CTP Synthetase Activity Research Articles

Differential roles of CTP synthetases CTPS1 and CTPS2 in cell proliferation.

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.

CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode.

Cytidine 5'-triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography-mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters Vmax and Km of CTPS and verified that an inhibition of the enzyme activity was induced after 3-deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis-Menten kinetics for lysates of resting cells were Km =280±310 μmol/L for UTP, Vmax =83±20 pmol/min and, for lysates of activated PBMCs, Km =230±280 μmol/L for UTP, Vmax =379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Regulation of Human Cytidine Triphosphate Synthetase 1 by Glycogen Synthase Kinase 3

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 GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]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.

Cyclopentenyl Cytosine (CPEC): An Overview of its in vitro and in vivo Activity

The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is an analogue of cytidine. Besides its antiviral effect, its potential use in the treatment of cancer has become an important area of research. CPEC is activated by intracellular phosphorylation ultimately forming its metabolite CPEC-TP. CPEC-TP is a non competitive inhibitor of cytidine-5'-triphosphate synthetase (CTP-synthetase), an important enzyme in the formation of CTP. Studies have shown that cancer cells have a high CTP synthetase activity, thus making them interesting targets for chemotherapy. CPEC has been preclinically studied in different malignancy models. In vitro results on leukemia show activity in the nanomolar range on several cell lines. However in vivo results are conflicting and the findings vary from increase in life span over 100% to only limited effectiveness. Interesting results have been obtained in colorectal and neuroblastoma cells. In several neuroblastoma cell lines incubation with CPEC in combination with cytarabine or gemcitabine has resulted in increased cell death compared to incubation with with only one of the agents. CPEC has been studied in a phase I trial in patients with solid tumors. In five of 26 patients unexplained cardiotoxicity (extreme hypotension) occurred. The cardiotoxic effects could not be reproduced in animal models. However, precautions should be taken when using this drug in future clinical trials. Low dosage of CPEC seems necessary and intensive cardiac monitoring is advisable. In this manuscript, it is demonstrated that CPEC has an anti-cancer effect in several tumor models: CPEC might be a potentially useful drug in anticancer treatment.

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C: IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION

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 PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) 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 Ser(462) and Thr(455) 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 Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION

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.

Cytidine triphosphate synthetase (CTP synthetase) as a druggable target in cancer

Cytidine triphosphate (CTP) synthetase is a key enzyme in the biosynthesis of pyrimidine ribonucleotides. The enzyme catalyzes the conversion of uridine triphosphate (UTP) to CTP and is the predominant pathway for the synthesis of CTP in proliferating and malignant tissues. Elevated CTP synthetase activity is seen in various malignancies, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), hepatoma, colon cancer and renal carcinoma, as well as non-Hodgkin's lymphoma (NHL). The high activity of the enzyme has led to the development of inhibitors as potential new therapeutic tools. The best explored inhibitor of CTP synthetase is cyclopentenyl cytosine (CPEC), which has been investigated in both preclinical and clinical studies. CPEC proved to profoundly inhibit CTP synthetase in vitro in colon carcinoma, ALL and AML cell lines. Moreover, CPEC has been shown to inhibit the growth of human and murine leukemia xenografts in vivo. A phase I clinical trial with CPEC as monotherapy showed that this compound inhibits CTP synthetase in surrogate tissues such as bone marrow. However, at the highest doses, CPEC proved to have severe cardiovascular toxicity, the mechanism of which has not been unraveled until recently. Additional preclinical studies did not show any cardiotoxicity in rats. There is probably a good rationale for using CPEC at lower doses, since it has been shown in preclinical models to display synergistic cytotoxic effects with other nucleoside analogues such as cytarabine or gemcitabine. In conclusion, CTP synthetase is a druggable target, especially for combination treatment of AML and ALL

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

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.

Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions

In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo- and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair. This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell. By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E. coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase. The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool. The other investigations in this work stem from the unexpected finding that E. coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E. coli mutant. Our data indicate that the regulatory roles are metabolic, not genetic, in nature.

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser36, Ser330, Ser354, and Ser454, contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser --> Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the Vmax of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the Vmax of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser36, Ser330, Ser354, and Ser454 residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.

Phosphorylation of Saccharomyces cerevisiae CTP Synthetase at Ser424 by Protein Kinases A and C Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.

Recovery of SK-N-BE(2)c cells from treatment with cyclopentenyl cytosine.

Neuroblastoma is one of the most common extra cranial solid tumours of childhood. The tumours are derived from the neural crest and have a high degree of heterogeneity1. Success of current therapies is modest. New pharmaceuticals have to be developed and their mechanism of action evaluated. As neuroblastoma cells are rapidly proliferating cells, they rely to a great extend on CTP synthetase for the synthesis of CTP. CTP synthetase catalyses the conversion of UTP into CTP and an increased CTP synthetase activity may cause an imbalance in the pyrimidine ribonucleotide pool2. The gene coding for CTP synthetase is located on chromosome 1p343, which is often deleted in neuroblastoma causing LOH. Despite 1p-deletion neuroblastoma cells have a high CTP synthetase activity, making CTP synthetase an attractive target for chemotherapy. Cyclopentenyl cytosine (CPEC) is a cytidine analogue that is readily metabolised to its active form: CPEC-triphosphate, which potently inhibits CTP synthetase causing rapid depletion of the intracellular cytidine nucleotides4 . In this study, we

In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia.

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

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/μg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/μg 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 μM 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.

Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae.

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.

Nucleotide-dependent Tetramerization of CTP Synthetase from Saccharomyces cerevisiae

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.

Increased activity of cytidine Triphosphate synthetase in pediatric acute lymphoblastic leukemia.

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).