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

Abstract

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...

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

“…CTP synthetase is phosphorylated by protein kinase A, and it is unknown whether the phosphorylation of CTP synthetase by protein kinase C affects the ability of the enzyme to be phosphorylated by protein kinase A and vice versa. Moreover, data suggest that CTP synthetase is phosphorylated by yet additional protein kinases (34). Thus, the regulation of CTP synthetase by phosphorylation is very complex.…”

“…These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33)(34)(35). In addition, phosphorylation facilitates the nucleotide-dependent tetramerization of the enzyme (29) and causes a decrease in the sensitivity of the enzyme to inhibition by CTP (34,35). Ser 424 has been identified as the target site for protein kinase A phosphorylation (see Fig.…”

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

“…The phosphorylation of purified URA7-encoded CTP synthetase by protein kinase A and protein kinase C results in the stimulation of enzyme activity by 190 % and 300 %, respectively [74,75]. In vivo labeling studies have shown that the phosphorylation of CTP synthetase by protein kinase A [74] and protein kinase C [75] is physiologically relevant. The stimulation of CTP synthetase by protein kinases A or C may be related to signal transduction pathways that direct the cell growth, proliferation, and biosynthesis of macromolecules.…”

“…The stimulation of CTP synthetase by protein kinases A or C may be related to signal transduction pathways that direct the cell growth, proliferation, and biosynthesis of macromolecules. Kinetic analyses show that the mechanisms for stimulation of CTP synthetase activity by the two different protein kinases are the same [74,75]. Phosphorylated CTP synthetase shows an increase in the V max with respect to the substrates UTP and ATP, a decrease in the K m value toward ATP, and a decrease in the positive cooperativity of the enzyme for ATP [74,75].…”

“…Protein kinase C is a lipid-dependent protein kinase required for S. cerevisiae cell cycle [68][69][70][71][72] and plays a role maintaining cell wall integrity [73]. The phosphorylation of purified URA7-encoded CTP synthetase by protein kinase A and protein kinase C results in the stimulation of enzyme activity by 190 % and 300 %, respectively [74,75]. In vivo labeling studies have shown that the phosphorylation of CTP synthetase by protein kinase A [74] and protein kinase C [75] is physiologically relevant.…”