Phosphorylation and regulation of CTP synthetase from Saccharomyces cerevisiae by protein kinase A.
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.
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903, USA. carman@aesop.rutgers.edu
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D018631:ras Proteins / Q000378:metabolism
D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D018631:ras Proteins; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase