Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C

Abstract

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.

Fig. 1

Reaction catalyzed by CTP synthetase. The figure shows the structures of UTP and CTP and the reaction catalyzed by CTP synthetase.

Fig. 2

Complementation of the CTP synthetase defect of the S. cerevisiae ura7Δ ura8Δ mutant by the human CTPS1 or CTPS2 gene. Strain SD195, a ura7Δ ura8Δ mutant carrying the URA7 gene on a URA3-based plasmid (pDO134), was transformed to leucine prototrophy with the empty LEU2-based plasmid (pDO105), or with the same plasmid containing the human CTP synthetase genes (pDO105-hCTPS1 or pDO105-hCTPS2). The transformants were streaked onto SC-leucine plates containing 5FOA and incubated for 3 days at 30 °C. The 5FOA-resistant colonies were produced only from the transformants carrying pDO105-hCTPS1 or pDO105-hCTPS2. Four independent colonies from 5FOA-resistant cells were patched onto SC-leucine plates and grown for 2 days at 30 °C. Strain SD195 carrying pDO105 was included in this analysis as a negative control. The patches were replica plated onto SC-leucine, SC-leucine + 5FOA, and SC-uracil plates. The plates were incubated for 3 days at 30 °C.

Fig. 3

Expression of human CTP synthetase 1 and human CTP synthetase 2 proteins in S. cerevisiae. The ura7Δ ura8Δ mutant bearing plasmids with the yeast URA7 gene (strain GHY52) or the human CTPS1 (strain GHY55) or CTPS2 (strain GHY56) genes were grown to the exponential phase of growth. Cell extracts were prepared and samples (20 μg of protein) were subjected to SDS-PAGE, followed by transfer to PVDF membrane. The membrane was probed with anti-His₆ antibodies to detect the His₆-tagged human CTP synthetase 1 (hCTPS1) and human CTP synthetase 2 (hCTPS2) proteins (left). The membrane was stripped of the anti-His₆ antibodies and reprobed with anti-Ura7p antibodies to detect the yeast URA7-encoded CTP synthetase (yCTPS) protein (middle). Prior to probing with the antibodies, the PVDF membrane was stained with Ponceau S to detect the total amount of protein on the membrane (right). The data shown were representative of two independent experiments.

Fig. 4

CTP synthetase activities and the synthesis of CTP in S. cerevisiae expressing human CTP synthetase 1 and human CTP synthetase 2. The ura7Δ ura8Δ mutant bearing plasmids with the yeast URA7 gene (strain GHY52) or the human CTPS1 (strain GHY55) or CTPS2 (strain GHY56) genes were grown to the exponential phase of growth. Panel A, The cytosolic fraction was isolated from the cells and assayed for CTP synthetase activity from the indicated yeast (yCTPS) or human CTP synthetase 1 (hCTPS1) and CTP synthetase 2 (hCTPS2) enzymes. Panel B, CTP was extracted from the cells and analyzed by high performance liquid chromatography. Each data point represents the average of duplicate determinations from two independent experiments ± S.D.

Fig. 5

Phosphorylation of human CTP synthetase 1 in S. cerevisiae. Panel A, the ura7Δ ura8Δ mutant bearing plasmids with the yeast URA7 gene (strain GHY52) or the human CTPS1 gene (strain GHY55) was grown in 50 ml of growth medium to the exponential phase of growth. Cells were harvested, resuspended in 5 ml of fresh medium containing ³²P_i (0.25 mCi/ml), and incubated for 3 h. Cell extracts were prepared from the labeled cells and incubated with Ni²⁺-NTA resin to bind the His₆-tagged human CTP synthetase 1. Proteins bound to the Ni²⁺-NTA resin were eluted with SDS-PAGE treatment buffer, followed by electrophoresis and transfer to PVDF membrane. The membrane was subjected to phosphorimaging (left) followed by immunoblot analysis using anti-His₆ antibodies (right). The arrow indicates the position of human CTP synthetase 1 (hCTPS1). Panel B, a PVDF membrane slice containing ³²P-labeled human CTP synthetase 1 was hydrolyzed with 6N HCl for 90 min at 110 °C, and the hydrolysate was separated by 2-dimensional electrophoresis. The positions of the standard phosphoamino acids phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) are indicated in the figure. Panel C, a PVDF membrane slice containing ³²P-labeled human CTP synthetase 1 was digested with trypsin. The resulting peptides were separated on cellulose thin layer plates by electrophoresis (from left to right) in the first dimension and by chromatography (from bottom to top) in the second dimension. The data shown in the three panels were representative of two independent experiments.

Fig. 6

Effects of protein kinase A activation on the phosphorylation of human CTP synthetase 1 and on the cellular concentration of CTP in S. cerevisiae. Panel A, cells expressing human CTP synthetase 1 (strain GHY55) were labeled with ³²P_i for 3 h in low phosphate YEPA medium. Following the labeling period, glucose was added to a final concentration of 5% to activate the Ras-cAMP pathway and protein kinase A activity. Human CTP synthetase 1 was precipitated from lysates with Ni²⁺-NTA resin, followed by SDS-PAGE and transfer of proteins to PVDF membrane. The membrane was subjected to phosphorimaging and immunoblot analyses followed by the quantification of the signals using ImageQuant software. Relative phosphorylation was calculated by dividing the signal intensity of ³²P-labeled human CTP synthetase 1 by that of enzyme protein. The extent of phosphorylation of human CTP synthetase 1 before glucose addition was set at 1. Panel B, in a separate experiment, the cellular concentration of CTP was measured from unlabeled cells that were activated in the Ras-cAMP pathway. The data are representative of two independent experiments.

Fig. 7

Phosphorylation of E. coli-expressed human CTP synthetase 1 by protein kinase A. Panel A, pure human CTP synthetase 1 (1 μg) was incubated with [γ-³²P]ATP (50 μM) and the indicated amounts of protein kinase A for 10 min. Panel B, pure human CTP synthetase 1 (1 μg) was incubated with protein kinase A (1 U/ml) and [γ-³²P]ATP (50 μM) for the indicated time intervals. Panel C, protein kinase A (1 U/ml) and [γ-³²P]ATP (50 μM) were incubated with the indicated concentrations of pure human CTP synthetase 1 for 10 min. Panel D, protein kinase A (1 U/ml) and pure human CTP synthetase 1 (1 μg) were incubated with the indicated concentrations of [γ-³²P]ATP for 10 min. Following the phosphorylation reactions, samples were subjected to SDS-PAGE. The SDS polyacrylamide gels were dried and the phosphorylated proteins were subjected to phosphorimaging analysis. The relative amounts of phosphate incorporated into human CTP synthetase 1 were quantified using ImageQuant software. Portions of the images with the phosphorylated human CTP synthetase 1 are shown above each graph. The data are representative of two independent experiments.