Characterization of a Novel dUTP-Dependent Activity of CTP Synthetase from<i>Saccharomyces cerevisiae</i>

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser 36 , Ser 330 , Ser 354 , and Ser 454 , 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 3 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 V max 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 V max of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser 330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser 36 , Ser 330 , Ser 354 , and Ser 454 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.In the yeast Saccharomyces cerevisiae CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (1, 2). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2-5). URA7 (6) and URA8 (7) are duplicate genes that code for CTP synthetase in S. cerevisiae. The yeast CTP synthetase enzymes (6, 7) contain a conserved glutamine amide transfer domain (see Fig. 1) common to CTP synthetases from other organisms (8 -16). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (17) and is responsible for the majority of the CTP synthesized in vivo (7). Neither the URA7 nor the URA8 gene is essential as long as cells possess one functional CTP synthetase gene (6, 7). CTP synthetase is an indispensable enzyme because its reaction product CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). The importance of understanding the regulation of CTP synthetase is emphasized by the fact that unregulated levels of its activity is a common property of various cancers in humans (19 -26).The yeast CTP synthetase is regulated by genetic and biochemical m...

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“…CTP was extracted (6), and its concentration was analyzed by high performance liquid chromatography as described by Pappas et al (56).…”

Section: Methodsmentioning

confidence: 99%

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

Park

O'Brien

Carman

2003

Journal of Biological Chemistry

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“…liquid chromatography using a Partisil 10 SAX column (250 ϫ 4.6 mm, inner diameter) with a SAX guard column (60). The column was equilibrated and eluted with 0.26 M ammonium phosphate/acetonitrile (10:1, pH 5.55) at a flow rate of 2 ml/min.…”

Section: Table 3 Oligonucleotides Used In This Workmentioning

confidence: 99%

“…The nucleotide extract (0.1 ml) was analyzed for CTP by high performance liquid chromatography (60).…”

Section: Sds-page and Immunoblot Analysis-sds-page (61) And Immunoblomentioning

confidence: 99%

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A

Choi

Carman

2007

Journal of Biological Chemistry

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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.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (1, 2). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (2, 3). The enzyme displays positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (2-10). The kinetic behavior with respect to UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme (2,8,11,12). In fact, it is the tetrameric form of the enzyme that is active (2-10, 12).CTP synthetase is an essential enzyme for life because it catalyzes the formation of the CTP required for the synthesis of nucleic acids and membrane phospholipids (13). In eukaryotic cells, CTP synthetase activity controls the balance of nucleotide pools (8, 14 -19) and regulates the synthesis of membrane phospholipids (19 -21). The importance of understanding the regulation of CTP synthetase is highlighted by the fact that an unregulated level of its activity is a phenotype common to leukemia cells (22-24) and rapidly growing tumors of liver (25), colon (26), and lung (27). Indeed, CTP synthetase is a target of antiproliferative drug development for cancer therapy (28 -32, 34) and a target for parasite-based (35) and virally based (36) diseases.Two important modes of CTP synthetase regulati...

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“…Indeed, the CTP synthetase tetramer is the active form of the enzyme (8, 21, 23, 24, 29 -33, 35). The enzyme may also utilize dUTP for the synthesis of dCTP (23,36). 2 An important mode of CTP synthetase regulation is feedback inhibition by CTP (8,21,(23)(24)(25).…”

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confidence: 99%

“…CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (8,21,24,25). dCTP does not substitute for CTP as a feedback inhibitor of CTP synthetase activity using dUTP or UTP as a substrate (23,36). A defect in CTP feedback inhibition results in abnormally high intracellular levels of CTP and dCTP (4,9,37), resistance to nucleotide analog drugs used in cancer chemotherapy (38 -41), and an increased rate of spontaneous mutations (5,39,41).…”

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

Han

Sreenivas

Choi

et al. 2005

Journal of Biological Chemistry

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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 ura7⌬ ura8⌬ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7⌬ ura8⌬ 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.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) catalyzes the final step in the pyrimidine biosynthetic pathway (1). The end product CTP is required for the synthesis of nucleic acids and membrane phospholipids (2). Thus, CTP synthetase is an essential enzyme for the growth and metabolism of all organisms (2). In eukaryotes, CTP synthetase activity regulates the balance of nucleotide pools (3-9) and influences the pathways by which membrane phospholipids are synthesized (9 -11). The importance of understanding the mode of action and regulation of CTP synthetase is further highlighted by the fact that elevated CTP synthetase activity is a common property of several cancers in humans (12)(13)(14)(15)(16)(17)(18)(19)(20).CTP synthetase has been purified and characterized from bacteria (21-23), Saccharomyces cerevisiae (8, 24), and rat liver (25). In addition, crystal structures for the Escherichia coli (26) and Thermus thermophilus (27) enzymes have been solved. The enzymological properties of CTP synthetase enzymes from various sources are similar, although some differences have been identified (23). The enzyme catalyzes a complex set of reactions involving the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (Fig. 1) (21, 28). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (21,29). CTP synthetase exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (8, 21, 23, 24, 29 -33). The positive c...

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Characterization of a Novel dUTP-Dependent Activity of CTP Synthetase fromSaccharomyces cerevisiae

Cited by 18 publications

References 47 publications

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

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

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A

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

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