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Thomas S. Stewart - One of the best experts on this subject based on the ideXlab platform.
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nucleotide variation in the Cytidine Triphosphate synthetase gene of giardia duodenalis
Journal of Eukaryotic Microbiology, 2007Co-Authors: Alexander Swarbrick, Jacqueline Upcroft, Thomas S. StewartAbstract:The Cytidine Triphosphate synthetase genes from three diverse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including divergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most divergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.
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Molecular cloning and characterization of the Plasmodium falciparum Cytidine Triphosphate synthetase gene
Biochimica et Biophysica Acta, 1998Co-Authors: Ed F. Hendriks, William J. O'sullivan, Thomas S. StewartAbstract:Abstract Using degenerate oligonucleotides derived from conserved amino acid regions of Cytidine Triphosphate synthetase, a fragment of the gene from the malarial parasite, Plasmodium falciparum , was isolated by polymerase chain reaction (PCR). This fragment was used as a probe in the isolation of genomic clones containing the entire pf CTP synthetase coding region (2580 bp). The gene encodes the largest CTP synthetase found in any organism to date due to the presence of two additional sequences which are part of the continuous open reading frame and are not introns as their presence in the mRNA was confirmed by reverse transcriptase-PCR. These features distinguish the parasite enzyme from that of the host making it an attractive target for structure based drug design.
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ISOLATION, CHARACTERIZATION AND EXPRESSION OF THE GENE ENCODING Cytidine Triphosphate SYNTHETASE FROM GIARDIA INTESTINALIS
Molecular and Biochemical Parasitology, 1996Co-Authors: William J. O'sullivan, Thomas S. StewartAbstract:Abstract The Cytidine Triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ. This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries. Two genomic clones were obtained one with a 3.6 kb Hin dIII DNA fragment, containing approximately three-quarters of the 5′-end of the synthetase gene and subsequently, a 5.8 kb Pst I DNA fragment which contained the whole gene. The intronless gene has a 1863 bp open reading frame encoding 620 amino acids ( M r of 68.3 kDa). A well conserved catalytic glutamine aminotransferase (GAT) domain was identified. In addition, three insert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes. The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb. The coding region of G. intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E. coli . This construct yielded a soluble and enzymatically active recombinant protein which was purified by a Ni-NTA affinity column. The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa. Kinetic studies of the partially purified recombinant G. intestinalis CTP synthetase gave apparent K m values of 0.1 mM and approximately 0.5 mM for the substrates UTP and l -glutamine respectively in accord with previously reported values for the native enzyme.
Lee M Graves - One of the best experts on this subject based on the ideXlab platform.
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regulation of human Cytidine Triphosphate synthetase 2 by phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Karen M Kassel, Da Ryung Au, Matthew J Higgins, Maria Hines, Lee M GravesAbstract:Abstract Cytidine Triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the Km and IC50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [32P]H3PO4 demonstrated that Ser568 and Ser571 were two major phosphorylation sites, and additional studies demonstrated that Ser568 was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser568 (S568A) but not Ser571 significantly increased hCTPS2 activity, demonstrating that Ser568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the Vmax of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser568 as a major site of CTPS2 regulation by phosphorylation.
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human Cytidine Triphosphate synthetase 1 interacting proteins
Nucleosides Nucleotides & Nucleic Acids, 2008Co-Authors: Matthew J Higgins, David Loiselle, Timothy A J Haystead, Lee M GravesAbstract:We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.
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regulation of human Cytidine Triphosphate synthetase 1 by glycogen synthase kinase 3
Journal of Biological Chemistry, 2007Co-Authors: Matthew J Higgins, Paul R Graves, Lee M GravesAbstract:Abstract Cytidine Triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3′-monoxime and GSK3β short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [32P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3′-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.
A B P Van Kuilenburg - One of the best experts on this subject based on the ideXlab platform.
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In vitro inhibition of Cytidine Triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia.
British Journal of Haematology, 2000Co-Authors: Arnauld Verschuur, Rutger Meinsma, A H Van Gennip, René Leen, P.a. Voûte, A B P Van KuilenburgAbstract: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
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Cyclopentenyl cytosine inhibits Cytidine Triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy
European Journal of Cancer, 2000Co-Authors: Arnauld Verschuur, A H Van Gennip, L Elzinga, René Leen, E.j. Muller, P.a. Voûte, A B P Van KuilenburgAbstract: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
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Cytidine Triphosphate synthase activity and mrna expression in normal human blood cells
Biological Chemistry, 1999Co-Authors: Arnauld Verschuur, A H Van Gennip, E.j. Muller, P.a. Voûte, P Vreken, A B P Van KuilenburgAbstract:: Cytidine Triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.
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increased activity of Cytidine Triphosphate synthetase in pediatric acute lymphoblastic leukemia
Advances in Experimental Medicine and Biology, 1998Co-Authors: Arnauld Verschuur, A H Van Gennip, E.j. Muller, P.a. Voûte, A B P Van KuilenburgAbstract:Children suffering from acute lymphoblastic leukemia (ALL) possess an increased concentration of Cytidine Triphosphate (CTP) in their lymphoblasts compared to resting lymphocytes.1 This might be due to either an enhanced flux through the pyrimidine “de novo” and/or uridine salvage pathway or to an increased flux through the Cytidine salvage pathway. By studying ribonucleotide fluxes in a MOLT-3 lymphoblastic cell line it has been shown that the increased CTP concentration is the result of an enhanced activity of CTP synthetase (CTPS).2 We now analyzed the “in vitro” CTPS activity in lymphoblasts of children with ALL. If increased, CTPS might be inhibited by drugs like cyclopentenylcytosine (CPEC).
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Cytidine Triphosphate ctp synthetase activity during cell cycle progression in normal and malignant t lymphocytic cells
European Journal of Cancer, 1995Co-Authors: A A Van Den Berg, H Van Lenthe, J B A Kipp, Dirk De Korte, A B P Van Kuilenburg, A H Van GennipAbstract: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.
A H Van Gennip - One of the best experts on this subject based on the ideXlab platform.
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In vitro inhibition of Cytidine Triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia.
British Journal of Haematology, 2000Co-Authors: Arnauld Verschuur, Rutger Meinsma, A H Van Gennip, René Leen, P.a. Voûte, A B P Van KuilenburgAbstract: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
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Cyclopentenyl cytosine inhibits Cytidine Triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy
European Journal of Cancer, 2000Co-Authors: Arnauld Verschuur, A H Van Gennip, L Elzinga, René Leen, E.j. Muller, P.a. Voûte, A B P Van KuilenburgAbstract: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
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Cytidine Triphosphate synthase activity and mrna expression in normal human blood cells
Biological Chemistry, 1999Co-Authors: Arnauld Verschuur, A H Van Gennip, E.j. Muller, P.a. Voûte, P Vreken, A B P Van KuilenburgAbstract:: Cytidine Triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.
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increased activity of Cytidine Triphosphate synthetase in pediatric acute lymphoblastic leukemia
Advances in Experimental Medicine and Biology, 1998Co-Authors: Arnauld Verschuur, A H Van Gennip, E.j. Muller, P.a. Voûte, A B P Van KuilenburgAbstract:Children suffering from acute lymphoblastic leukemia (ALL) possess an increased concentration of Cytidine Triphosphate (CTP) in their lymphoblasts compared to resting lymphocytes.1 This might be due to either an enhanced flux through the pyrimidine “de novo” and/or uridine salvage pathway or to an increased flux through the Cytidine salvage pathway. By studying ribonucleotide fluxes in a MOLT-3 lymphoblastic cell line it has been shown that the increased CTP concentration is the result of an enhanced activity of CTP synthetase (CTPS).2 We now analyzed the “in vitro” CTPS activity in lymphoblasts of children with ALL. If increased, CTPS might be inhibited by drugs like cyclopentenylcytosine (CPEC).
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Cytidine Triphosphate ctp synthetase activity during cell cycle progression in normal and malignant t lymphocytic cells
European Journal of Cancer, 1995Co-Authors: A A Van Den Berg, H Van Lenthe, J B A Kipp, Dirk De Korte, A B P Van Kuilenburg, A H Van GennipAbstract: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.
Matthew J Higgins - One of the best experts on this subject based on the ideXlab platform.
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regulation of human Cytidine Triphosphate synthetase 2 by phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Karen M Kassel, Da Ryung Au, Matthew J Higgins, Maria Hines, Lee M GravesAbstract:Abstract Cytidine Triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the Km and IC50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [32P]H3PO4 demonstrated that Ser568 and Ser571 were two major phosphorylation sites, and additional studies demonstrated that Ser568 was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser568 (S568A) but not Ser571 significantly increased hCTPS2 activity, demonstrating that Ser568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the Vmax of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser568 as a major site of CTPS2 regulation by phosphorylation.
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human Cytidine Triphosphate synthetase 1 interacting proteins
Nucleosides Nucleotides & Nucleic Acids, 2008Co-Authors: Matthew J Higgins, David Loiselle, Timothy A J Haystead, Lee M GravesAbstract:We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.
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regulation of human Cytidine Triphosphate synthetase 1 by glycogen synthase kinase 3
Journal of Biological Chemistry, 2007Co-Authors: Matthew J Higgins, Paul R Graves, Lee M GravesAbstract:Abstract Cytidine Triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3′-monoxime and GSK3β short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [32P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3′-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.
