Phosphorylation of CTP synthetase on Ser36, Ser330, Ser354, and Ser454 regulates the levels of CTP and phosphatidylcholine synthesis in Saccharomyces cerevisiae.
The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser36, Ser330, Ser354, and Ser454, 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 --> 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 Vmax 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 Vmax of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser330 affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser36, Ser330, Ser354, and Ser454 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.
Park
Tae-Sik
TS
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
O'Brien
Daniel J
DJ
Carman
George M
GM
D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D012694:Serine; D003570:Cytidine Triphosphate; D011493:Protein Kinase C; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase