Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A.
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.
Han
Gil-Soo
GS
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
Sreenivas
Avula
A
Choi
Mal-Gi
MG
Chang
Yu-Fang
YF
Martin
Shelley S
SS
Baldwin
Enoch P
EP
Carman
George M
GM
D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis* / Q000737:chemistry*; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D015151:Immunoblotting; D008956:Models, Chemical; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D019606:Phosphoamino Acids / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013997:Time Factors
D010455:Peptides; D019606:Phosphoamino Acids; D010743:Phospholipids; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase