Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphorylation and regulation of the URA7-encoded CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by cAMP-dependent 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 Km 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 Vmax of the reaction with respect to UTP and ATP, a decrease in the Km 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.
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PMID:Phosphorylation and regulation of CTP synthetase from Saccharomyces cerevisiae by protein kinase A. 891 May 20

The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized. CTP synthetase existed as a dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer. Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer. The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity. The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions. ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme. The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization. CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme. Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization. Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme. This correlated with the inactivation of CTP synthetase activity. Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme. This tetramerization correlated with the partial restoration of CTP synthetase activity. Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme.
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PMID:Nucleotide-dependent tetramerization of CTP synthetase from Saccharomyces cerevisiae. 963 43

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.
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PMID:Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae. 1039 61

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 ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta 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.
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PMID:Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A. 1617 39