Pleiotropic mutants of Chinese hamster cells with altered cytidine 5?-triphosphate synthetase

Chu, Ernest H. Y.; McLaren, James; Li, I-Chian; Lamb, Barbara

doi:10.1007/bf00485854

Cited by 24 publications

(24 citation statements)

References 34 publications

(26 reference statements)

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“…Concluding Discussion-The level of CTP synthetase activity plays an important role in the growth and metabolism of S. cerevisiae (5,6,9), as well as in higher eukaryotic organisms (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). In this study, we demonstrated that the URA7-encoded CTP synthetase was phosphorylated and stimulated by protein kinase A.…”

Section: Figmentioning

confidence: 61%

Phosphorylation and Regulation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase A

Yang

Carman

1996

Journal of Biological Chemistry

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The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by cAMPdependent protein kinase (protein kinase A) were examined. Protein kinase A is the principal mediator of signals transmitted through the RAS/cAMP pathway in S. cerevisiae. The results of labeling experiments indicated that the phosphorylation of CTP synthetase was mediated by the RAS/cAMP pathway in vivo. In vitro, protein kinase A phosphorylated CTP synthetase at a serine residue with a stoichiometry consistent with one phosphorylation site per CTP synthetase subunit. Protein kinase A activity was dose-and time-dependent using CTP synthetase as a substrate. The dependence of protein kinase A activity on CTP synthetase was cooperative (n ‫؍‬ 1.8) and the K m value for CTP synthetase was 73 nM. Phosphorylation of CTP synthetase with protein kinase A resulted in the stimulation (190%) of activity. The mechanism of this stimulation included an increase in the V max of the reaction with respect to UTP and ATP, a decrease in the K m for ATP, and a decrease in the cooperative kinetic behavior of the enzyme. Phosphorylated CTP synthetase was less sensitive to product inhibition by CTP. Protein kinase C also phosphorylates and activates CTP synthetase. Phosphorylation of CTP synthetase with protein kinases A and C together resulted in an increase in CTP synthetase activity that was slightly greater than that obtained when the enzyme was phosphorylated with either protein kinase alone.CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADPforming)) is a glutamine aminotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (1, 2). GTP activates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2, 3). In the yeast Saccharomyces cerevisiae, CTP synthetase catalyzes the final step in the pyrimidine biosynthetic pathway (4). CTP synthetase is encoded by the URA7 and URA8 genes (4, 5). The deduced amino acid sequences of the open reading frames of the URA7 and URA8 genes show 78% identity and have predicted molecular masses of 64.7 and 64.5 kDa, respectively (4, 5). Purified native URA7-and URA8-encoded CTP synthetases exist as dimers that oligomerize to tetramers in the presence of their substrates, UTP and ATP (6, 7). Neither one of the URA7 and URA8 genes is essential provided that cells possess one functional gene encoding for CTP synthetase (4, 5). Phenotypic analysis of ura7 and ura8 mutants (5) and the characterization of the enzymological properties of the pure URA7-and URA8-encoded enzymes (6, 7) have shown that the two CTP synthetases are not functionally identical. In addition, the URA7-encoded CTP synthetase is 2-fold more abundant when compared with the URA8-encoded enzyme (7) and is responsible for the majority of the CTP synthesized in vivo (5).The product of the CTP synthetase reaction, CTP, is required for the synthesis of RNA, DNA, sialogl...

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Section: Figmentioning

confidence: 61%

Phosphorylation and Regulation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase A

Yang

Carman

1996

Journal of Biological Chemistry

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“…We found that two V79 lines obtained from separate sources differed greatly in the size of their dCTP pools and that the line with the large pool (now named V79/dC) lacked the ability to deaminate dCMP. Several earlier reports have demonstrated the absence or low activity of the enzyme dCMP deaminase in hamster cells (4,5,12).…”

Section: Discussionmentioning

confidence: 92%

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

Bianchi¹,

Pontis²,

Reichard³

1987

Mol. Cell. Biol.

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A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.A network of biosynthetic and catabolic reactions regulates the size of deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells so that the production of DNA precursors is adjusted to their utilization during DNA replication and repair. It has been appreciated for some time that the enzyme ribonucleotide reductase plays a pivotal role in this connection. It not only directs the total flow of metabolites into DNA, but also, via an exquisite allosteric control mechanism, divides the flow into four separate channels so that proper amounts of each dNTP are available for DNA synthesis (14, 18a, 21). More recently it has become apparent that catabolic reactions also control the size of dNTP pools. This first became apparent in connection with two inborn errors of metabolism arising from the lack of adenosine deaminase or purine nucleoside phosphorylase, enzymes catabolizing purine nucleosides (16). In both instances purine dNTPs accumulate and, via disturbances of the allosteric control of ribonucleotide reductase, interfere with the synthesis of DNA of lymphocytes, resulting in immunodeficiency syndromes.Catabolic reactions also contribute to the regulation of pyrimidine dNTP pools (2,3,18). Mouse 3T6 fibroblasts in tissue culture normally catabolize a fraction of pyrimidine dNTPs synthesized de novo to deoxynucleosides that are excreted into the medium. However, cells also use deoxynucleosides present in the medium for the synthesis of dNTPs. We proposed that the balance between import and export is set by the intracellular concentration of deoxynucleosides, which is regulated by substrate cycles. Such cycles operate via phosphorylation of deoxynucleosides by kinases and dephosphorylation of their 5'-phosphates by nucleotidases. The relative rates of these opposing reactions are influenced by both the rate of de novo synthesis of dNTPs and the rate of DNA synthesis. Inhibition of d...

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“…A number of mammalian mutant cell lines possess CTP synthetase activity which is insensitive to inhibition by CTP. As a result, these cell lines display complex phenotypes which include increased intracellular pools of CTP and dCTP (13,14), resistance to nucleotide analog drugs used in cancer chemotherapy (15)(16)(17)(18), and an increased rate of spontaneous mutations (14 -16). In addition, elevated CTP synthetase activity is a common property of leukemic cells (19) and rapidly growing tumors found in liver (20), colon (21), and lung (22).…”

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confidence: 99%

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

Ostrander¹,

O'Brien²,

Gorman³

et al. 1998

Journal of Biological Chemistry

125

142

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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 Glu 161 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 Glu 161 3 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.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is a cytosolic-associated glutamine amidotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (1, 2). This enzyme plays an essential role in the synthesis of all membrane phospholipids in eukaryotic cells (3, 4). Its reaction product CTP is the direct precursor of the activated, energyrich phospholipid pathway intermediates CDP-DG 1 (5), CDPcholine (6), and CDP-ethanolamine (6) (Fig. 1). CDP-DG is the source of the phosphatidyl moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as PI, phosphatidylglycerol, and cardiolipin (3, 4). CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the Kennedy pathways, respectively (3, 4). Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and its impact on phospholipid metabolism. CTP synthetase is encoded by ...

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Pleiotropic mutants of Chinese hamster cells with altered cytidine 5?-triphosphate synthetase

Cited by 24 publications

References 34 publications

Phosphorylation and Regulation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase A

Phosphorylation and Regulation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase A

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

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

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