Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C.
Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.
Yang
W L
WL
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903, USA.
Carman
G M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D019731:Carbon-Nitrogen Ligases; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine; D013912:Threonine
D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase