Phosphorylation of Saccharomyces cerevisiae CTP Synthetase at Ser424 by Protein Kinases A and C Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway*

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser 424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser 424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent K m values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser 424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser 424 was also a target site for protein kinase C. Phosphorylation of Ser 424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3 H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser 424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.
CTP synthetase is an essential enzyme in all organisms. The essential nature of this enzyme emanates from the fact that the product of its reaction CTP is required for the synthesis of nucleic acids and membrane phospholipids (1). The enzyme catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (2, 3). GTP stimulates the reaction by accelerating the formation of a co-valent glutaminyl enzyme catalytic intermediate (3)(4)(5)(6). In eukaryotic cells, regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (6 -12) and in the synthesis of membrane phospholipids (12)(13)(14). The importance of understanding the regulation of CTP synthetase is further emphasized by the fact that unregulated levels of CTP synthetase activity is a common property of various human cancers (15)(16)(17)(18)(19)(20)(21)(22).
We utilize the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and the impact of this regulation on phospholipid synthesis (Fig. 1). In yeast, CTP synthetase is encoded by the URA7 (10) and URA8 (11) genes. The yeast CTP synthetases (10, 11) contain a conserved glutamine amide transfer domain common to CTP synthetases from other organisms (24 -33). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (34) and is responsible for the majority of the CTP synthesized in vivo (11). Like CTP synthetase from mammalian cells (35), the yeast enzymes are allosterically regulated by their substrates and product CTP (6,34).
The S. cerevisiae URA7-encoded CTP synthetase is also regulated by phosphorylation. In vivo, CTP synthetase is phosphorylated on multiple serine residues (36). In vitro studies have shown that CTP synthetase is a substrate for protein kinase A (37) and for protein kinase C (36,38). In S. cerevisiae, protein kinase A is the principal mediator of signals transmitted through the Ras-cAMP pathway (39,40) whereas protein kinase C is required for the cell cycle (41)(42)(43)(44)(45) and plays a role maintaining cell wall integrity (46). Independently, the phosphorylation of CTP synthetase by protein kinase A (37) and by protein kinase C (36,38) results in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover and decreases enzyme sensitivity to CTP product inhibition.
In this work, we addressed the question of whether the phosphorylation of CTP synthetase by protein kinase A affects the phosphorylation by protein kinase C. Amino acid residue Ser 424 has been identified as the target site for protein kinase A phosphorylation in CTP synthetase (47). Therefore, we utilized a S424A mutant CTP synthetase enzyme for our studies. The S424A mutant enzyme is not phosphorylated in response to the activation of protein kinase A in vivo, and the mutant enzyme is not phosphorylated and stimulated by protein kinase A in vitro (47). This mutant enzyme exhibits lower catalytic activity and greater sensitivity to CTP product inhibition when compared with the wild type enzyme (47). These properties are consistent with the effects that protein kinase A phosphorylation has on the activity of wild type CTP synthetase (37,47). We showed here that the S424A mutation reduced the ability of CTP synthetase to be a substrate for protein kinase C. An explanation for this effect was that Ser 424 was also a target site for protein kinase C phosphorylation. We also showed that cells bearing the S424A mutant CTP synthetase enzyme exhibited a decrease in the synthesis of the membrane phospholipid PC 1 via the CDP-choline pathway.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were purchased from Difco Laboratories. Nucleotides, L-glutamine, phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, pepstatin, histone, casein, choline, phosphocholine, CDP-choline, and bovine serum albumin were purchased from Sigma. PVDF paper was from Amersham Biosciences. Protein kinase C (rat brain) and protein kinase A catalytic subunit (bovine heart) were purchased from Promega. Protein assay reagent, electrophoresis reagents, immunochemical reagents, and protein molecular mass markers were purchased from Bio-Rad. Phosphocellulose filters were purchased from Pierce. Radiochemicals were purchased from PerkinElmer Life Sciences. Scintillation counting supplies and acrylamide solutions were from National Diagnostics. Phospholipids were from Avanti Polar Lipids. Silica Gel 60 thin-layer chromatography plates and cellulose thinlayer glass plates were purchased from EM Science. The peptide SLGRKDSHSA was synthesized and purified commercially by Bio-Synthesis, Inc.
Strain and Growth Conditions-The wild type URA7 and mutant URA7 S424A alleles coding for CTP synthetase were expressed from multicopy (pTP1 and pTP2, respectively) and single copy (pTP3 and pTP4, respectively) plasmids in the ura7 ura8 double mutant strain OK8 (47). Methods for growth and analysis of yeast were performed as described previously (48,49). Yeast cultures were grown in complete synthetic medium minus inositol (50) containing 2% glucose at 30°C. Yeast cell numbers were determined by microscopic examination with a hemacytometer or spectrophotometrically at an absorbance of 600 nm.
Purification of Wild Type and S424A Mutant CTP Synthetases-Cells expressing the wild type and S424A mutant CTP synthetases from multicopy plasmids were used for enzyme purification. The enzymes were purified by the method of Yang et al. (6) with the following modifications. The Sephacryl 300 HR chromatography step was replaced by dialysis and the Superose 6 chromatography step was replaced by Mono Q chromatography. This procedure resulted in the isolation of nearly homogeneous enzyme preparations as evidenced by SDS-PAGE.
Enzyme Assays and Protein Determination-CTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M Ϫ1 cm Ϫ1 , respectively) by following the increase in absorbance at 291 nm on a recording spectrophotometer (3). The standard reaction mixture contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 10 mM 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM GTP, 2 mM ATP, 2 mM UTP, and an appropriate dilution of enzyme protein in a total volume of 0.1 ml. Enzyme assays were performed in triplicate with an average standard deviation of Ϯ 3%. All assays were linear with time and protein concentration. A unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 mol of product/min. Protein concentration was estimated by the method of Bradford (51) using bovine serum albumin as the standard.
Phosphorylation Reactions-Phosphorylation reactions were routinely measured for 10 min at 30°C in a total volume of 40 l. The indicated concentrations of purified wild type and S424A mutant CTP synthetase enzymes or synthetic peptide were phosphorylated with the indicated concentrations of protein kinase C in a reaction mixture that contained 50 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl 2 , 10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375 mM EGTA, 1.7 mM CaCl 2 , 20 M diacylglycerol, 50 M phosphatidylserine, and 50 M [␥-32 P]ATP (5,000 cpm/pmol). CTP synthetase was phosphorylated with protein kinase A (0.2 unit/ml) in a reaction mixture that contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , and 50 M [␥-32 P]ATP (5,000 cpm/pmol). Samples containing 32 P-labeled CTP synthetase were treated with an equal volume of 4ϫ Laemmli sample buffer (52) followed by SDS-PAGE, transfer to PVDF paper, and visualized by phosphorimaging. The extent of phosphorylation was analyzed using ImageQuant software. Phosphorylation signals were in the linear range of detectability. Reactions containing the synthetic peptide were terminated by spotting an aliquot of the reaction mixture onto phosphocellulose filters. The filters were washed with 75 mM phosphoric acid and subjected to scintillation counting. Phosphorylation reactions were performed in triplicate.
Tryptic Digestion and Two-dimensional Peptide Mapping-Pieces of PVDF paper containing 32 P-labeled CTP synthetase were subjected to digestion with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin and two-dimensional peptide mapping analysis as described by MacDonald and Kent (54). Electrophoresis (1% ammonium bicarbonate buffer at 1000 volts for 20 min) and ascending chromatography (n-butyl alcohol/glacial acetic acid/pyridine/water, 10:3:12:15) were performed on cellulose thin-layer glass plates. Dried plates were then subjected to phosphorimaging analysis.
Analysis of Phospholipids-Phospholipids were labeled with 32 P i and [methyl-3 H]choline as described previously (14,55,56). Phospholipids were extracted from cells by the method of Bligh and Dyer (57) as described previously (58). Phospholipids were separated by two-dimensional thin-layer chromatography using silica gel 60 thin-layer chromatography plates. The solvent systems for dimensions one and two were chloroform/methanol/glacial acetic acid (65:25:10, v/v) and chloroform/ methanol/88% formic acid (65:25:10, v/v), respectively (59). 32 P-Labeled phospholipids were visualized by phosphorimaging analysis. The positions of the labeled lipids on chromatography plates were compared with standard phospholipids after exposure to iodine vapor. The amount of each labeled phospholipid was determined by liquid scintillation counting of the corresponding spots on the chromatograms.
Analysis of CDP-choline Pathway Intermediates-Labeling of the CDP-choline pathway intermediates with [methyl-3 H]choline was performed as described by McDonough et al. (14). Choline, phosphocholine, and CDP-choline were isolated from whole cells following lipid extraction (57). The aqueous phase was neutralized and dried in vacuo, and the residue was dissolved in deionized water. Samples were centrifuged for 3 min at 12,000 ϫ g to remove insoluble material. The CDP-choline pathway intermediates were separated by thin-layer chromatography with silica gel 60 plates using the solvent system methanol/0.5% sodium chloride/ammonia (50:50:1) (60). The intermediates were detected on chromatograms by fluorography using EN 3 HANCE and compared with standards. Liquid scintillation counting was used to quantify the amounts of the intermediates.
Data Analyses-Kinetic data were analyzed according to the Michaelis-Menten equation using the EZ-FIT enzyme kinetic modelfitting program (61). Statistical analyses were performed with SigmaPlot 5.0 software.

Effect of the S424A Mutation on the Phosphorylation and Stimulation of CTP Synthetase by Protein Kinase C-The
URA7-encoded CTP synthetase is phosphorylated by protein kinase A (37) and by protein kinase C (36,38). Residue Ser 424 in the enzyme has been identified as the protein kinase A target site (47). We questioned whether phosphorylation at Ser 424 affects phosphorylation of the enzyme by protein kinase C. Accordingly, we analyzed the phosphorylation of the S424A mutant CTP synthetase by protein kinase C. Protein kinase C was incubated with [␥-32 P]ATP and various concentrations of the purified S424A mutant and wild type CTP synthetase enzymes. After the phosphorylation reactions, samples were subjected to SDS-PAGE and transferred to PVDF paper, followed by phosphorimaging analysis. Protein kinase C activity was dependent on the concentration of both the wild type and mutant forms of CTP synthetase (Fig. 2). The S424A mutation caused a decrease in enzyme phosphorylation at each CTP synthetase concentration (Fig. 2). The apparent K m value for the S424A mutant enzyme (50 g/ml) was 20-fold higher than that of the wild type enzyme (2.5 g/ml). The effect of the S424A mutation on the dependence of protein kinase C activity on ATP concentration was also examined (Fig. 3). The extent of CTP synthetase phosphorylation at each ATP concentration was reduced for the S424A mutant enzyme when compared with the wild type enzyme. The apparent K m value for the mutant enzyme (25 M) was 2-fold higher when compared with the wild type enzyme (K m ϭ 12.5 M). The phosphorylation reactions using the S424A mutant CTP synthetase as a substrate were performed for different time intervals and with various concentrations of protein kinase C. The phosphorylation of the S424A mutant CTP synthetase by protein kinase C was time-dependent ( Fig. 4) and dose-dependent (Fig. 5), but the rate and extent of phosphorylation was reduced by about 50% when compared with the wild type enzyme.
The effect of the S424A mutation on the stimulation of CTP synthetase activity by protein kinase C was examined. The purified S424A mutant and wild type CTP synthetase enzymes were phosphorylated with various concentrations of protein kinase C for 10 min. Following the phosphorylation reactions, samples were assayed for CTP synthetase activity using subsaturating concentrations of UTP and ATP. These assay conditions were used to accentuate the effect of phosphorylation on the stimulation of CTP synthetase activity (36,38). As described previously (36,38), protein kinase C phosphorylation of the wild type enzyme resulted in a dose-dependent stimulation of CTP synthetase activity (Fig. 6). The activity of the S424A mutant enzyme was also stimulated by protein kinase C phosphorylation; however, the extent of stimulation was much reduced ( Fig. 6). At the highest protein kinase C concentration, the stimulation of CTP synthetase activity was reduced by 80%.
A CTP Synthetase Synthetic Peptide Containing Amino Acid Residue Ser 424 Is a Substrate for Protein Kinase C-The CTP synthetase peptide SLGRKDS 424 HSA, which contains amino acid residue Ser 424 , was synthesized based on the protein sequence of CTP synthetase. This synthetic peptide has been shown to be a substrate for protein kinase A (47). We examined whether this peptide could also serve as a substrate for protein kinase C. Protein kinase C catalyzed the phosphorylation of the peptide in a dose-dependent manner (Fig. 7). Analysis of the data yielded a K m value of 1 mM. The K m value for this peptide for protein kinase A phosphorylation is 30 M (47). Thus, based on this assay, Ser 424 was a better target site for protein kinase A phosphorylation when compared with protein kinase C.

Effect of the S424A Mutation on the Phosphopeptide Map of CTP Synthetase Phosphorylated by Protein Kinase C-
We examined the effect of the S424A mutation on the phosphorylation of CTP synthetase by protein kinase C using phosphopeptide mapping analysis. The purified S424A mutant and wild type CTP synthetase enzymes were phosphorylated with protein kinase C and 32 P-labeled ATP followed by SDS-PAGE and transfer to PVDF paper. Papers containing the phosphorylated mutant and wild type enzymes were digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin and subjected to two-dimensional phosphopeptide mapping analysis. About equal amounts of the 32 P-labeled peptides derived from the wild type and mutant proteins were applied to the cellulose plates for comparison of the phosphopeptide maps. Five major phosphopeptides (labeled 1 through 5) were present in the phosphopeptide map of the wild type CTP synthetase enzyme (Fig. 8A). Quantification of the 32 P label showed that phosphopeptides 1 through 5 accounted for 26, 17, 24, 23, and 10%, respectively, of the 32 P-label incorporated into the wild type enzyme. For the S424A mutant CTP synthetase, phosphopeptides 1 through 4 were detected like the wild type enzyme, but phosphopeptide 5 was not detected (Fig. 8B). The loss of phosphopeptide 5 in the S424A mutant enzyme indicated that Ser 424 was contained in this phosphopeptide. Phosphopeptide mapping analysis of the wild type CTP synthetase phosphorylated by protein kinase A showed one major phosphopeptide at the same position of phosphopeptide 5 (Fig. 8C). Protein kinase A does not catalyze the incorporation of the ␥-phosphate of 32 P-labeled ATP into the purified S424A mutant CTP synthetase (47). Therefore, these results indicated that Ser 424 was the phosphorylation site for both protein kinase A and protein kinase C.

Effect of the S424A Mutation in CTP Synthetase on Phospholipid Composition and on the Composition of the CDP-choline
Pathway Intermediates-The effect of the S424A mutation on phospholipid composition was examined. The rationale for this experiment was that CTP, product of the CTP synthetase reaction, is required for the synthesis of PC, the most abundant membrane phospholipid in S. cerevisiae (62)(63)(64)(65). PC is synthesized from CTP by two different pathways (62-65) (Fig. 1). In one pathway, PC is synthesized from CTP via CDP-choline, whereas in the other pathway, PC is synthesized from CTP via CDP-diacylglycerol (62-65) (Fig. 1). The synthesis of PC from CDP-choline is direct whereas the synthesis from CDP-diacylglycerol is indirect (62-65) (Fig. 1).
Cells bearing the wild type and S424A mutant CTP synthetase enzymes expressed from the single copy plasmids were grown in complete synthetic medium without inositol and choline to exclude regulatory effects that these precursors have on phospholipid synthesis (62,64,66). As described previously (47), immunoblot analysis showed that the S424A mutation did not affect the levels of the CTP synthetase protein. When grown in the absence of choline, wild type cells synthesize PC by both the CDP-diacylglycerol-dependent and CDP-cholinedependent pathways (12,14,(67)(68)(69). The choline required for the CDP-choline pathway is derived from the phospholipase D-mediated turnover of PC that is synthesized by way of the CDP-diacylglycerol pathway (69,70). The composition of phospholipids was examined by labeling cells to steady state with both 32 P i and [methyl-3 H]choline. 32 P i is incorporated into phospholipids that are synthesized by way of the CDP-diacylglycerol-dependent and CDP-choline-dependent pathways (12,14). Labeled choline is only incorporated into PC that is synthesized by way of the CDP-choline-dependent pathway (12,14,71). The concentration of choline added to the growth medium from the radioactive label was 0.1 M, a concentration too low to affect the rate of synthesis of PC by the CDP-choline pathway (71).
The major effect of the S424A mutation on phospholipid composition was a 24% decrease for PC (Fig. 9A). This was PC synthesized by both the CDP-diacylglycerol-dependent and CDP-choline-dependent pathways. The amounts of the other major membrane phospholipids were not significantly affected by the S424A mutation (Fig. 9A). As indicated in Fig. 9B, [methyl-3 H]choline was incorporated into PC by way of the CDP-choline pathway (12,14). Plotting the data as the ratio of the cpm of [methyl-3 H]choline incorporated into PC to the cpm of 32 P i incorporated into PC was used to determine whether the S424A mutation affected the pathways by which cells synthesized PC (12,14). Cells carrying the S424A mutation in CTP synthetase exhibited a 46% decrease in the ratio of 3 H/ 32 P incorporated into PC when compared with the control cells (Fig. 9B). These data indicated that the S424A mutation caused a decrease in the utilization of the CDP-choline-dependent pathway for PC synthesis.
The intermediates for the synthesis of PC by the CDP-cholinedependent pathway are phosphocholine and CDP-choline (72) (Fig. 1). The effect of the S424A mutation on the composition of these intermediates was examined by labeling cells with [methyl-3 H]choline. The amounts of phosphocholine and CDP-choline were reduced by 48 and 32%, respectively, in cells carrying the S424A mutation in CTP synthetase when compared with cells carrying the wild type enzyme (Fig. 10). The reduction in these intermediates was consistent with the conclusion that the S424A mutation caused a decrease in utilization of the CDPcholine pathway for PC synthesis. DISCUSSION CTP synthetase is an essential enzyme in S. cerevisiae (10,11), because it provides the CTP that is required for the synthesis of nucleic acids and membrane phospholipids (1). Proper regulation of CTP synthetase in human cells is underscored by the fact that a number of cancers are characterized by unregulated levels of CTP synthetase activity (15)(16)(17)(18)(19)(20)(21)(22). Covalent modification by phosphorylation is a major mechanism by which the activity of an enzyme may be regulated (73,74). Previous studies have shown that the S. cerevisiae URA7-encoded CTP synthetase is phosphorylated by protein kinase A (37) and by protein kinase C (36,38). These protein kinases have a major impact on the growth and metabolism of yeast (39,(41)(42)(43)(44)(45)(46). The phosphorylation of CTP synthetase by protein kinases A and C results in the stimulation of CTP synthetase activity. Interest- Purified wild type (WT) and S424A mutant CTP synthetases (0.8 g each) were incubated with protein kinase C (PKC) and [␥-32 P]ATP for 10 min (panels A and B, respectively). Panel C, purified wild type CTP synthetase (0.8 g) was incubated with protein kinase A (PKA) and [␥-32 P]ATP for 10 min. Following the phosphorylation reactions, samples were subjected to SDS-PAGE followed by transfer to PVDF paper. Paper slices containing the 32 P-labeled proteins were digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-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 major phosphopeptides derived from the phosphorylation by protein kinase C are labeled 1 through 5 in panels A and B. The position of the phosphopeptide that was absent in the S424A mutant CTP synthetase and was present in wild type enzyme phosphorylated by protein kinase C and by protein kinase A is indicated by the ellipse in the figure. The data shown are representative of two independent experiments. ingly, the combined effect of protein kinase A and protein kinase C phosphorylation on wild type CTP synthetase activity is only slightly greater than that caused by either one of the protein kinases alone (37). In the present study, we questioned whether the phosphorylation of CTP synthetase by protein kinase A affected the phosphorylation of the enzyme by protein kinase C. Protein kinase A phosphorylates CTP synthetase at only one site, namely Ser 424 (47), and thus, our studies were facilitated by the use of the S424A mutant CTP synthetase enzyme.
The S424A mutation resulted in about a 50% reduction in the phosphorylation of CTP synthetase by protein kinase C and an 80% reduction in the stimulatory effect that protein kinase C had on CTP synthetase activity. These effects could be attributed to a decrease in the rate of phosphorylation. In addition, the S424A mutation caused increases in the apparent K m values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. Because protein kinase C phosphorylated CTP synthetase on multiple sites, the meaning of these effects is not straightforward. The decrease in the ability of the S424A mutant CTP synthetase to be phosphorylated by protein kinase C was not because of any major effects on the overall structure of the enzyme. The S424A mutation does not affect the behavior of CTP synthetase during purification, the nucleotide-dependent tetramerization of the purified enzyme, or the stability of the enzyme to temperature (47). Moreover, the S424A mutant enzyme is functional in vivo as evidenced by the suppression of the lethal phenotype of the ura7 ura8 double mutant (47).
An explanation for the decreased ability of the S424A mutant enzyme to be phosphorylated by protein kinase C was that Ser 424 was a target site for both protein kinase A and protein kinase C. Indeed, the phosphopeptide that was present in the phosphopeptide map of the protein kinase C-phosphorylated wild type enzyme but absent from the map of the protein kinase C-phosphorylated S424A mutant enzyme was the same phosphopeptide derived from the wild type enzyme phosphorylated by protein kinase A. Phosphorylation of Ser 424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. In addition, the CTP synthetase synthetic peptide containing Ser 424 that is a substrate for protein kinase A (47) was also a substrate for protein kinase C. That Ser 424 was phosphorylated by protein kinases A and C provides a plausible explanation why the combined effect of these phosphorylations on CTP synthetase activity is small (37). Overall, the data support the hypothesis that phosphorylation of Ser 424 by protein kinase A (or by protein kinase C) was required for maximum phosphorylation and stimulation of the enzyme by protein kinase C.
We addressed the physiological relevance of the phosphorylation of Ser 424 with respect to phospholipid synthesis using cells carrying the S424A mutant CTP synthetase. The in vivo labeling experiments showed that the S424A mutation resulted in a decrease for PC. The decrease in PC content could be attributed to a decrease in the utilization of the CDP-choline pathway, because the ratio of the cpm of [methyl-3 H]choline incorporated into PC to the cpm of 32 P i incorporated into PC was reduced in cells with the S424A mutation. This conclusion was further supported by the reduced amounts of phosphocholine and CDP-choline in cells with the S424A mutant enzyme. The reason for the decrease in phosphocholine was unclear. One would expect that the level of phosphocholine would accumulate, because CTP is the direct precursor of CDP-choline in the pathway (Fig. 1). The reduction for phosphocholine may be attributed to the down-regulation of the choline kinase enzyme in response to the mutation in CTP synthetase. The reduced amount of PC in cells with the S424A mutation did not appear to be from a reduction in utilization of the CDP-diacylglyceroldependent pathway. The amounts of the phospholipids (e.g. phosphatidylserine and phosphatidylethanolamine) in this pathway were not significantly affected by the S424A mutation. Thus, the phosphorylation of CTP synthetase at Ser 424 , whether it is mediated by protein kinase A and/or by protein kinase C, plays a role in the regulation of PC synthesis via the CDP-choline pathway. Differentiating between which protein kinase phosphorylates Ser 424 and regulates phospholipid synthesis in vivo will require additional studies.
Protein kinase C phosphorylated CTP synthetase at sites other than Ser 424 . Recent studies have shown that Ser 36 , Ser 330 , Ser 354 , and Ser 454 are also target sites for protein kinase C phosphorylation (23). Whereas Ser 424 was phosphorylated by protein kinases A and C, Ser 36 , Ser 330 , Ser 354 , and Ser 454 are not phosphorylated by protein kinase A. Protein kinase A only phosphorylates Ser 424 (47). Phosphorylation of CTP synthetase by protein kinase C is not straightforward. For example, phosphorylation at Ser 330 affects the phosphorylation of CTP synthetase at other protein kinase C sites (23). Moreover, phosphorylation at the different protein kinase C target sites has different effects on CTP synthetase activity (23). It is unknown whether the phosphorylation of CTP synthetase at Ser 36 , Ser 330 , Ser 354 , and Ser 454 by protein kinase C affects phosphorylation by protein kinase A. Studies are in progress to address this question. Clearly, the phosphorylation and regulation of CTP synthetase is complex. This complex regulation is likely to represent a mechanism by which various signal transduction pathways mediate CTP synthetase activity and its roles in cell physiology.