Regulation of Yeast CTP Synthetase Activity by Protein Kinase C

Abstract

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent V max of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is an allosterically regulated enzyme that is essential for the growth and metabolism of cells. The product of its reaction, CTP, is required for the synthesis of RNA, DNA, phospholipids, and sialoglycoproteins (1). CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (2, 3). GTP activates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (3,4). Genes encoding for CTP synthetase have been isolated from Escherichia coli (5), Chlamydia trachomatis (6), Bacillus subtilis (7), Saccharomyces cerevisiae (8, 9), and human cells (10). The deduced amino acid sequences of the cloned CTP synthetase genes have a relatively high degree of identity, including a conserved glutamine amide transfer domain characteristic of glutamine amidotransferases (5-10).CTP synthetases have been purified to apparent homogeneity from E. coli (3, 11), S. cerevisiae (12, 13), and rat liver cells (14). Purified CTP synthetases exist as dimers that oligomerize to tetramers in the presence of their substrates UTP and ATP (3,(12)(13)(14)(15). CTP synthetases from E. coli (3, 4, 16 -19) and S. cerevisiae (12, 13) exhibit positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP. The positive cooperative kinetics toward UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme (3, 15).A characteristic common to the pure CTP synthetases is the inhibition of their activities by the product CTP (3,(12)(13)(14). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (12)(13)(14). Regulation of CTP synthetase activity by CTP in...

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

“…CTP synthetase (1 g) was incubated for 15 min with 50 mM Tris-HCl (pH 8.0), 50 M [␥-32 P]ATP (4 Ci/nmol), 10 mM MgCl 2 , 10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375 mM EGTA, 1.7 mM CaCl 2 , 20 M diacylglycerol, 50 M phosphatidylserine, and 0.1 nmol/min/ml protein kinase C (35). At the end of the phosphorylation reactions, samples were treated with an equal volume of 2ϫ Laemmli sample buffer (52) followed by SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose paper.…”

“…The ZZ tag facilitates purification of protein kinase C but does not alter the biochemical properties of the enzyme (50,51). The enzyme was purified by Q-Sepharose chromatography and IgG-Sepharose chromatography as described by Antonsson et al (50) with the modifications of Yang et al (35).…”

“…The URA7-encoded CTP synthetase is phosphorylated on multiple serine residues in vivo (33). In vitro studies show that CTP synthetase is a substrate for protein kinases A (34) and C (33,35). These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33)(34)(35).…”

“…The ratio of counts/min of 32 P incorporated into CTP synthetase to the counts of 14 C incorporated into CTP synthetase was used to examine the extent of phosphorylation in vivo (35). The wild type and mutant CTP synthetases were isolated by immunoprecipitation, and the amount of each label incorporated into the enzymes was determined.…”

“…These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33-35). 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.…”