. The increase in phospholipid synthesis correlated with increased activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylEthanolamine methyltransferase, and phospholipid methyltransferase. However, other enzyme activities, including phosphatidylinositol synthase and phosphatidate phosphatase, were not affected in the cki1Δ eki1Δ mutant. For phosphatidylserine synthase, the enzyme catalyzing the committed step in the pathway, activity was regulated by increases in the levels of mRNA and protein. Decay analysis of CHO1 mRNA indicated that a dramatic increase in transcript stability was a major component responsible for the elevated level of phosphatidylserine synthase. These results revealed a novel mechanism that controls phospholipid synthesis in yeast.
Yuki Nakamura - One of the best experts on this subject based on the ideXlab platform.
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The Four Arabidopsis Choline/Ethanolamine Kinase Isozymes Play Distinct Roles in Metabolism and Development
Plant physiology, 2020Co-Authors: Ying-chen Lin, Galileo Estopare Araguirang, Anh H. Ngo, Kui-ting Lin, Artik Elisa Angkawijaya, Yuki NakamuraAbstract:Phosphatidylcholine and phosphatidylEthanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or Ethanolamine (Etn) catalyzed by either choline or Ethanolamine Kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited Kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho Kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific Kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn Kinases and Cho Kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.
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the four arabidopsis choline Ethanolamine Kinase isozymes play distinct roles in metabolism and development
Plant Physiology, 2020Co-Authors: Ying-chen Lin, Galileo Estopare Araguirang, Anh H. Ngo, Kui-ting Lin, Artik Elisa Angkawijaya, Yuki NakamuraAbstract:Phosphatidylcholine and phosphatidylEthanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or Ethanolamine (Etn) catalyzed by either choline or Ethanolamine Kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited Kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho Kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific Kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn Kinases and Cho Kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.
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arabidopsis choline Ethanolamine Kinase 1 cek1 is a primary choline Kinase localized at the endoplasmic reticulum er and involved in er stress tolerance
New Phytologist, 2019Co-Authors: Ying-chen Lin, Yuki Nakamura, Kazue KaneharaAbstract:Choline Kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline Kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) encodes functional CEK that prefers choline than Ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline Kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline Kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.
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Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) is a primary choline Kinase localized at the endoplasmic reticulum (ER) and involved in ER stress tolerance
The New phytologist, 2019Co-Authors: Ying-chen Lin, Kazue Kanehara, Yuki NakamuraAbstract:Choline Kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline Kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) encodes functional CEK that prefers choline than Ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline Kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline Kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.
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The Choline/Ethanolamine Kinase Family in Arabidopsis: Essential Role of CEK4 in Phospholipid Biosynthesis and Embryo Development
The Plant cell, 2015Co-Authors: Ying-chen Lin, Yu-chi Liu, Yuki NakamuraAbstract:Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylEthanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoEthanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/Ethanolamine Kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis.
Chieko Aoyama - One of the best experts on this subject based on the ideXlab platform.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.
The Biochemical journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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expression and characterization of the active molecular forms of choline Ethanolamine Kinase α and β in mouse tissues including carbon tetrachloride induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.
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Expression and characterization of the active molecular forms of choline/Ethanolamine Kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Biochemical Journal, 2002Co-Authors: Chieko Aoyama, Akiko Ohtani, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (ChoK/EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α/β hetero-oligomers, with relatively small parts of activity expressed by α/α and β/β homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2–4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was α, not β. The level of α mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the α isoform. Consequently, the composition of α/α homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.
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structure and characterization of the genes for murine choline Ethanolamine Kinase isozymes α and β
Journal of Lipid Research, 2000Co-Authors: Chieko Aoyama, Naoshi Yamazaki, Hiroshi Terada, Kozo IshidateAbstract:Choline/Ethanolamine Kinase (CK/EK) is the first enzyme in phosphatidylcholine/phosphatidylethanol- amine biosynthesis in all animal cells. The highly purified CKs from mammalian sources and their recombinant gene products so far were all shown to have EK activity also, in- dicating that both activities reside on the same protein. CK/EK in most animal cells exists as several isoforms, for two of which ( a and b ) their cDNAs have been cloned from both the rat and mouse, and they are found to be separate gene products. The physiological significance for the existence of more than one CK/EK enzyme, however, remains to be clarified. In this study, we isolated mouse genes encoding both types of CK/EK isozyme and deter- mined their entire structure. The 5 9 -flanking promoter re- gions were found to have quite different features from each other, indicating that their expression could be un- der distinct control. Comparison of the nucleotide se- quence between the corresponding coding exons showed the best homology (75%) residing on exon VIII. A search of the database resulted in the possible existence of 17 different origins of eukaryotic CK and/or EK, each of which presumably contained the entire amino acid se- quence. Multialignment of their putative amino acid sequences led to an identification of the novel consensus sequence possibly required for the expression of either CK or EK activity, which corresponded to the sequence within exons VII and VIII of CK/EK- a and - b genes from the mouse. This sequence was localized in close proximity to the C-terminal region of the general (Brenner's) phos- photransferase concensus sequence which was also com- pletely conserved in all of the putative eukaryotic CK/EK proteins. The results demonstrated that, while both CK/EK- a and - b genes were composed of 11 major exons, the size of their genes was quite different: 40 kb for CK/EK- a , whereas it was only 3.5 kb for CK/EK- b . —Aoyama, C., N. Yamazaki, H. Terada, and K. Ishidate. Structure and char- acterization of the genes for murine choline/ethanol- amine Kinase isozymes a and b . J. Lipid Res. 2000. 41: 452- 464.
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The Four Arabidopsis Choline/Ethanolamine Kinase Isozymes Play Distinct Roles in Metabolism and Development
Plant physiology, 2020Co-Authors: Ying-chen Lin, Galileo Estopare Araguirang, Anh H. Ngo, Kui-ting Lin, Artik Elisa Angkawijaya, Yuki NakamuraAbstract:Phosphatidylcholine and phosphatidylEthanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or Ethanolamine (Etn) catalyzed by either choline or Ethanolamine Kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited Kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho Kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific Kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn Kinases and Cho Kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.
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the four arabidopsis choline Ethanolamine Kinase isozymes play distinct roles in metabolism and development
Plant Physiology, 2020Co-Authors: Ying-chen Lin, Galileo Estopare Araguirang, Anh H. Ngo, Kui-ting Lin, Artik Elisa Angkawijaya, Yuki NakamuraAbstract:Phosphatidylcholine and phosphatidylEthanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or Ethanolamine (Etn) catalyzed by either choline or Ethanolamine Kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited Kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho Kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific Kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn Kinases and Cho Kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.
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arabidopsis choline Ethanolamine Kinase 1 cek1 is a primary choline Kinase localized at the endoplasmic reticulum er and involved in er stress tolerance
New Phytologist, 2019Co-Authors: Ying-chen Lin, Yuki Nakamura, Kazue KaneharaAbstract:Choline Kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline Kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) encodes functional CEK that prefers choline than Ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline Kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline Kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.
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Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) is a primary choline Kinase localized at the endoplasmic reticulum (ER) and involved in ER stress tolerance
The New phytologist, 2019Co-Authors: Ying-chen Lin, Kazue Kanehara, Yuki NakamuraAbstract:Choline Kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline Kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/Ethanolamine Kinase 1 (CEK1) encodes functional CEK that prefers choline than Ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline Kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline Kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.
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The Choline/Ethanolamine Kinase Family in Arabidopsis: Essential Role of CEK4 in Phospholipid Biosynthesis and Embryo Development
The Plant cell, 2015Co-Authors: Ying-chen Lin, Yu-chi Liu, Yuki NakamuraAbstract:Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylEthanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoEthanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/Ethanolamine Kinase (CEK) family catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in Arabidopsis. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type Arabidopsis increased the levels of PtdCho in seedlings and mature siliques and of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in Arabidopsis.