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George M Carman - One of the best experts on this subject based on the ideXlab platform.

  • CTP Synthetase and its role in phospholipid synthesis in the yeast saccharomyces cerevisiae
    Progress in Lipid Research, 2008
    Co-Authors: Yufang Chang, George M Carman
    Abstract:

    Abstract CTP Synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae , the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP Synthetase in S. cerevisiae , and the URA7 gene is responsible for the majority of CTP synthesized in vivo . The CTP Synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7 -encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP Synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP Synthetase enzymes are functionally expressed in S. cerevisiae , and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP Synthetase activity. The expression in yeast has revealed that the human CTPS1 -encoded enzyme is also phosphorylated and regulated by protein kinases A and C.

  • phosphorylation of human CTP Synthetase 1 by protein kinase a identification of thr455 as a major site of phosphorylation
    Journal of Biological Chemistry, 2007
    Co-Authors: Yufang Chang, Shelley S Martin, Enoch P Baldwin, George M Carman
    Abstract:

    Abstract Phosphorylation of human CTP Synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP Synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP Synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP Synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP Synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1R398A-encoded protein kinase C resulted in a 50% increase for human CTP Synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Δ ura8Δ mutant lacking yeast CTP Synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser462 and Thr455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP Synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP Synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser462 and Thr455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP Synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP Synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367–5377). These data indicated that protein kinase C phosphorylation at Ser462 stimulates human CTP Synthetase 1 activity, whereas phosphorylation at Thr455 inhibits activity.

  • phosphorylation of human CTP Synthetase 1 by
    2007
    Co-Authors: Malgi Choi, George M Carman
    Abstract:

    CTP Synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP Synthetase 1 by protein kinase A. CTP Synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7 ura8 double mutant that lacks CTP Synthetase activity. Using purified CTP Synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP Synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr 455 was a substrate for protein kinase A. A Thr 455 to Ala (T455A) mutation in CTP Synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7 ura8 mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP Synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP Synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.

  • Phosphorylation of human CTP Synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation.
    The Journal of biological chemistry, 2006
    Co-Authors: Malgi Choi, George M Carman
    Abstract:

    CTP Synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP Synthetase 1 by protein kinase A. CTP Synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP Synthetase activity. Using purified CTP Synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP Synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP Synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP Synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP Synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.

  • expression of human CTP Synthetase in saccharomyces cerevisiae reveals phosphorylation by protein kinase a
    Journal of Biological Chemistry, 2005
    Co-Authors: Avula Sreenivas, Malgi Choi, Yufang Chang, Shelley S Martin, Enoch P Baldwin, George M Carman
    Abstract:

    CTP Synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP Synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP Synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP Synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP Synthetase proteins, the measurement of CTP Synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP Synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP Synthetase 1 and the cellular level of CTP. Human CTP Synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP Synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP Synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP Synthetase phosphorylation.

Lars Thelander - One of the best experts on this subject based on the ideXlab platform.

Albert H Van Gennip - One of the best experts on this subject based on the ideXlab platform.

  • Retinoic acid reduces the cytotoxicity of cyclopentenyl cytosine in neuroblastoma cells
    FEBS Letters, 2002
    Co-Authors: Jörgen Bierau, Albert H Van Gennip, René Leen, Huib N. Caron, Andre B P Van Kuilenburg
    Abstract:

    Abstract In this paper, it is demonstrated that all-trans, 9-cis and 13-cis retinoic acid (RA) decreased the sensitivity of SK-N-BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine-5′-triphosphate Synthetase. Retinoic acid attenuated CPEC-induced apoptosis as reflected by a decreased caspase-3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [3H]uridine showed a strongly decreased biosynthesis of CTP via CTP Synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP Synthetase.

  • identification of a cdna encoding an isoform of human CTP Synthetase
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Andre B P Van Kuilenburg, Rutger Meinsma, P Vreken, Hans R Waterham, Albert H Van Gennip
    Abstract:

    Abstract A full-length cDNA clone encoding an isoform of human CTP Synthetase (type II) was isolated. A 1761-nucleotide open reading frame which corresponds to a protein of 586 amino acids with a predicted molecular mass of 65678 Da was identified. The predicted protein sequence showed 74% identity with the translation product of a previously identified human CTP Synthetase cDNA clone (type I). The function of the human cDNA encoding type II CTP Synthetase was verified by successful complementation of the cytidine-requiring CTP Synthetase deficient mutant JF618 of Escherichia coli . The gene encoding type II CTP Synthetase has been localized on chromosome Xp22.

  • kinetic properties of CTP Synthetase from hl 60 cells
    Advances in Experimental Medicine and Biology, 1998
    Co-Authors: Andre B P Van Kuilenburg, L Elzinga, Albert H Van Gennip
    Abstract:

    CTP Synthetase (EC 6.3.4.2) catalyses the formation of CTP from UTP with the concomitant deamination of glutamine to glutamate: $${\rm{UTP + ATP + glutamine }}\buildrel {{\rm{GTP,M}}{{\rm{g}}^{2 + }}} \over \longrightarrow {\rm{CTP + ADP + }}{{\rm{P}}_{\rm{i}}} + {\rm{glutamate}}$$

  • determination of CTP Synthetase activity in crude cell homogenates by a fast and sensitive non radiochemical assay using anion exchange high performance liquid chromatography
    Journal of Chromatography B: Biomedical Sciences and Applications, 1997
    Co-Authors: Andre B P Van Kuilenburg, A A Van Den Berg, L Elzinga, R J Slingerland, Arnauld Verschuur, Albert H Van Gennip
    Abstract:

    Abstract A non-radiochemical assay procedure for CTP Synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2×106 cells was already sufficient to determine the specific activity of CTP Synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.

  • the imp dehydrogenase inhibitor mycophenolic acid antagonizes the CTP Synthetase inhibitor 3 deazauridine in molt 3 human leukemia cells a central role for phosphoribosyl pyrophosphate
    Biochemical Pharmacology, 1995
    Co-Authors: A A Van Den Berg, Andre B P Van Kuilenburg, H Van Lenthe, Petra A W Mooyer, E H Stet, Ronney A De Abreu, Albert H Van Gennip
    Abstract:

    Mycophenolic acid, an inhibitor of the enzyme IMP dehydrogenase, antagonizes the CTP Synthetase inhibitor 3-deazauridine in its anti-proliferative effects on MOLT-3 human T leukemia cells. No depletion of CTP occurred, and decreased amounts of 3-deazauridine-triphosphate were measured in cells incubated with mycophenolic acid and 3-deazauridine. Most probably, these phenomena are related to the increased amounts of PRPP observed, which can result in an increased pyrimidine biosynthesis de novo and, as a consequence, a decreased metabolism of 3-deazauridine via the salvage pathway.

A H Van Gennip - One of the best experts on this subject based on the ideXlab platform.

  • Cyclopentenyl Cytosine Induces Apoptosis and Secondary Necrosis in a T-Lymphoblastic Leukemic Cell-Line
    Advances in Experimental Medicine and Biology, 2020
    Co-Authors: Arnauld Verschuur, A H Van Gennip, René Leen, P.a. Voûte, A B P Van Kuilenburg
    Abstract:

    Cyclopentenyl cytosine (CPEC) induced a depletion of CTP and dCTP in cell-lines of hematological malignancies by inhibiting the enzyme CTP Synthetase , which was accompanied by a decreased synthesis of DNA and RNA . CPEC proved to have a cytostatic effect in Molt-4 and L1210 leukemic cells 1,3 and increased the life-span in mice suffering from lymphocytic leukemia 1. We have shown that a high activity of CTP Synthetase is present in malignant blasts of children suffering from acute (non)-lymphocytic leukemia 4,5 and have demonstrated that CPEC depleted in vitro the concentrations of CTP and dCTP in these samples 4,5 . So far, the mechanism of cytotoxicity of CPEC has been less elucidated. Therefore, in this study we analyzed whether CPEC could induce apoptosis and/or (secondary) necrosis in a Molt-3 lymphocytic leukemic cell-line.

  • In vitro inhibition of cytidine triphosphate Synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia.
    British Journal of Haematology, 2000
    Co-Authors: Arnauld Verschuur, Rutger Meinsma, A H Van Gennip, René Leen, P.a. Voûte, A B P Van Kuilenburg
    Abstract:

    Cytidine triphosphate (CTP) Synthetase is a key enzyme for the synthesis of cytosine (deoxy)ribonucleotides, catalysing the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in several malignancies. In this preclinical study, the enzyme activity and mRNA expression of the enzyme and (deoxy)ribonucleotide concentrations were analysed in leukaemic cells of 57 children suffering from acute lymphocytic leukaemia (ALL). In addition, in vitro experiments were performed with the CTP Synthetase inhibitor cyclopentenyl cytosine (CPEC). A significantly higher activity of CTP Synthetase (6.5 +/- 3.9 nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1.8 +/- 0.9 nmol CTP/mg/h, P < 0.001) that was independent of white blood cell (WBC) count, blast percentage, age, gender or type of ALL. The enzyme activity was not correlated with the CTP Synthetase mRNA expression. The activity of CTP Synthetase in ALL cells compared with non-malignant CD34+ bone marrow controls (5.6 +/- 2.4 nmol CTP/mg/h) was not statistically different. In vitro treatment of ALL cells with CPEC induced a dose-dependent decrease of the CTP concentration. The lowest concentration of CPEC (0.63 microM) induced a depletion of CTP of 41 +/- 20% and a depletion of dCTP of 27 +/- 21%. The degree of CTP depletion of ALL cells after treatment with CPEC was positively correlated with the activity of CTP Synthetase. The inhibition of CTP Synthetase in situ was confirmed by flux studies using radiolabelled uridine. From these results, it can be expected that CPEC has a cytostatic effect on lymphoblasts of children with ALL

  • Cyclopentenyl cytosine inhibits cytidine triphosphate Synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy
    European Journal of Cancer, 2000
    Co-Authors: Arnauld Verschuur, A H Van Gennip, L Elzinga, René Leen, E.j. Muller, P.a. Voûte, A B P Van Kuilenburg
    Abstract:

    Cytidine triphosphate (CTP) Synthetase is a key enzyme in the anabolic pathways of cytosine and uracil ribonucleotide metabolism. The enzyme catalyses the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in various malignancies, which has led to the development of inhibitors of CTP Synthetase for therapeutic purposes. We studied both CTP Synthetase activity and ribonucleotide concentrations in leukaemic cells of 12 children suffering from acute non-lymphocytic leukaemia (ANLL), and performed incubation experiments with cyclopentenyl cytosine (CPEC), a nucleoside analogue that is capable of inhibiting CTP Synthetase. The CTP Synthetase activity in ANLL cells (5.1+/-2.3 nmol CTP/mg/h) was significantly higher compared with granulocytes of healthy controls (0.6+/-0.4 nmol CTP/mg/h, P=0.0002), but was not different from the CTP Synthetase activity in non-malignant CD34+ bone marrow cells (5. 6+/-2.4 nmol CTP/mg/h). Major shifts were observed in the various ribonucleotide concentrations in ANLL cells compared with granulocytes: the absolute amount of ribonucleotides was increased with a substantial rise of the CTP (2.4 versus 0.4 pmol/microg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/microg protein, P=0. 0007) concentrations in ANLL cells compared with granulocytes. Treatment of ANLL cells in vitro with CPEC induced a major depletion (77% with 2.5 microM of CPEC) in the concentration of CTP, whilst the concentrations of the other ribonucleotides remained unchanged. Therefore, the high activity of CTP Synthetase in acute non-lymphocytic leukaemic cells can be inhibited by CPEC, which provides a key to a new approach for the treatment of ANLL

  • cytidine triphosphate CTP Synthetase activity during cell cycle progression in normal and malignant t lymphocytic cells
    European Journal of Cancer, 1995
    Co-Authors: A A Van Den Berg, H Van Lenthe, J B A Kipp, Dirk De Korte, A B P Van Kuilenburg, A H Van Gennip
    Abstract:

    Abstract The role of cytidine triphosphate (CTP) Synthetase (EC 6.3.4.2.) m the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP Synthetase in situ , measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP Synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP Synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP Synthetase of MOLT-3 cells is a cell cycleindependent feature, whereas among normal T lymphocytes, the increase in activity of CTP Synthetase that arises after a growth stimulus is more prominent in the S-phase.

  • inhibition of CTP Synthetase induces differentiation of hl 60 cells and down regulation of the c myc oncogene
    Advances in Experimental Medicine and Biology, 1995
    Co-Authors: A B P Van Kuilenburg, A A Van Den Berg, R J Slingerland, J R Meinsma, A H Van Gennip
    Abstract:

    CTP Synthetase and IMP dehydrogenase are the two ‘key-enzymes’ for the de novo biosynthesis of CTP and GTP, respectively. Inhibition of IMP dehydrogenase depletes the intracellular guanine nucleotide pools and induces granulocytic differentiation of HL-60 cells. Furthermore, the induction of HL-60 differentiation to granulocytes is associated with specific changes in the expression of protooncogenes1, 2. These findings have suggested that guanine ribonucleotides play an important role in the regulation of the differentiation of leukemic-myeloblastic cells. So far, the role of CTP Synthetase and cytosine nucleotides in the process of differentiation and regulation of oncogene expression in HL-60 cells has hardly been studied. Therefore, we studied the pattern of c-myc expression in HL-60 cells after differentiation induction with either DMSO or with 3’-deazauridine (DAU) an inhibitor of CTP Synthetase.

Anders Hofer - One of the best experts on this subject based on the ideXlab platform.