Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation.
CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.
Choi
Mal-Gi
MG
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, USA.
Carman
George M
GM
D019943:Amino Acid Substitution; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013997:Time Factors
D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase