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

Abstract

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...

Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome

“…Cells were grown at 37°C in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0). For the selection of cells carrying plasmids for the expression of proteins, the growth medium was supplemented with ampicillin (100 g/ml) and chloramphenicol (34 g/ml) (59). The expression of proteins in cells bearing derivatives of plasmid pET-15b was induced with 1 mM isopropyl ␤-D-1-thiogalactopyranoside.…”

“…Purification of Enzymes, Proteasomes, and ␣-Synuclein-All steps were performed at 4°C. His 6 -tagged wild type and mutant forms of Pah1, His 6 -tagged Pho85-Pho80 protein kinase complex, His 6 -tagged human lipin 1 ␣, ␤, and ␥ isoforms, and His 6 -tagged human ␣-synuclein expressed in E. coli were purified by affinity chromatography with nickel-nitrilotriacetic acid-agarose as described by Han et al (59). For phosphorylation experiments, the affinity-purified Pah1 was further purified by ion-exchange chromatography with Q-Sepharose (54).…”

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

“…The human and S. cerevisiae CTP synthetase enzymes have a relatively high degree of amino acid sequence identity (~ 53%). Indeed, the human CTP synthetase genes (i.e., CTPS1 and CTPS2) are functionally expressed in S. cerevisiae [79]. Moreover, the CTPS1 or CTPS2 genes rescue the lethal phenotype of the S. cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity [79].…”

“…Moreover, the extent of enzyme phosphorylation nearly doubles when yeast cells are activated for the Ras-cAMP pathway and protein kinase A activity [79] and when cells are activated for protein kinase C activity [80]. Indeed, the human CTP synthetase expressed and purified from S. cerevisiae ura7Δ ura8Δ cells is phosphorylated by mammalian forms of protein kinase A [81] and protein kinase C [80].…”

“…In vivo labeling experiments with S. cerevisiae cells have revealed that the human CTPS1-encoded CTP synthetase is phosphorylated [79]. Moreover, the extent of enzyme phosphorylation nearly doubles when yeast cells are activated for the Ras-cAMP pathway and protein kinase A activity [79] and when cells are activated for protein kinase C activity [80].…”