Regulation of yeast CTP synthetase activity by protein kinase C.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent V max of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is an allosterically regulated enzyme that is essential for the growth and metabolism of cells. The product of its reaction, CTP, is required for the synthesis of RNA, DNA, phospholipids, and sialoglycoproteins (1). CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (2, 3). GTP activates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (3,4). Genes encoding for CTP synthetase have been isolated from Escherichia coli (5), Chlamydia trachomatis (6), Bacillus subtilis (7), Saccharomyces cerevisiae (8,9), and human cells (10). The deduced amino acid sequences of the cloned CTP synthetase genes have a relatively high degree of identity, including a conserved glutamine amide transfer domain characteristic of glutamine amidotransferases (5)(6)(7)(8)(9)(10).
A characteristic common to the pure CTP synthetases is the inhibition of their activities by the product CTP (3,(12)(13)(14). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (12)(13)(14). Regulation of CTP synthetase activity by CTP inhibition plays an important role in vivo. For example, mutant mammalian cell lines with CTP synthetase activity insensitive to CTP inhibition exhibit abnormally high intracellular levels of CTP and dCTP (20,21), resistance to nucleotide analog drugs used in cancer chemotherapy (22)(23)(24)(25), and an increased rate of spontaneous mutations (23,25,26). In addition, elevated CTP synthetase activity is a common property of leukemic cells (27) and rapidly growing tumors found in liver (28), colon (29), and lung (30).
In S. cerevisiae, CTP synthetase is encoded by the URA7 and URA8 genes (8,9). Comparison of the nucleotide and deduced amino acid sequences of the open reading frames of the URA7 and URA8 genes show 70 and 78% identity, respectively (8,9). Biochemical characterization of the purified enzymes (12,13) and phenotypic analysis of ura7 and ura8 mutants (9) have shown that the two CTP synthetases are not functionally identical. Our studies on the pure URA7-encoded enzyme have revealed that CTP synthetase is also regulated by protein kinase C phosphorylation (31). Protein kinase C is a transducer of lipid second messengers (32)(33)(34) and plays a central role in the regulation of a host of cellular functions including cell growth and proliferation (35)(36)(37). Rat brain protein kinase C phosphorylates the yeast CTP synthetase in vitro on serine and threonine residues, which results in an activation of CTP synthetase activity (31). In this study we demonstrated that CTP synthetase was a substrate for the S. cerevisiae protein kinase C (Pkc1p). Our data also demonstrated that this phosphorylation regulated CTP synthetase activity by changing the kinetic properties of the enzyme and its sensitivity to inhibition by CTP.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Nucleotides, L-glutamine, phenylmethanesulfonyl fluoride, benzamide, aprotinin, leupeptin, pepstatin, nitrocellulose paper, phosphoamino acids, and bovine serum albumin were purchased from Sigma. Phosphatidylserine and diacylglycerol were purchased from Avanti Polar Lipids. Radiochemicals were purchased from DuPont NEN. Scintillation counting supplies were purchased from National Diagnostics. Protein assay reagent, molecular mass standards for SDS-polyacrylamide gel electrophoresis, and electrophoresis reagents were purchased from Bio-Rad. IgG-Sepharose was purchased from Pharmacia Biotech Inc. DE53 (DEAE-cellulose) was purchased from Whatman, Inc. Cellulose thin layer sheets were from EM Science. Phosphocellulose papers for protein kinase C assays were obtained from Pierce.
Strains and Growth Conditions-CTP synthetase was purified from strain OK8 (MAT␣ leu2 trp1 ura3 ura7⌬::TRP1 ura8) bearing the * This work was supported by United States Public Health Service Grant GM-50679 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. This is New Jersey Agricultural Experiment Station Publication D-10581-1-96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. multicopy plasmid pFL44S-URA7 (8,9). Plasmid pFL44S-URA7 directs a 10-fold overexpression of CTP synthetase (12). Cells were grown in complete synthetic medium (38) without uracil. Strain DL101 (MATa leu2-3, 112 ura3-52 trp1-1 his4 can1 r ) was used as a representative wild-type for Pkc1p expression. Strain DL376 (MATa leu2-3, 112 ura3-52 trp1-1 his4 can1 r pkc1⌬::LEU2) is a null allele pkc1 mutant defective in the expression of Pkc1p (39). Strain DL105 is DL376 bearing the PKC1 gene (40) on the multicopy plasmid YEp352. Strain DL868 is DL376 bearing the plasmid pGY82. Plasmid pGY82 is a multicopy plasmid carrying a PKC1-ZZ fusion gene (41). This fusion gene carries two (ZZ) repeats of the 60-amino acid IgG-binding domain of Staphylococcus aureus protein A (41). The plasmids in strains DL105 and DL868 direct the overexpression of Pkc1p. 1 DL101, DL376, and DL105 were grown in low phosphate medium (42) containing 1 M sorbitol (39) and used to examine the effect of Pkc1p expression on the phosphorylation of CTP synthetase. DL868 cells were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) and used to purify ZZ-tagged Pkc1p. Cell numbers were determined by microscopic examination with a hemacytometer or by absorbance at 660 nm. All cells were grown at 30°C. Strains DL101, DL376, DL105, and DL868 were obtained from David E. Levin (The Johns Hopkins University, Baltimore, MD).
Purification of CTP Synthetase-URA7-encoded CTP synthetase was purified to homogeneity by ammonium sulfate fractionation of the cytosol followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel Blue, and Superose 6 as described by Yang et al. (12). The specific activity of the pure enzyme was 2.5 mol/min/mg.
Purification of ZZ-tagged Pkc1p-ZZ-tagged Pkc1p was purified by DE53 chromatography and IgG-Sepharose chromatography as described by Antonsson et al. (41) with minor modifications. These modifications were the disruption of cells with glass beads (44) and the elution of ZZ-tagged Pkc1p from the IgG-Sepharose column with 0.1 M glycine-HCl (pH 3.0). Fractions from the IgG-Sepharose column were neutralized with 0.5 M Tris-HCl buffer (pH 8.0). Pkc1p was stored in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM EGTA, and 50% glycerol at Ϫ20°C. The properties of our preparation of Pkc1p were similar to those previously described (41,45). The activity of Pkc1p reported in the present study was based on using the Bck1-Ser939 peptide (LKRGNSKRVVSSTSAAD) as a substrate (45).
Phosphorylation of CTP Synthetase with Pkc1p-Phosphorylation reactions were measured for 10 min at 30°C in a total volume of 40 l. CTP synthetase (0.8 g) was incubated with 50 mM Tris-HCl (pH 8.0), 50 M [␥-32 P]ATP (4 Ci/nmol), 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 0.3 nmol/min/ml Pkc1p. At the end of the phosphorylation reactions, samples were treated with 2 ϫ Laemmli's sample buffer (46) followed by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. The density of the CTP synthetase bands on autoradiograms was determined by scanning densitometry. Phosphorylation reactions were also performed with unlabeled ATP. Following incubation with Pkc1p, the reaction mixtures were diluted 5-fold, and CTP synthetase activity was measured spectrophotometrically as described below.
Phosphoamino Acid Analysis-CTP synthetase was phosphorylated with Pkc1p and [␥-32 P]ATP and then subjected to SDS-polyacrylamide gel electrophoresis. Gel slices containing 32 P-labeled CTP synthetase were treated with 50 mM ammonium bicarbonate (pH 8.0) and 0.1% SDS at 37°C for 30 h to elute the enzyme. Bovine serum albumin (50 g) was added to the samples as carrier protein, and trichloroacetic acid was added to a final concentration of 20%. After incubation for 30 min at 4°C, protein precipitates were collected by centrifugation. Proteins were washed three times with cold acetone and dried in vacuo. Samples were then subjected to acid hydrolysis with 6 N HCl at 100°C for 4 h. The hydrolysates were dried in vacuo and applied to 0.1-mm cellulose thin-layer chromatography plates with 2.5 g of phosphoserine, 2.5 g of phosphothreonine, and 5 g of phosphotyrosine as carrier phosphoamino acids in water. Phosphoamino acids were separated by twodimensional electrophoresis (48). Following electrophoresis, the plates were dried, sprayed with 0.25% ninhydrin in acetone to visualize carrier phosphoamino acids, and subjected to phosphor imaging analysis.
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 0.2 ml. Pkc1p activity was measured for 10 min at 30°C by following the phosphorylation of the Bck1-Ser939 peptide with [␥-32 P]ATP (3,000 -4,000 cpm/pmol) as described previously (45). The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.2 mg/ml Bck1-Ser939 peptide, 50 M ATP, 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 an appropriate dilution of enzyme protein in a total volume of 50 l. Reactions were terminated by loading samples onto phosphocellulose filter paper. The filters were washed with 75 mM phosphoric acid and subjected to scintillation counting. Enzyme assays were performed in triplicate with an average standard deviation of Ϯ3%. 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 unless otherwise indicated. Protein was determined by the method of Bradford (49) using bovine serum albumin as the standard.
Analysis of Kinetic Data-Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT Enzyme Kinetic Model Fitting Program (50). EZ-FIT uses the Nelder-Mead simplex and Marquardt/Nash nonlinear regression algorithms sequentially and tests for the best fit of the data among different kinetic models.

Phosphorylation of CTP Synthetase by
Pkc1p-Our studies were performed with protein kinase C (Pkc1p) from S. cerevisiae (41,45). Pkc1p is the product of the PKC1 gene, which is required for progression through the cell cycle (40). Pkc1p has a substrate specificity similar to that of the ␣, ␤, and ␥ isoforms of mammalian protein kinase C (41,45). To examine if CTP synthetase phosphorylation was mediated by Pkc1p in vivo, the extent of enzyme phosphorylation was measured in cells that expressed different levels of the PKC1 gene. Cells were incubated with 32 P i to detect phosphorylated CTP synthetase and [ 14 C(U)]L-amino acids to normalize for the amount of CTP synthetase isolated. CTP synthetase was immunoprecipitated from wild-type cells, pkc1 mutant cells, and cells that overexpressed Pkc1p. The amount of each label incorporated into CTP synthetase was determined. The data shown in Fig. 1 are plotted as the ratio of the cpm of 32 P incorporated into CTP synthetase to the cpm of 14 C incorporated into CTP synthetase. If Pkc1p mediated phosphorylation of CTP synthetase in vivo, then the ratio of the labels found in CTP synthetase would be expected to change in response to Pkc1p levels. Indeed, this ratio was reduced in pkc1 mutant cells and elevated in cells that overexpressed Pkc1p when compared with cells that were wild-type for Pkc1p expression (Fig. 1). However, the fact that CTP synthetase was still phosphorylated to some extent in pkc1 mutant cells suggested that the enzyme was a substrate for other protein kinase(s) in vivo.
We examined if purified CTP synthetase was a substrate for Pkc1p in vitro. These studies were performed with a purified preparation of a ZZ-tagged Pkc1p (45). The ZZ-tag facilitates the purification of Pkc1p but does not alter the biochemical properties of the enzyme (41,45). We examined the phosphorylation of CTP synthetase with Pkc1p under the conditions used to phosphorylate the enzyme with rat brain protein kinase C (31). Pkc1p catalyzed the incorporation of the ␥-phosphate of 32 P-labeled ATP into CTP synthetase ( Fig. 2A, lane 2). The omission of the protein kinase C cofactors calcium, diacylglycerol, and phosphatidylserine from the phosphorylation reaction resulted in a 70% decrease in the phosphorylation of CTP synthetase (Fig. 2A, lane 4). 2 CTP synthetase was phosphorylated on serine and threonine residues (Fig. 2B), and the stoichiometry of phosphorylation was 0.4 mol of phosphate/mol enzyme.
The effect of phosphorylation on CTP synthetase activity was measured. Phosphorylation of the enzyme resulted in a dosedependent activation (3-fold) of CTP synthetase activity (Fig.  3). Maximum activation of activity occurred when the enzyme was measured with subsaturating concentrations of UTP and ATP (Fig. 3). When CTP synthetase activity was measured with saturating concentrations of UTP and ATP, Pkc1p did not have a significant effect on CTP synthetase activity (Fig. 3). Overall, these results were similar to those reported for rat brain protein kinase C phosphorylation of yeast CTP synthetase (31). This indicated that the yeast Pkc1p and the rat brain protein kinase C phosphorylated and activated yeast CTP synthetase in a similar manner.
Effect of Phosphorylation on the Kinetics of CTP Synthetase Activity with Respect to UTP and ATP-Kinetic analyses were performed to further characterize the effects of phosphorylation of CTP synthetase on its activity in vitro. The CTP synthetase that we purified is phosphorylated to some extent (31). We were unable to perform kinetic studies on the dephosphorylated form of CTP synthetase, however, because alkaline phosphatase treatment of the purified enzyme results in the loss of its activity (31). Previous studies have shown that yeast CTP synthetase exhibits positive cooperative kinetics toward UTP and ATP (12). Therefore, we investigated whether phos-phorylation of the enzyme influenced these kinetic properties. These experiments were performed with saturating concentrations of glutamine, GTP, and magnesium ions. We first examined the effect of phosphorylation on the dependence of CTP synthetase activity on UTP using subsaturating and saturating concentrations of ATP. At the subsaturating ATP concentration, the phosphorylation of CTP synthetase altered the apparent V max of the reaction and the positive cooperative kinetic behavior of the enzyme (Fig. 4A and Table I). Under these conditions, the apparent V max of the phosphorylated enzyme was 2.75-fold greater than the apparent V max of the native enzyme. Phosphorylation of CTP synthetase resulted in the loss of the enzyme's positive cooperative kinetic behavior toward UTP when compared with the native enzyme (n ϭ 1.1 and 1.4, respectively). At a saturating ATP concentration, phosphorylation had a small effect on the apparent V max of the reaction FIG. 3. Activation of CTP synthetase activity by Pkc1p. CTP synthetase was incubated with the indicated amounts (U ϭ nmol/min) of Pkc1p for 10 min. Following the incubations, samples were diluted 5-fold, and CTP synthetase activity was measured as described in the text using subsaturating (q) concentrations of ATP (0.5 mM) and UTP (0.2 mM) and saturating (E) concentrations of ATP (2 mM) and UTP (2 mM). The concentrations of glutamine, GTP, and MgCl 2 were maintained at 2, 0.1, and 10 mM, respectively.
( Fig. 4B and Table I). Phosphorylation of CTP synthetase did not affect the apparent K m values for UTP whether activity was measured with subsaturating or saturating concentrations of ATP ( Fig. 4 and Table I).
We next examined the effect of phosphorylation on the dependence of CTP synthetase activity on ATP using subsaturating and saturating concentrations of UTP. At the subsaturating UTP concentration, the phosphorylation of CTP synthetase altered the apparent K m of the reaction and the positive cooperative kinetic behavior of the enzyme (Fig. 5A and Table I). Under these conditions, the apparent K m value for ATP of the phosphorylated enzyme was 2.5-fold lower than the apparent K m value for the native enzyme. Phosphorylated CTP synthetase exhibited a decrease in its positive cooperative kinetic behavior toward ATP when compared with the native enzyme (n ϭ 1.4 and 2.5, respectively). At a saturating UTP concentration, phosphorylation of CTP synthetase resulted in a 5.3-fold reduction in its apparent K m value for ATP and a loss of its positive cooperative kinetic behavior with respect to ATP (Fig.  5B and Table I). Phosphorylation of CTP synthetase resulted in a modest increase in the apparent V max of the reaction when activity was measured with subsaturating or saturating concentrations of UTP ( Fig. 5 and Table I).
Effect of Phosphorylation on the Kinetics of CTP Synthetase Activity with Respect to Glutamine and GTP-GTP stimulates yeast CTP synthetase activity by increasing the V max of the reaction and decreasing the K m for glutamine (12). The enzyme also exhibits negative cooperative kinetics toward glutamine (12). We examined if phosphorylation influenced these kinetic properties. These experiments were performed with saturating concentrations of UTP, ATP, and magnesium ions. Phosphorylation of the enzyme did not have a significant effect on the apparent V max and K m values of the enzyme with respect to glutamine when measured in the absence (Fig. 6A) or the presence (Fig. 6B) of GTP (Table I). However, the phosphorylation of CTP synthetase abolished the negative cooperative behavior of the enzyme with respect to glutamine ( Table I). The effect of phosphorylation on the activation of CTP synthetase activity by GTP was examined using saturating concentrations of all substrates (Fig. 7). Phosphorylation of CTP synthetase resulted in a small decrease in the apparent activation constant (K a ) for GTP (Table I).
Effect of Phosphorylation on the Inhibition of CTP Synthetase Activity by CTP-We questioned what effect phosphorylation of CTP synthetase had on the inhibition of CTP synthetase activity by CTP. The phosphorylated and native forms of CTP synthetase were inhibited by CTP in a dose-dependent manner (Fig. 8). When enzyme activity was measured using 0.5 mM ATP, phosphorylated CTP synthetase was less sensitive to CTP inhibition when compared with the native enzyme ( Fig. 8A and Table I). On the other hand, when CTP synthetase activity was measured using 1 mM ATP, the sensitivities of both enzyme FIG. 4. Effect of phosphorylation on the kinetics of CTP synthetase activity with respect to UTP. Phosphorylated (q) and native (E) CTP synthetase activities were measured as a function of the concentration of UTP using 0.5 (A) and 2 mM ATP (B). The concentrations of glutamine, GTP, and MgCl 2 were maintained at 2, 0.1, and 10 mM, respectively. forms to CTP were similar ( Fig. 8B and Table I).
As described previously (12), the presence of CTP in the assay for the native enzyme caused an increase in the positive cooperativity toward UTP, an increase in the apparent K m for UTP, and a decrease in the apparent V max (Fig. 9). We questioned what effect phosphorylation had on the dependence of CTP synthetase activity on UTP in the presence of CTP. In these experiments the concentration of ATP was held constant at 1 mM, and the other substrates in the reaction were saturating. The apparent V max value of the phosphorylated enzyme measured in the presence of CTP was greater than the apparent V max value of the native enzyme measured in the presence of CTP ( Fig. 9 and Table I). However, enzyme phosphorylation did not have a significant effect on the cooperative kinetic behavior of the enzyme toward UTP nor the apparent K m value for UTP ( Fig. 9 and Table I).

DISCUSSION
Phosphorylation/dephosphorylation is a major mechanism by which the activity of an enzyme is regulated (51,52). The aim of this work was to examine the regulation of yeast CTP synthetase activity by protein kinase C. We demonstrated that purified CTP synthetase was a substrate for yeast Pkc1p and that Pkc1p mediated the phosphorylation of the enzyme in vivo. This does not, however, demonstrate that CTP synthetase is a substrate for Pkc1p in vivo. An alternate explanation could be that Pkc1p modulates the activity of another kinase that is responsible for phosphorylating CTP synthetase. We do know on the basis of our labeling experiments with pkc1 mutant cells that another kinase phosphorylated the enzyme in vivo.
Phosphorylation of the enzyme in vitro resulted in the acti-vation of CTP synthetase activity. This regulation of activity involved changes in the kinetic properties of the enzyme. Pkc1p phosphorylation of CTP synthetase activated the enzyme by increasing the apparent V max of the reaction. The change in the apparent V max was most evident when the kinetics of the enzyme toward UTP was measured using a subsaturating concentration of ATP. The phosphorylated enzyme also showed a greater affinity for ATP when compared with the native enzyme. This was reflected in the apparent K m values for ATP. Another striking effect of Pkc1p phosphorylation of CTP synthetase was the decrease in the positive cooperativity toward UTP and ATP. This was most evident when activity was measured with respect to ATP.
Pkc1p phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. This indicated that phosphorylation did not affect the reaction involving the formation of the glutaminyl enzyme intermediate nor the role GTP plays as an enzyme activator (3,4). Phosphorylated CTP synthetase was less sensitive to inhibition by CTP when activity was measured with a subsaturating concentration of ATP. However, enzyme phosphorylation did not affect the mechanism by which CTP inhibited CTP synthetase activity. The cooperative kinetic behavior toward UTP and the apparent K m value for UTP were not significantly affected by Pkc1p phosphorylation of the enzyme. The only kinetic parameter affected by phosphorylation was the apparent V max .
CTP synthetase is one of several allosteric enzymes whose kinetic properties are changed by phosphorylation. For example, phosphorylation changes the kinetic behavior of glycogen phosphorylase (53,54) and phosphofructokinase (55) with respect to their substrates, activators, and inhibitors. Moreover, these changes in kinetic behavior play a role in the regulation of glycogen phosphorylase and phosphofructokinase activities in vivo (1).
How could the phosphorylation of CTP synthetase by Pkc1p affect activity in vivo? The steady-state cellular concentrations of UTP (0.75 mM) and ATP (2.3 mM) are saturating for CTP synthetase activity (8,12). Under these conditions Pkc1p phosphorylation of CTP synthetase would not be expected to affect activity in vivo. At the same time, enzyme phosphorylation would not affect the regulation of CTP synthetase activity by CTP inhibition. On the other hand, if the cellular concentrations of UTP and ATP were to decrease to subsaturating concentrations for the enzyme, Pkc1p phosphorylation would affect CTP synthetase activity. For example, the cellular concentration of ATP can vary between 0.57 to 2.3 mM depending on growth conditions (56). These concentrations fall within the range of the subsaturating to saturating ATP concentrations for CTP synthetase activity (12). It was the subsaturating ATP concentrations that had the major effect on the kinetic properties of the phosphorylated enzyme. In addition, CTP synthetase activity was less sensitive to product inhibition by CTP when ATP levels were subsaturating. The activation of CTP synthetase activity by Pkc1p phosphorylation may be a mechanism by which the cell regulates CTP synthesis when cellular ATP levels are limiting. At the present time it is not known what role phosphorylation plays in the function of CTP synthetase under different growth conditions. Future studies will address this question.
Our interest in CTP synthetase originates from the role CTP plays in the regulation of the pathways by which phosphatidylcholine is synthesized (57). Phosphatidylcholine is the essential end product of phospholipid synthesis and the major phospholipid found in S. cerevisiae (58,59). CTP is required for the synthesis of phosphatidylcholine through the CDP-cholineand CDP-diacylglycerol-based pathways (58,59). In mammalian cells, phosphatidylcholine plays a major role in lipid signal transduction pathways (60). The diacylglycerol derived from the receptor-mediated hydrolysis of phosphatidylcholine is responsible for the sustained activation of protein kinase C (60). The phosphorylation and activation of CTP synthetase by Pkc1p in yeast may represent a mechanism by which lipid signal transduction pathways are coordinately regulated to CTP synthesis and cell growth.