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

Abstract

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...

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

“…Moreover, in vivo studies using these mutants have shown that the regulatory effects of the phosphorylations at specific sites have an impact on the pathways by which membrane phospholipids are synthesized. Phosphorylations at Ser 36 , Ser 354 , and Ser 454 correlate with an increase in PC synthesis via the Kennedy pathway [77,78]. In contrast, phosphorylation at Ser 330 correlates with a decrease in the utilization of the Kennedy pathway [77].…”

“…A combination of biochemical and molecular approaches has been used to identify protein kinase A and protein kinase C sites in the URA7-encoded CTP synthetase [76][77][78]. Ser 424 is a target site for both protein kinase A and protein kinase C [76,78].…”

“…The phosphorylation of this site is required to maintain optimum CTP synthetase activity in vivo [76,78]. Protein kinase C also phosphorylates Ser 36 , Ser 330 , Ser 354 , and Ser 454 [77]. Biochemical and physiological analyses of Ser-to-Ala mutations have shown that phosphorylations at all sites, with the exception of Ser 330 , result in the stimulation of CTP synthetase activity [77,78].…”

“…Protein kinase C also phosphorylates Ser 36 , Ser 330 , Ser 354 , and Ser 454 [77]. Biochemical and physiological analyses of Ser-to-Ala mutations have shown that phosphorylations at all sites, with the exception of Ser 330 , result in the stimulation of CTP synthetase activity [77,78]. The phosphorylation of Ser 330 results in the inhibition of activity [77].…”

“…Biochemical and physiological analyses of Ser-to-Ala mutations have shown that phosphorylations at all sites, with the exception of Ser 330 , result in the stimulation of CTP synthetase activity [77,78]. The phosphorylation of Ser 330 results in the inhibition of activity [77]. Moreover, in vivo studies using these mutants have shown that the regulatory effects of the phosphorylations at specific sites have an impact on the pathways by which membrane phospholipids are synthesized.…”