Phosphorylation of CTP Synthetase on Ser 36 , Ser 330 , Ser 354 , and Ser 454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae *

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinase C. We examined the hypothesis that Ser, Ser, Ser, and Ser, contained in a protein kinase C sequence motif in CTP synthetase, were target sites for the kinase. Synthetic peptides containing a phosphorylation motif at these serine residues served as substrates for protein kinase C in vitro. Ser 3 Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase were constructed by site-directed mutagenesis and expressed normally in a ura7 ura8 double mutant that lacks CTP synthetase activity. The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. Kinetic analysis of purified mutant enzymes showed that the S36A and S354A mutations caused a decrease in the Vmax of the reaction. This regulation could be attributed in part by the effects phosphorylation has on the nucleotide-dependent oligomerization of CTP synthetase. In contrast, CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of purified enzyme showed that the S330A mutation caused an elevation in the Vmax of the reaction. In vitro data indicated that phosphorylation of CTP synthetase at Ser affected the phosphorylation of the enzyme at another site. The phosphorylation of CTP synthetase at Ser, Ser, Ser, and Ser residues was physiologically relevant. Cells bearing the S36A, S354A, and S454A mutations had reduced CTP levels, whereas cells with the S330A mutation had elevated CTP levels. The alterations in CTP levels correlated with the regulatory effects CTP has on the pathways responsible for the synthesis of the membrane phospholipid phosphatidylcholine.

In the yeast Saccharomyces cerevisiae CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (1,2). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2)(3)(4)(5). URA7 (6) and URA8 (7) are duplicate genes that code for CTP synthetase in S. cerevisiae. The yeast CTP synthetase enzymes (6,7) contain a conserved glutamine amide transfer domain (see Fig. 1) common to CTP synthetases from other organisms (8 -16). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (17) and is responsible for the majority of the CTP synthesized in vivo (7). Neither the URA7 nor the URA8 gene is essential as long as cells possess one functional CTP synthetase gene (6,7). CTP synthetase is an indispensable enzyme because its reaction product CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). The importance of understanding the regulation of CTP synthetase is emphasized by the fact that unregulated levels of its activity is a common property of various cancers in humans (19 -26).
The yeast CTP synthetase is regulated by genetic and biochemical mechanisms. Like many enzymes involved in macromolecular synthesis, CTP synthetase is regulated by growth phase. CTP synthetase mRNA and protein levels are highest in the exponential phase of growth and decline as cells enter the stationary phase (28). CTP synthetase activity is allosterically regulated by its substrates and product CTP. The enzyme exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (5,28). The positive cooperative kinetics of the URA7-encoded enzyme with respect to UTP and ATP are due to the nucleotide-dependent oligomerization of an inactive dimeric form to an active tetrameric form of the enzyme (29). A major form of CTP synthetase regulation is mediated by CTP product inhibition (5,28). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (5). Amino acid residue Glu 161 has been identified as being involved in this regulation (see Fig. 1) (30).
Phosphorylation is a major mechanism by which enzymes are regulated (31,32), and indeed, the yeast CTP synthetase is regulated by phosphorylation. The URA7-encoded CTP synthetase is phosphorylated on multiple serine residues in vivo (33). In vitro studies show that CTP synthetase is a substrate for protein kinases A (34) and C (33,35). These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (33)(34)(35). In addition, phosphorylation facilitates the nucleotide-dependent tetramerization of the enzyme (29) and causes a decrease in the sensitivity of the enzyme to inhibition by CTP (34,35). Ser 424 has been identified as the target site for protein kinase A phosphorylation (see Fig. 1) (36). However, the site(s) of phosphorylation for protein kinase C is unknown. In this study we examined the hypothesis that amino acid residues Ser 36 , Ser 330 , Ser 354 , and Ser 454 within a protein kinase C phosphorylation motif in the URA7-encoded CTP synthetase are target sites of phosphorylation (Fig. 1). We showed that S36A, S330A, S354A, and S454A mutant CTP synthetase enzymes exhibited alter-ations in their catalytic properties and that cells carrying the mutant enzymes exhibited alterations in the cellular levels of CTP and in the synthesis of the membrane phospholipid PC. 1

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase with 5Ј and 3Ј exonuclease activity and the DNA size ladder used for agarose gel electrophoresis were purchased from New England Biolabs. Oligonucleotides were prepared commercially by Genosys Biotechnologies, Inc. The QuikChange site-directed mutagenesis kit was purchased from Stratagene. The Prism DyeDeoxy DNA sequencing kit was obtained from Applied Biosystems. Nucleotides, L-glutamine, 5-fluroorotic acid, phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, pepstatin, nitrocellulose paper, casein, and bovine serum albumin were purchased from Sigma. Peptides were synthesized and purified commercially by Bio-Synthesis, Inc. Protein assay, electrophoresis reagents, and protein markers were purchased from Bio-Rad. Superose 6, Mono Q, ECF Western blotting kit, and 2Ј, 3Ј-dideoxynucleotide triphosphates were purchased from Amersham Biosciences. IMMUNOcatcher immunoprecipitation kit was purchased from CytoSignal Research Products. Centricon-10 concentration filters were purchased from Amicon. Phosphocellulose filter kits and dialysis cassettes 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 were purchased from EM Science.
Strains, Plasmids, and Growth Conditions-The strains and plasmids used in this work are listed in Table I. Wild type and mutant alleles of the URA7-encoded CTP synthetase were expressed in the ura7 ura8 double mutant strain SDO195 (30). Growth of this strain is dependent on a plasmid bearing either the URA7 or the URA8 gene (30). Methods for growth and analysis of yeast were performed as described previously (37,38). Yeast cultures were grown in complete synthetic medium minus inositol (39) containing 2% glucose at 30°C. Plasmid maintenance and amplifications were performed in Escherichia coli strain DH5␣. E. coli cells were grown in LB medium (1% Tryptone, 0.5% yeast extract, 1% NaCl (pH 7.4)) at 37°C. Ampicillin (100 g/ml) was added to cultures carrying plasmids. Media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar for growth on plates. Yeast cell numbers in liquid media were determined by microscopic examination with a hemacytometer or spectrophotometrically at an absorbance of 600 nm.
DNA Manipulations, Amplification of DNA by PCR, and DNA Sequencing-Plasmid DNA preparation, restriction enzyme digestion, and DNA ligations were performed by standard methods (38). Transformation of yeast (40,41) and E. coli (38) were performed as described previously. Conditions for PCR reactions were optimized as described previously (42). DNA sequencing reactions were performed by the dideoxy method using Taq DNA polymerase (38).
Constructions of Plasmids-The codons for Ser 36 , Ser 330 , Ser 354 , and Ser 454 in the URA7-encoded CTP synthetase were changed to alanine codons by site-directed mutagenesis. The URA7 S36A (primers: 5Ј-CCC-TCGGTTTAAAGGTTACcgCcATTAAAATTGACCCTTATATGA-3Ј and 5Ј-TCATATAAGGGTCAATTTTAATgGcgGTAACCTTTAAACCGAGG-G-3Ј), URA7 S330A (primers: 5Ј-GCATTGGAACATTCAgCaATGAAGTG-TCGTCGTAAG-3Ј and 5Ј-CTTACGACGACACTTCATtGcTGAATGTT-CCAATGC-3Ј), URA7 S354A (primers: 5Ј-GGAACCTGAAGCACAAGAA-gcCAACAAAACTAAATTTCATG-3Ј and 5Ј-CATGAAATTTAGTTTTG-TTGgcTTCTTGTGCTTCAGGTTCC-3Ј), and URA7 S454A (primers: 5Ј-G-GAAACCATGGGGGGCgCAATGAGATTAGGTTT-3Ј and 5Ј-AAACCT-AATCTCATTGcGCCCCCCATGGTTTCC-3Ј) mutations ere constructed by PCR with the QuikChange site-directed mutagenesis kit using plasmid pDO178 as the template. Plasmid pDO178 contains the URA7 coding sequence in pBlueScript II (30). The lowercase letters in the primers refer to nonhomologous sequences used for mutagenesis or for incorporating restriction enzyme sites. The primers for the S36A mutation incorporated a BstEII restriction site, and the primers for the S330A and S454A mutations incorporated a BsrDI site. These silent mutations were used to identify the plasmids with correct mutations by restriction enzyme analysis. Plasmids with the S354A mutation were verified by DNA sequencing. All of the mutant alleles were completely sequenced to verify that no additional mutations were made. The wild type and mutant alleles of URA7 were released from the plasmids by digestion with NotI/PstI. These 1.8-kilobase fragments of the URA7encoding sequences were inserted into the same restriction enzyme sites of multicopy plasmid pDO105 in which the expression of URA7 was under the control of the ADH1 promoter (30). The same wild type and mutant allele fragments were inserted into single copy plasmid pDO120 containing the ADH1 promoter. Plasmid pDO120 was constructed by subcloning the 1.58-kilobase EcoRI/PstI fragment of pDO105 into the single copy plasmid YCpLac111 (43).
Strain SDO195, bearing pFL44S-URA7 (7), was utilized for the expression of the S36A, S330A, S354A, and S454A mutant alleles of URA7. Plasmid pFL44S-URA7 was subsequently selected against 5-fluroorotic acid by plasmid shuffle (44). Cells were then examined to verify that they regained uracil auxotrophy. To verify the presence of the ADH1 expression vectors containing the wild type and mutant alleles of URA7, plasmids were rescued from yeast, amplified in E. coli, and subjected to restriction enzyme analysis. We used the ADH1 promoter for enzyme expression to preclude regulation mediated by the native URA7 promoter. The single copy plasmid was used to approximate normal expression of the enzymes and to examine the effects of the mutations in growing cells. The multicopy plasmid was used to overexpress CTP synthetase for enzyme purification. The mutant and wild type alleles were expressed on multicopy and single copy plasmids in the ura7 ura8 double mutant to avoid effects due to the native URA7and URA8-encoded enzymes.
In Vivo Labeling of CTP Synthetase-Cells bearing single copy plasmids containing the wild type and the S36A, S330A, S354A, and S454A mutant URA7 alleles were used to examine the phosphorylation state of CTP synthetase. Exponential phase cells grown in YEPD medium were labeled in low phosphate medium (45) with 32 P i (0.3 mCi/ml) and U-14 C-labeled L amino acids (5 Ci/ml) for 3 h. Labeled cells were harvested by centrifugation and washed with phosphate-buffered saline. Cells were disrupted with glass beads in radioimmune precipitation lysis buffer (46) containing a mixture of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 g/ml aprotinin, 5 g/ml leupeptin, and 5 g/ml pepstatin) and phosphatase inhibitors (10 mM NaF, 5 mM ␤-glycerophosphate, and 1 mM sodium vanadate). CTP synthetase was immunoprecipitated from the cell lysate with anti-URA7-encoded CTP synthetase IgG antibodies (5) as described previously (46). CTP synthetase was dissociated from the enzyme-antibody complex (46), and the amount of radiolabel incorporated into the enzyme was determined by scintillation counting.
Purification of Wild Type and Mutant CTP Synthetases-Cells overexpressing the wild type and mutant CTP synthetases were used for enzyme purification. The enzymes were purified by a modification of the method described by Yang et al. (5). All steps were performed at 4°C. Cells (10 g wet weight) were disrupted with glass beads with a Bead-  Beater in buffer A (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 20 mM L-glutamine, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 5 mg/ml pepstatin) as described previously (47). Unbroken cells and glass beads were removed by centrifugation at 1500 ϫ g for 5 min. The cytosol was obtained by centrifugation at 100,000 ϫ g for 1.5 h and diluted to a final protein concentration of 4.5 mg/ml with buffer A. Enzyme-grade ammonium sulfate was added to the cytosol to 45% saturation with slow stirring. After stirring for 2 h, precipitated protein was collected by centrifugation at 12,000 ϫ g for 20 min and dissolved in a minimum volume of buffer B (50 mM Tris-HCl (pH 8.0), 4 mM L-glutamine, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol). The ammonium sulfate fraction was applied to a Superose 6 column (1 ϫ 24 cm) that was equilibrated with buffer B. CTP synthetase was eluted from the column in 0.5-ml fractions with buffer B at a flow rate of 15 ml/h. This step served to desalt the preparation and to enrich for the CTP synthetase enzyme. The most active fractions were pooled and applied to a Mono Q column (0.5 ϫ 5 cm) equilibrated with buffer B at a flow rate of 30 ml/min. The column was washed with 5 column volumes of buffer B followed by elution of CTP synthetase in 1-ml fractions with 50 column volumes of a linear NaCl gradient (0 -1.5 M) in buffer B. The peak of CTP synthetase activity eluted from the column at a NaCl concentration of about 0.3 M. The active fractions were stored at Ϫ80°C in buffer B with the glycerol concentration raised to 25%. The enzyme preparations were stable for at least 6 months when stored at Ϫ80°C. For peptide-mapping analyses, the wild type and mutant CTP synthetases were purified to electrophoretic homogeneity by SDS-polyacrylamide gel electrophoresis. The CTP synthetase proteins were visualized with 0.3 M CuCl 2 (48) and eluted from gel slices using a Bio-Rad Electroeluter (Model 422). The enzymes were concentrated by filtration with Amicon Centricon 10 filters and then dialyzed against buffer B to remove SDS (49).
Purification of Protein Kinase C-The PKC1-encoded protein kinase C was purified from cells bearing the PKC1-ZZ fusion gene on multicopy plasmid (50). This fusion gene carries two (ZZ) repeats of the 60-amino acid IgG binding domain of Staphylococcus aureus protein A (50). The ZZ tag facilitates purification of protein kinase C but does not alter the biochemical properties of the enzyme (50,51). The enzyme was purified by Q-Sepharose chromatography and IgG-Sepharose chromatography as described by Antonsson et al. (50) with the modifications of Yang et al. (35).
Electrophoresis and Immunoblotting-Standard SDS-polyacrylamide gel electrophoresis (52) was performed with 10% slab gels. Molecular mass standards were phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa). Proteins on SDS-polyacrylamide gels were stained with Coomassie Blue or with silver. Immunoblot assays were performed with anti-URA7-encoded CTP synthetase antibodies (5) as described previously (53). The density of the CTP synthetase bands on immunoblots was quantified by scanning densitometry. Immunoblot signals were in the linear range of detectability.
Oligomerization of CTP Synthetase-The ATP/UTP-dependent oligomerization of CTP synthetase (5) from the dimeric to the tetrameric forms of the enzyme was analyzed by Superose 6 gel filtration chromatography (29). The column was calibrated with blue dextran 2000 (for the void volume), thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa). The relative amounts of the dimeric and tetrameric forms of the CTP synthetase in the column fractions were quantified by scanning densitometry of silver-stained SDS-polyacrylamide gels (29).
Phosphorylation of CTP Synthetase and Synthetic Peptides with Protein Kinase C-Phosphorylation of CTP synthetase was performed in a total volume of 40 l at 30°C. CTP synthetase (1 g) was incubated for 15 min 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 0.1 nmol/min/ml protein kinase C (35). At the end of the phosphorylation reactions, samples were treated with an equal volume of 2ϫ Laemmli sample buffer (52) followed by SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose paper. The phosphorylated enzyme was visualized by phosphorimaging, and the extent of phosphorylation was quantified using ImageQuant software. Reactions containing synthetic peptides were terminated by spotting an aliquot of the reaction mixture onto phosphocellulose filters. The filters were washed with 75 mM phosphoric acid and then subjected to scintillation counting.
CNBr Digestion and One-dimensional Phosphopeptide Mapping-The wild type and mutant CTP synthetases were phosphorylated with protein kinase C and [␥-32 P]ATP for 15 min. The phosphorylated enzymes were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper. The CTP synthetase on the nitrocellulose paper was isolated and digested with 200 l of 100 mg/ml CNBr in 70% formic acid for 1.5 h (54). The mixture was centrifuged, and the supernatant was collected and dried in vacuo. The samples were suspended in 0.5 ml of deionized water, dried in vacuo, and then suspended in 20 l of Laemmli sample buffer (52). Phosphopeptides were separated by SDS-polyacrylamide gel electrophoresis using a 24% slab gel as described by Luo et al. (54). Phosphorimaging was used to identify phosphopeptides in the polyacrylamide gel.
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 (2). 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. Enzyme assays were performed in triplicate with an average S.D. 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. Protein concentration was estimated  Analysis of CTP-Cells expressing the wild type and the S36A, S330A, S354A, and S454AS424A mutant CTP synthetases from single copy plasmids were grown to the exponential phase of growth. CTP was extracted (6), and its concentration was analyzed by high performance liquid chromatography as described by Pappas et al. (56).
Analysis of Phospholipids-Phospholipids were labeled with 32 P i and [methyl-3 H]choline as described previously (17,57,58). Phospholipids were extracted from labeled cells by the method of Bligh and Dyer (59) as described previously (60). Phospholipids were analyzed by two-dimensional thin-layer chromatography on silica gel 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 (61). The radiolabeled 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.
Analyses of Data-Kinetic data were analyzed with the EZ-FIT enzyme kinetic model-fitting program (62). Statistical analyses were performed with SigmaPlot software.

CTP Synthetase Synthetic Peptides Containing a Protein Kinase C Sequence Motif Are Substrates for Protein Kinase C-
Analysis of the deduced sequence of CTP synthetase revealed that the protein has potential serine (Ser 36 , Ser 330 , Ser 354 , and Ser 454 ) and threonine (Thr 90 , Thr 303 , Thr 514 , and Thr 539 ) phosphorylation sites within a protein kinase C sequence motif. Peptides containing the serine (LKVTS36IKIDP, LEH-SS330MKCRR, EAQES354NKTKF, and TMGGS454MRLGL) and threonine (HNITT90GKIYS, ESMET303VKIRL, GKDDT-514GKRCE, and HPEYT539SKVLD) target sites, respectively, were synthesized based on the protein sequence of CTP synthetase. The four peptides containing sequences for the potential serine phosphorylation sites served as substrates for protein kinase C in a concentration-dependent manner (Fig. 2). Of the four peptides, the Ser 330 peptide was the best substrate for protein kinase C (Fig. 2). None of the peptides containing the potential threonine phosphorylation sites was a substrate for protein kinase C (data not shown).
Construction and Characterization of CTP Synthetase S36A, S330A, S354A, and S454A Mutants-Mutagenesis of Ser 36 , Ser 330 , Ser 354 , and Ser 454 within the CTP synthetase was performed to further examine the hypothesis that these sites might be targets for protein kinase C. CTP synthetases with serine to alanine (S36A, S330A, S354A, and S454A) mutations were constructed by site-directed mutagenesis and expressed on single copy and multicopy plasmids in the ura7⌬ ura8⌬ double mutant. Cells bearing the wild type and mutant alleles of the URA7 gene exhibited similar growth rates when grown vegetatively at 30°C in minimal synthetic media and in rich YEPD. No morphological differences were observed in cells bearing the mutant enzymes. Antibodies directed against the wild type CTP synthetase (63) recognized the mutant enzymes (Fig. 3A). Scanning densitometry of the immunoblot shown in Fig. 3A showed that there were no major differences in the expression of the wild type and mutant CTP synthetases in cells bearing the single copy plasmids. Thus, the S36A, S330A, S354A, and S454A mutations in the URA7 gene did not affect the functional expression of the enzyme.
Cells expressing the wild type and mutant CTP synthetase alleles on the single-copy plasmid were labeled with 32 P i to detect phosphorylated CTP synthetase and with U-14 C-labeled L-amino acids to normalize for the amount of CTP synthetase isolated. The ratio of counts/min of 32 P incorporated into CTP synthetase to the counts of 14 C incorporated into CTP synthetase was used to examine the extent of phosphorylation in vivo (35). The wild type and mutant CTP synthetases were isolated by immunoprecipitation, and the amount of each label incorporated into the enzymes was determined. The phosphorylation state of the S36A, S354A, and S454A mutant enzymes was reduced by 37, 37, and 30%, respectively, when compared with the wild type control enzyme (Fig. 3B). On the other hand, the S330A mutation did not have a significant effect on the phosphorylation state of CTP synthetase (Fig. 3B).
We next examined the levels of CTP synthetase activity in cells bearing the mutations. The specific activity in cells with the S36A, S354A, and S454A mutations was reduced by 53, 63, and 10%, respectively, when compared with cells bearing the wild type enzyme (Fig. 3C). In contrast, the CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated by 64% (Fig. 3C). CTP synthetase specific activity was based on the total protein concentration in cell extracts.
Partial followed by chromatography with Superose 6 and with Mono Q. The Superose 6 chromatography step purified the enzyme and served as a convenient desalting step. The Mono Q chromatography step purified the enzyme and at the same time was effective in concentrating the enzyme. The mutant enzymes behaved similarly to the wild type enzyme during each step of the purification. Analysis by SDS-polyacrylamide gel electrophoresis showed that the purification scheme resulted in highly purified preparations of the wild type and mutant CTP synthetases (Fig. 4). The yield (17-27%) and degree of purifi-cation (20 -26-fold) of the mutant enzymes were similar to that of the wild type control enzyme (Table II). The specific activities of the partially purified enzyme preparations ranged from 0.66 to 0.2 unit/mg. For reference, the specific activity of pure wild type CTP synthetase is typically 2.3-2.5 units/mg (5,36).
Effects of the S36A, S330A, S354A, and S454A Mutations on the Phosphorylation of CTP Synthetase by Protein Kinase C in Vitro-The effects of the S36A, S330A, S354A, and S454A mutations on the phosphorylation of CTP synthetase by protein kinase C were examined. The SDS gel-purified CTP synthetase proteins were used for these experiments. In control experiments, we showed that this procedure did not affect the phosphorylation of the enzyme by protein kinase C. Samples of the wild type and mutant enzymes were incubated with protein kinase C and 32 P-labeled ATP. After the phosphorylation reactions, samples were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and phosphorimaging analysis. The phosphorylation of CTP synthetase by protein kinase C was most affected by the S330A mutation. The S330A mutation caused a 70% decrease for label incorporated into the enzyme (Fig. 5A). The S36A, S354A, and S454A mutations did not have a great effect on the ability of protein kinase C to phosphorylate CTP synthetase in vitro. These results were consistent with the observation that protein kinase C activity was 100-fold greater using the synthetic peptide containing the phosphorylation motif at Ser 330 when compared with the other peptide substrates.
The phosphorylated wild type and mutant CTP synthetase enzymes were subjected to CNBr cleavage and one-dimensional phosphopeptide mapping analysis (Fig. 5B). Six phosphopeptides (labeled a through f) were derived from the wild type and mutant enzymes. The most heavily phosphorylated peptides  3. Effects of the S36A, S330A, S354A, and S454A mutations on the expression, the state of phosphorylation, and activity of CTP synthetase. Cells expressing wild type (WT) and the indicated S36A, S330A, S354A, and S454A mutant CTP synthetase enzymes from the single-copy plasmids were grown to the exponential phase of growth. Panel A, cell extracts were prepared and subjected to immunoblot analysis using anti-CTP synthetase antibodies. A portion of the immunoblot shows the expression of wild type and mutant CTP synthetase enzymes. Relative amounts of CTP synthetase protein were determined by densitometry scanning. The amount of CTP synthetase protein found in cells bearing the wild type enzyme was set as 1. The position of CTP synthetase is indicated. Panel B, cells were labeled with 32 P i and U-14 C-labled L-amino acids. The CTP synthetase proteins were immunoprecipitated from cell extracts using anti-CTP synthetase antibodies. CTP synthetase was dissociated from the enzyme-antibody complex, and the amount of the label incorporated into CTP synthetase was determined by scintillation counting. The values are reported as the cpm of 32 P incorporated into CTP synthetase relative to the cpm of 14 C incorporated into CTP synthetase. The values reported were the averages of three separate experiments Ϯ S.D. Panel C, cell extracts were prepared and assayed for CTP synthetase activity. The specific activity (mol/min/mg) was determined using the total protein concentration in cell extracts. The values reported were the averages of three separate experiments Ϯ S.D. derived from the wild type CTP synthetase were c and d. Of the four mutations, the S330A mutation had the greatest effect on the phosphopeptide map of CTP synthetase. The major effects of the S330A mutation were decreases in the amounts of phosphopeptides c and d and an increase for phosphopeptide f. The amount of phosphopeptide f was also elevated in the S354A mutant enzyme. Based on the predicted sizes of the CNBr cleavage products, Ser 330 should be contained in phosphopeptide d.
Effects of the S36A, S330A, S354A, and S454A Mutations on the Enzymic Properties of CTP Synthetase-A kinetic analysis was performed to further characterize the effects of the S36A, S330A, S354A, and S454A mutations on CTP synthetase activity. Equal amounts (as determined by densitometry scanning of SDS-polyacrylamide gels) of the wild type and mutant proteins were used for these experiments. The dependence of CTP synthetase activity on UTP (Fig. 6A) and ATP (Fig. 6B) was examined using a subsaturating concentration of UTP and ATP, respectively. Under these conditions, we could more readily observe stimulatory or inhibitory effects of the mutations on enzyme activity. The S36A and S354A mutations caused a decrease in CTP synthetase activity when measured with respect to UTP and with respect to ATP. Conversely, the S330A mutation caused an increase in activity when measured with respect to each substrate. The S36A and S354A mutations caused the inhibition of CTP synthetase activity by a mechanism that primarily affected the apparent V max values with respect to UTP and ATP (Table III). The S330A mutation caused the stimulation of activity by a mechanism that primarily affected the apparent V max with respect to UTP and ATP (Table III). The S454A mutation did not have a significant effect on CTP synthetase activity except for a small decrease in the apparent K m value for ATP (Table III). The mutations did not affect the Hill number (n ϭ 1.4) for UTP.
The activity of the wild type CTP synthetase enzyme is inhibited by CTP (5). Because protein kinase C phosphorylation of pure wild type CTP synthetase results in a decrease in the enzyme sensitivity to product inhibition by CTP (35), we examined the effects of the mutations on this property. As described previously (5), the IC 50 for CTP inhibition of wild type CTP synthetase activity was 0.3 mM. None of the mutations had a significant effect on this property. This suggests that the role of protein kinase C phosphorylation on this enzyme property may be more complicated than the phosphorylation of any one site. We also examined the effects of the S36A, S330A, S354A, and S454A mutations on the pH optimum of the CTP synthetase reaction. As described previously (5), the pH optimum for wild type CTP synthetase activity was 8.0, and the mutations did not have an effect on this property.
Effects of the S36A, S330A, S354A, and S454A Mutations on the Oligomerization of CTP Synthetase-In vitro, wild type CTP synthetase exists as an inactive dimer that oligomerizes to an active tetramer in the presence of its substrates ATP and UTP (5). Data indicate that this process is facilitated by protein FIG. 5. Effects of the S36A, S330A, S354A, and S454A mutations on the phosphorylation of CTP synthetase by protein kinase C in vitro. Panel A, samples of purified wild type (WT) and the indicated S36A, S330A, S354A, and S454A mutant CTP synthetases were incubated with protein kinase C and 32 P-labeled ATP for 15 min. After incubation, samples were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose paper. The phosphorylated proteins were subjected to phosphorimaging analysis, and the relative amounts of phosphate incorporated were quantified using Im-ageQuant software. The relative phosphorylation of the wild type CTP synthetase protein was set as 1. The position of CTP synthetase is indicated in the figure. Panel B, the labeled proteins were excised from the nitrocellulose paper and subjected to CNBr cleavage. The resulting peptides were subjected to SDS-polyacrylamide gel electrophoresis using a 24% low bis-Tricine gel and then visualized by phosphorimaging analysis. The positions of peptide markers and the phosphopeptides labeled a through f are indicated in the figure. kinase C phosphorylation of the enzyme (29). Accordingly, we questioned whether the S36A, S330A, S354A, and S454A mutations in CTP synthetase affected this property of the enzyme. The purified preparations of the wild type and mutant enzymes were subjected to Superose 6 chromatography in the absence and presence of subsaturating concentrations of ATP and UTP. Under these conditions, we could more readily observe the effects of the mutations on the oligomerization of CTP synthetase. The amount of the inactive dimeric form of the S36A and S354A mutant CTP synthetase enzymes was 54 and 98% greater, respectively, when compared with the wild type enzyme (Fig. 7). The S330A and S454A mutations did not have a major effect on the oligomerization of CTP synthetase.
Effects of the S36A, S330A, S354A, and S454A Mutations in CTP Synthetase on the Cellular Concentration of CTP-We questioned whether the changes in CTP synthetase activity that were brought about by the S36A, S330A, S354A, and S454A mutations would affect the levels of CTP in vivo. Cells bearing the wild type and mutant CTP synthetases expressed from the single-copy plasmid were grown to the exponential phase of growth, and CTP was extracted and analyzed by high performance liquid chromatography. The concentration of CTP in cells with the S36A, S354A, and S454A mutant enzymes was reduced by 40, 50 and 11%, respectively, whereas the concentration of CTP in cells with the S330A mutant enzyme was elevated by 38% (Fig. 8).
Effects of the S36A, S330A, S354A, and S454A Mutations in CTP Synthetase on Phospholipid Composition-CTP is essential for the synthesis of PC, the major membrane phospholipid in S. cerevisiae (64 -67). There are two pathways by which PC is synthesized from CTP. In one pathway, PC is synthesized from CTP via CDP-choline, whereas in the other pathway PC is synthesized from CTP via CDP-diacylglycerol (64 -67). The synthesis of PC from CDP-choline is direct. The synthesis of PC from CDP-diacylglycerol is indirect and occurs by the reaction sequence CDP-diacylglycerol 3 phosphatidylserine 3 phosphatidylethanolamine 3 3 3 phosphatidylcholine. We questioned whether the S36A, S330A, S354A, and S454A mutations in CTP synthetase would affect phospholipid composition. Cells bearing the wild type and mutant enzymes expressed from the single copy plasmids were grown in complete synthetic medium without inositol and choline to preclude the regulatory effects these precursors have on phospholipid synthesis (64,66,68). In the absence of exogenous choline, wild type cells synthesize PC by both the CDP-choline and CDP-diacylglycerol pathways (17, 30, 69 -71). The choline required for the CDP-choline pathway is derived from the phospholipase D-mediated turnover of PC synthesized via the CDP-diacylglycerol pathway (71,72). We examined the composition of phospholipids by labeling cells with both 32 P i and [methyl-3 H]choline. The 32 P i will be incorporated into phospholipids synthesized by both the CDP-choline and CDP-diacylglycerol pathways, whereas the labeled choline will only be incorporated into PC synthesized via the CDP-choline pathway. 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 (73).
The 32 P-labeling experiments showed that the S36A, S330A, S354A, and S454A mutations did not have any major effects on phospholipid composition. The 3 H-labeled choline was incorporated into PC during the labeling experiments, indicating that PC was synthesized via the CDP-choline pathway (17,30). The data shown in Fig. 9 were plotted as the ratio of the cpm of 3 H incorporated into PC to the cpm of 32 P incorporated into PC. This allowed us to determine whether the mutations affected the pathways by which cells synthesized PC (17,30). The 3 H/ 32 P ratio decreased in cells bearing the S36A, S354A, and S454A mutations by 35, 20, and 19%, respectively, whereas the 3 H/ 32 P ratio in cells with the S330A mutation increased by 24%. These data indicated that the S36A, S354A, and S454A mutations in CTP synthetase resulted in decreased utilization of the CDP-choline pathway for PC synthesis, whereas the S330A mutation had the opposite effect.

DISCUSSION
CTP synthetase, the rate-limiting enzyme in CTP synthesis, plays a major role in the growth and metabolism of eukaryotic and prokaryotic organisms (18). Regulation of this enzyme is critical because the product of its reaction CTP is essential for the synthesis of nucleic acids and membrane phospholipids (18). One of the mechanisms by which CTP synthetase activity is regulated in S. cerevisiae is by phosphorylation via protein kinase C (33,35). Protein kinase C is a lipid-dependent protein kinase required for S. cerevisiae cell cycle (74 -78) and plays a role maintaining cell wall integrity (79). In mammalian cells, protein kinase C plays a central role in the transduction of lipid second messengers generated by receptor-mediated hydrolysis of membrane phospholipids (80 -82). Phosphorylation of CTP synthetase by protein kinase C in yeast may represent a mechanism by which lipid signaling transduction pathways are coordinately regulated to CTP synthesis and cell growth. Identification of the protein kinase C phosphorylation sites in CTP synthetase is necessary for gaining information about the physiological significance of this phosphorylation.
Peptides with sequences for potential serine and threonine protein kinase C phosphorylation sites in CTP synthetase were synthesized and examined for their ability to serve as substrates for protein kinase C in vitro. The peptides containing a phosphorylation motif at Ser 36 , Ser 330 , Ser 354 , and Ser 454 served as substrates for protein kinase C. This assay provided confidence that these residues might be phosphorylation sites for protein kinase C. Based on this information and previous data indicating that only serine residues are phosphorylated in vivo (33), we constructed Ser 3 Ala (S36A, S330A, S354A, and S454A) mutations in CTP synthetase to further examine the hypothesis that these sites were targets of protein kinase C. The mutant enzymes were functional in vivo as evidenced by the suppression of the lethal phenotype (7) of the ura7 ura8 double mutant, and immunoblot analysis showed that the phosphorylation site mutations did not affect the expression of CTP synthetase. Moreover, the mutations did not have a major effect on the overall structure of the CTP synthetase protein. The mutant enzymes behaved normally during purification and exhibited the property of nucleotide-dependent tetramerization.
Consistent with the loss of a phosphorylation site, the phosphorylation state of CTP synthetase in vivo was reduced in cells carrying the S36A, S354A, and S454A mutations. On the other hand, the phosphorylation state of the S330A mutant enzyme was not altered in vivo. An explanation for this result is that the loss of Ser 330 as a phosphorylation site resulted in the phosphorylation of the enzyme at another site. This phosphorylation could be due to protein kinase C or another protein kinase. That protein kinase C may be involved in vivo was supported by the in vitro phosphorylation experiment where the S330A mutation caused a decrease in phosphopeptides c and d and an increase in phosphopeptide f.
The CTP synthetase activity in extracts from cells bearing the S36A, S354A, and S454A mutant enzymes was reduced when compared with cells bearing the wild type enzyme. In addition, the kinetic analysis of the purified mutant enzymes revealed that the S36A and S354A mutations caused a decrease in the V max of the reaction. These results were consistent with previous data showing that protein kinase C phosphorylation stimulates the activity of the pure wild type CTP synthetase (33,35). Interestingly, the CTP synthetase activity in cells bearing the S330A mutant enzyme was elevated, and kinetic analysis of the purified enzyme showed that the S330A mutation caused an elevation in the V max of the reaction. These data raised the suggestion that the phosphorylation of Ser 330 causes the inhibition of CTP synthetase activity. Thus, the effect of phosphorylation on CTP synthetase activity in vivo might be governed by which site(s) is phosphorylated.
Previous data using the purified wild type CTP synthetase indicated that protein kinase C phosphorylation facilitates the ATP/UTP-dependent oligomerization of the enzyme (29). Of the four mutations, only the S36A and the S354A mutations affected the dimer to tetramer conversion of CTP synthetase. These mutations caused a greater amount of the enzyme to exist in its inactive dimeric form when compared with the wild type control enzyme. Thus, the lower activity exhibited by the S36A and S354A mutant enzymes may be attributed to the effect of phosphorylation of Ser 36 and Ser 354 on the nucleotidedependent oligomerization of CTP synthetase. This analysis indicated that phosphorylation of Ser 330 and Ser 454 did not have a major effect on the oligomerization of the enzyme.
We initiated studies to examine the physiological consequences of the S36A, S330A, S354A, and S454A mutations in CTP synthetase. The concentration of CTP in cells bearing the S36A, S354A, and S454A mutant enzymes was reduced by 40, 50, and 11%, respectively, whereas the CTP concentration in cells bearing the S330A mutation was elevated by 38%. These data were consistent with the effects of the mutations on CTP synthetase activity and provided further support that Ser 36 , Ser 330 , Ser 354 , and Ser 454 are phosphorylation sites in CTP synthetase. Previous studies show that the cellular levels of CTP affect the pathways by which PC is synthesized (17,30). For example, PC synthesis via the CDP-choline pathway is stimulated when CTP levels are elevated due to the misregulation of CTP synthetase activity by CTP product inhibition (30). Given the fact that the cellular levels of CTP were altered in cells bearing the S36A, S330A, S354A, and S454A mutations, we examined the effects of the mutations on phospholipid composition. The in vivo labeling experiments showed that the amount of PC synthesized via the CDP-choline pathway was reduced in cells with the S36A, S354A, and S454A mutations, whereas the amount of PC synthesized via this pathway was elevated in cells with the S330A mutation. The mechanism for this regulation may be attributed to the availability of CTP for phosphocholine cytidylyltransferase (17), the rate-limiting enzyme in the CDP-choline pathway (67,73,83). The cellular concentrations of CTP in cells with the wild type and the S36A, FIG. 9. Effects of the S36A, S330A, S354A, and S454A mutations in CTP synthetase on PC synthesis. Cells expressing the wild type (WT) and the indicated S36A, S330A, S354A, and S454A mutant CTP synthetases from the single-copy plasmids were grown for five to six generations with 32 P i (5 Ci/ml) and [methyl-3 H]choline (0.5 Ci/ml). The incorporation of 32 P i and [methyl-3 H]choline into total phospholipids and into phosphatidylcholine was about 4,000 cpm/10 7 cells and 30,000 cpm/10 7 cells, respectively. Phospholipids were extracted and analyzed by two-dimensional thin-layer chromatography. The data were reported as the cpm of 3 H incorporated into PC relative to the cpm of 32 P incorporated into PC and were the average of three separate experiments Ϯ S.D. S330A, S354A, and S454A mutant CTP synthetase enzymes were within the range of the K m value (1.4 mM) of CTP for the phosphocholine cytidylyltransferase enzyme (84). Thus, phosphocholine cytidylyltransferase activity would be sensitive to the changes in CTP levels brought about by the mutations in CTP synthetase. The CDP-diacylglycerol synthase enzyme may also be sensitive to the cellular concentrations of CTP because its K m value for CTP is 1 mM (85). However, in contrast to the phosphocholine cytidylyltransferase reaction, the synthesis of CDP-diacylglycerol is not a rate-limiting step in the CDPdiacylglycerol pathway for PC synthesis (86). Moreover, the synthesis of CDP-diacylglycerol is several steps upstream in the CDP-diacylglycerol-dependent pathway for the synthesis of PC (64 -67).
Several proteins are phosphorylated on multiple sites by one or more protein kinases (87)(88)(89)(90)(91)(92). For example, the phosphorylation of one site can affect the phosphorylation at another site (i.e. hierarchical phosphorylation (87)). Data indicated that phosphorylation of CTP synthetase at one site affected the phosphorylation of the enzyme at another site. Moreover, the phosphorylation of CTP synthetase at different sites had opposite effects on enzyme activity. CTP synthetase is phosphorylated by protein kinase A, and it is unknown whether the phosphorylation of CTP synthetase by protein kinase C affects the ability of the enzyme to be phosphorylated by protein kinase A and vice versa. Moreover, data suggest that CTP synthetase is phosphorylated by yet additional protein kinases (34). Thus, the regulation of CTP synthetase by phosphorylation is very complex. In this work, we addressed some of this complex regulation by identifying sites that are phosphorylated by protein kinase C and show that this phosphorylation is physiologically relevant.