©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of CTP Synthetase from Saccharomyces cerevisiae by Protein Kinase C (*)

Weng-Lang Yang , George M. Carman (§)

From the (1)Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae by protein kinase C was examined. Using pure CTP synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.


INTRODUCTION

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is the enzyme responsible for the synthesis of CTP(1, 2) . CTP synthetase is a glutamine amidotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to C-4 of UTP to form CTP ().

On-line formulae not verified for accuracy

REACTION 1

On-line formulae not verified for accuracy

GTP is an allosteric effector that accelerates the formation of a covalent glutaminyl enzyme catalytic intermediate(2, 3) . Since CTP is used in the synthesis of nucleic acids (4) and membrane phospholipids (5), regulation of CTP synthetase should play a major role in growth and metabolism.

In Saccharomyces cerevisiae, CTP synthetase is encoded by two duplicate genes named URA7(6) and URA8(7) . The deduced amino acid sequences of the CTP synthetases encoded by the URA7 and URA8 genes show 78% identity and have predicted molecular masses of 64.7 and 64.5 kDa, respectively(6, 7) . The proteins encoded by these yeast genes also show a high degree of homology with the deduced amino acid sequence of the human CTP synthetase. Neither one of the URA7 and URA8 genes is essential provided that cells possess one functional gene encoding for the enzyme(6, 7) . The simultaneous presence of null alleles for both URA7 and URA8 is lethal(7) . Based on the codon bias values for the URA7 and URA8 genes and the cellular concentrations of CTP in null allele mutants for each of these genes, the URA7 gene product appears to be responsible for the majority of the CTP synthesized in vivo(7) .

The URA7-encoded CTP synthetase has been purified to homogeneity(8) . The minimum subunit molecular mass of the purified enzyme is 68 kDa. Native CTP synthetase exists as a dimer that oligomerizes to a tetramer in the presence of its substrates UTP and ATP(8) . CTP synthetase displays positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP(8) . The product of the reaction, CTP, inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP(8) . This inhibition of activity by CTP is physiologically relevant in S. cerevisiae(8) .

To gain further insight into the regulation of CTP synthetase activity from S. cerevisiae, we have examined the consequence of phosphorylation of the enzyme by protein kinase C. Protein kinase C is a lipid-dependent protein kinase (9, 10, 11) required for the S. cerevisiae cell cycle(12) . 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(13, 14, 15) . In this study we demonstrated that CTP synthetase was phosphorylated by protein kinase C. The phosphorylation of CTP synthetase by protein kinase C resulted in an activation in CTP synthetase activity. We also provided evidence that CTP synthetase was phosphorylated in vivo by protein kinase C.


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, histone (type III-S), phosphoamino acids, TPCK-trypsin,()alkaline phosphatase-agarose, and bovine serum albumin were purchased from Sigma. Protein kinase C (rat brain) was purchased from Promega. PS and DG 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. Protein A-Sepharose CL-4B was purchased from Pharmacia Biotech Inc. Cellulose thin layer sheets were obtained from EM Science.

Strain and Growth Conditions

S. cerevisiae strain OK8 (MAT leu2 trp1 ura3 ura7::TRP1 ura8) bearing the multicopy plasmid pFL44S-URA7(6, 7) was used for the purification of CTP synthetase and in vivo labeling of CTP synthetase. Plasmid pFL44S-URA7 directs a 10-fold overexpression of CTP synthetase(8) . Strain OK8 bears a mutation in the URA8 gene, which is a duplicate gene encoding for CTP synthetase(7) . For enzyme purification, cells were grown in complete synthetic medium (16) without uracil. For labeling experiments, cells were grown to the exponential phase of growth in low phosphate medium (17) at 30 °C. Cell numbers were determined by microscopic examination with a hemacytometer or by absorbance at 660 nm.

Purification of CTP Synthetase

CTP synthetase was purified to homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel blue, and Superose 6(8) . The specific activity of the pure enzyme was 2.5 µmol/min/mg at 30 °C.

Electrophoresis and Immunoblotting

SDS-polyacrylamide gel electrophoresis (18) was performed with 10% slab gels. Immunoblot analysis (19) was performed with anti-CTP synthetase IgG antibodies (8). Immunoblot signals were in the linear range of detectability.

Phosphorylation of CTP Synthetase with Protein Kinase C

Phosphorylation reactions were measured for 10 min at 30 °C in a total volume of 40 µl. CTP synthetase (1.0 µg) was incubated with 50 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl, 10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375 mM EGTA, 1.7 mM CaCl, 20 µM DG, 50 µM PS, 50 µM [-P]ATP (4 µCi/nmol), and the indicated concentrations of protein kinase C. At the end of the phosphorylation reactions, samples were treated with 2 Laemmli's sample buffer (18) followed by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. The incorporation of phosphate into CTP synthetase was determined by scintillation counting of phosphorylated enzyme excised from immunoblots. Alternatively, the protein kinase C phosphorylation reactions were performed with unlabeled ATP. Following incubation with protein kinase C, the reaction mixtures were diluted 5-fold, and CTP synthetase activity was measured spectrophotometrically as described below.

In Vivo Labeling of CTP Synthetase

Exponential phase cells were labeled with P (1 mCi/ml) for 2 h. The labeled cells were harvested by centrifugation and washed with phosphate-buffered saline. Cells were disrupted with glass beads in radioimmune precipitation lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin) and phosphatase inhibitors (10 mM NaF, 5 mM -glycerophosphate, and 1 mM sodium vanadate) as described previously(20) . CTP synthetase was immunoprecipitated from the cell lysate with anti-CTP synthetase IgG antibodies (8) as described previously(20) . CTP synthetase was dissociated from the enzyme-antibody complex (20) and subjected to SDS-polyacrylamide gel electrophoresis. Gels were dried and subjected to autoradiography.

Phosphoamino Acid Analysis

P-Labeled CTP synthetase preparations that were phosphorylated in vitro and in vivo were subjected to SDS-polyacrylamide gel electrophoresis. P-Labeled enzymes on SDS-polyacrylamide gels were located by autoradiography and eluted with 50 mM ammonium bicarbonate (pH 8.0) and 0.1% SDS at 37 °C for 30 h. Bovine serum albumin (50 µg) 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 3 times with cold acetone and dried in vacuo. The protein 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 µg of phosphoserine, 2.5 µg of phosphothreonine, and 5 µg of phosphotyrosine as carrier phosphoamino acids in water. Phosphoamino acids were separated by two-dimensional electrophoresis(21) . Following electrophoresis, the plates were dried, sprayed with 0.25% ninhydrin in acetone to visualize carrier phosphoamino acids, and subjected to PhosphorImager analysis.

Tryptic Digestion and Two-dimensional Peptide Mapping

SDS-polyacrylamide gel slices containing P-labeled CTP synthetase phosphorylated in vitro and in vivo were subjected to proteolysis using TPCK-trypsin (0.15 mg/ml) in 50 mM ammonium bicarbonate for 16 h at 37 °C(22) . The mixture was subjected to centrifugation and the supernatant was removed and retained. Fresh TPCK-trypsin was added to the gel slices for an additional 11 h of proteolysis. The digest was subjected to centrifugation and the supernatant was again removed and retained. The gel slices were then incubated in water for 1 h at 37 °C. The mixture was then centrifuged and the supernatant was collected and added to the previously retained supernatants. The combined supernatants were dried in vacuo. Samples were resuspended in 1 ml of water and dried again. This process was repeated four times. The samples were resuspended in 10 µl of 1% ammonium carbonate, clarified by centrifugation, and spotted on cellulose thin layer chromatography plates(23) . Separation of phosphopeptides was accomplished by electrophoresis in 1% ammonium bicarbonate at 1000 V for 35 min, followed by ascending chromatography (n-butyl alcohol/glacial acetic acid/pyridine/water, 10:3:12:15) for 7 h(23) . Dried plates were then subjected to PhosphorImager analysis.

Dephosphorylation of CTP Synthetase

Alkaline phosphatase attached to beaded agarose was used to dephosphorylate CTP synthetase. A 0.4-ml column of alkaline phosphatase-agarose (1,000 µmol/min/ml of resin) was equilibrated with 5 ml of chromatography buffer (50 mM Tris-HCl buffer (pH 8.0), 1 mM MgCl, 1 mM ZnCl, and 10% glycerol) containing 0.1 mg/ml ovalbumin. Ovalbumin was included in the chromatography buffer to block nonspecific protein binding sites on the column. The column was then washed with 5 ml of chromatography buffer without ovalbumin. CTP synthetase was applied to the column and incubated for 10 min. The column was then washed with chromatography buffer to elute CTP synthetase.

Enzyme Assay and Protein Determination

CTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M cm, 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, 10 mM 2-mercaptoethanol, 2 mML-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 standard deviation of ±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 µmol of CTP/min under the assay conditions described above. Specific activity was defined as units/mg of protein. Protein was determined by the method of Bradford (24) using bovine serum albumin as the standard.


RESULTS

Phosphorylation of CTP Synthetase by Protein Kinase C

Phosphorylation of CTP synthetase by protein kinase C was examined with a protein kinase preparation isolated from rat brain. This rat brain protein kinase C preparation contained a mixture of the , , and isoforms of protein kinase C. We used rat brain protein kinase C in our studies because S. cerevisiae protein kinase C (25, 26) has catalytic properties characteristic of the , , and isoforms of the rat brain enzyme(9, 27) . The protein kinase C preparation used here was judged to be pure as determined by SDS-polyacrylamide gel electrophoresis. Protein kinase C phosphorylated histone with the activity stated by the manufacturer under the assay conditions used here.

To determine if CTP synthetase was a target for phosphorylation by protein kinase C, we examined whether protein kinase C catalyzed the incorporation of the -phosphate of P-labeled ATP into purified CTP synthetase. After the phosphorylation reaction, samples were subjected to SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose paper. Autoradiography of the nitrocellulose paper showed that CTP synthetase was a substrate for protein kinase C (Fig. 1). The position of P-labeled CTP synthetase on the nitrocellulose paper was confirmed by immunoblot analysis. When histone is used as a substrate for protein kinase C, maximum activity is dependent on calcium, DG, and PS as cofactors(9, 27) . We questioned if protein kinase C showed the same activity dependences when CTP synthetase was used as a substrate. The omission of either calcium, DG, or PS from the standard protein kinase C assay resulted in 67-73% decreases in protein kinase C activity (Fig. 1). When all three cofactors were omitted from the standard protein kinase C assay, there was a 92% decrease in protein kinase C activity (Fig. 1). Protein kinase C activity was linear with respect to the concentration of protein kinase C (Fig. 2A) and time (Fig. 2B) using CTP synthetase as a substrate.


Figure 1: Effect of calcium, DG, and PS on the phosphorylation of CTP synthetase by protein kinase C. Panel A, CTP synthetase (1 µg) was incubated with protein kinase C (4 pmol/min/ml) and [-P]ATP for 10 min in the presence of 1.7 mM calcium, 20 µM DG, and 50 µM PS, where indicated. Following the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. The lastlane to the farright of the figure shows the C-labeled protein molecular mass standards (from top to bottom): phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa. Panel B, the incorporation of phosphate into CTP synthetase was determined by scintillation counting of phosphorylated enzyme excised from the immunoblot. The various additions of protein kinase C cofactors are indicated in the figure.




Figure 2: Dose- and time-dependent phosphorylation of CTP synthetase by protein kinase C. Panel A, pure CTP synthetase (1 µg) was incubated with the indicated amounts (U = pmol/min) of protein kinase C and [-P]ATP for 10 min. Panel B, pure CTP synthetase (1 µg) was incubated with protein kinase C (4 pmol/min/ml) and [-P]ATP for the indicated time intervals. Following the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. The incorporation of phosphate into CTP synthetase was determined by scintillation counting of phosphorylated enzyme excised from the immunoblot.



The dependence of protein kinase C activity on the concentration of CTP synthetase was examined. Protein kinase C activity did not follow simple saturation kinetics with respect to the CTP synthetase concentration (Fig. 3). Instead, the kinetics of the protein kinase C reaction was complex, showing two apparent saturation patterns. The reaction first saturated at CTP synthetase concentrations of 5 µg/ml. Protein kinase C activity increased again with increased CTP synthetase concentration. The reaction saturated a second time at a CTP synthetase concentration of 27 µg/ml. This complex kinetic behavior prevented the determination of a kinetic constant for CTP synthetase.


Figure 3: Dependence of protein kinase C activity on CTP synthetase concentration. Panel A, various concentrations (indicated in panel B) of CTP synthetase were incubated with protein kinase C (4 pmol/min/ml) and [-P]ATP for 10 min. Following the incubations, samples was subjected to SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. A portion of an autoradiogram showing the position of CTP synthetase is shown. The firstlane to the farleft of the figure shows the C-labeled protein molecular mass standards (from top to bottom): phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa. Panel B, the incorporation of phosphate into CTP synthetase was determined by scintillation counting of phosphorylated enzyme excised from the immunoblot.



Phosphoamino Acid Analysis and Two-dimensional Phosphopeptide Mapping of CTP Synthetase Phosphorylated by Protein Kinase C

Protein kinase C is a serine/threonine-specific protein kinase (9, 27). We examined which amino acid residues of CTP synthetase were targets for phosphorylation. CTP synthetase was phosphorylated with protein kinase C and the P-labeled enzyme was subjected to phosphoamino acid analysis. Protein kinase C phosphorylated CTP synthetase on both serine and threonine residues (Fig. 4A). P-Labeled CTP synthetase was subjected to digestion with TPCK-trypsin followed by thin layer electrophoresis and chromatographic analysis. Fig. 5A showed that protease digestion of the protein kinase C-phosphorylated enzyme resulted in the appearance of several phosphopeptides.


Figure 4: Phosphoamino acid analysis of P-labeled CTP synthetase phoshorylated in vitro and in vivo. CTP synthetase (1 µg) was phosphorylated with protein kinase C (panel A) using [-P]ATP. CTP synthetase was immunoprecipitated from cell extracts of cells labeled with P (panel B). SDS-polyacrylamide gel slices containing P-labeled CTP synthetase were subjected to phosphoamino acid analysis as described in the text. The positions of the carrier standard phosphoamino acids are indicated in the figure. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine.




Figure 5: Phosphopeptide mapping analysis of P-labeled CTP synthetase phoshorylated in vitro and in vivo. CTP synthetase (1 µg) was phosphorylated with protein kinase C (panel A) using [-P]ATP. CTP synthetase was immunoprecipitated from cell extracts of cells labeled with P (panel B). SDS-polyacrylamide gel slices containing P-labeled CTP synthetase were digested with TPCK-trypsin. The resulting peptides were separated on cellulose thin layer sheets by electrophoresis (from left to right) in the first dimension and by chromatography (from bottom to top) in the second dimension.



Stoichiometry of Protein Kinase C Phosphorylation of CTP Synthetase

To determine the stoichiometry of the phosphorylation of CTP synthetase by protein kinase C, the phosphorylation reaction was carried out to completion. At the point of maximum phosphorylation, protein kinase C catalyzed the incorporation of 0.4 mol of phosphate/mol of CTP synthetase (Fig. 6). Given the results of the phosphopeptide mapping experiment, the stoichiometry of the reaction was low. We considered the possibility that the pure CTP synthetase preparation used in our studies contained a population of phospho and dephospho forms of the enzyme. CTP synthetase was treated with alkaline phosphatase to dephosphorylate the enzyme. The alkaline phosphatase-treated enzyme was then phosphorylated with protein kinase C, and the stoichiometry of the reaction was examined again. Protein kinase C catalyzed the incorporation of 2.2 mol of phosphate/mol of the alkaline phosphatase-treated CTP synthetase (Fig. 6).


Figure 6: Stoichiometry of protein kinase C phosphorylation of CTP synthetase. Native CTP synthetase and alkaline phosphatase (AP)-treated CTP synthetase were incubated with protein kinase C (4 pmol/min/ml) and [-P]ATP for 60 min. Following the incubations, samples were subjected to SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and autoradiography. The incorporation of phosphate into CTP synthetase was determined by scintillation counting of phosphorylated enzyme excised from the immunoblot.



Effects of Protein Kinase C and Alkaline Phosphatase on CTP Synthetase Activity

If the phosphorylation of CTP synthetase by protein kinase C was physiologically relevant, it might be expected that phosphorylation by protein kinase C would affect CTP synthetase activity. This question was examined by first phosphorylating CTP synthetase with protein kinase C followed by the measurement of CTP synthetase activity. In these experiments, CTP synthetase activity was measured with subsaturating concentrations (8) of ATP and UTP. In this manner we could simultaneously monitor for stimulatory or inhibitory effects of phosphorylation on CTP synthetase activity. Phosphorylation of CTP synthetase by protein kinase C resulted in a dose-dependent activation of CTP synthetase activity (Fig. 7). Incubation of CTP synthetase with 8 pmol/min/ml protein kinase C resulted in a 3-fold stimulation of CTP synthetase activity (Fig. 7).


Figure 7: Dose-dependent stimulation of CTP synthetase activity by protein kinase C. CTP synthetase was incubated with the indicated amounts (U = pmol/min) of protein kinase C for 10 min. Following the incubations, samples were diluted 5-fold, and CTP synthetase activity was measured as described in the text using 0.5 mM ATP and 0.2 mM UTP as substrates.



We questioned whether alkaline phosphatase treatment of CTP synthetase would result in an inactivation of enzyme activity. CTP synthetase was dephosphorylated with alkaline phosphatase bound to agarose beads. The dephosphorylation of the enzyme resulted in a an 80% decrease in CTP synthetase activity (Fig. 8). It was necessary to treat CTP synthetase with the bound form of alkaline phosphatase because phosphatase inhibitors used to inactivate alkaline phosphatase also inhibited CTP synthetase activity. Although the alkaline phosphatase-treated CTP synthetase could be rephosphorylated with protein kinase C (Fig. 6), only 40% of the native enzyme activity was recovered by rephosphorylation (Fig. 8). Other protein kinase phosphorylations may be necessary for the full activation of CTP synthetase activity by protein kinase C(28, 29) .


Figure 8: Inhibition of CTP synthetase activity by alkaline phosphatase. CTP synthetase activity was determined using native enzyme, alkaline phosphatase (AP)-treated enzyme, and alkaline phosphatase-treated enzyme that was rephosphorylated with protein kinase C (PKC) as indicated. CTP synthetase activity was measured as described in the text using 0.5 mM ATP and 0.2 mM UTP as substrates.



Phosphorylation of CTP Synthetase in Vivo

We addressed the question of whether CTP synthetase was phosphorylated in vivo. Cells were labeled with P followed by the immunoprecipitation of CTP synthetase from cell extracts with anti-CTP synthetase IgG antibodies. SDS-polyacrylamide gel electrophoresis of the immunoprecipitate, transfer to nitrocellulose paper, and autoradiographic analysis revealed that CTP synthetase was indeed phosphorylated in vivo. The identity of CTP synthetase in the immunoprecipitate was confirmed by immunoblot analysis. Immunoprecipitated P-labeled CTP synthetase was subjected to phosphoamino acid analysis. CTP synthetase was phosphorylated primarily on serine residues in vivo (Fig. 4B). TPCK-trypsin digestion of the enzyme phosphorylated in vivo yielded several phosphopeptides upon phosphopeptide mapping analysis (Fig. 5B). The phosphopeptide map of CTP synthetase phosphorylated in vivo was similar to the phosphopeptide map of the enzyme phosphorylated in vitro (Fig. 5A). There were also phosphopeptides in the map of CTP synthetase phosphorylated in vitro that were not present in the phosphopeptide map of the enzyme phosphorylated in vivo (Fig. 5).


DISCUSSION

CTP synthetase is an essential enzyme in S. cerevisiae(7) . The enzyme also plays an important role in the growth and metabolism of mammalian cells(30, 31, 32) . S. cerevisiae CTP synthetase (8) as well as mammalian (33, 34) and bacterial (2, 35) forms of the enzyme are activated by GTP and inhibited by CTP. To our knowledge, no other mechanisms have been described for the regulation of CTP synthetase activity. In this study, we showed that CTP synthetase from S. cerevisiae was phosphorylated by protein kinase C. Protein kinase C required calcium, DG, and PS as cofactors for maximum activity when purified CTP synthetase was used as a substrate. The protein kinase C reaction was dose- and time-dependent and dependent on the concentration of CTP synthetase. These results indicated that CTP synthetase was a substrate for protein kinase C.

The phosphorylation of CTP synthetase by protein kinase C in vitro was accompanied by a 3-fold stimulation of CTP synthetase activity. This extent of stimulation of CTP synthetase activity would be an underestimate of the overall effect protein kinase C phosphorylation had on activity if the enzyme was already partially phosphorylated(36) . The fact that the enzyme was shown to be phosphorylated in vivo suggested that this was the case. This was further supported by the relatively low stoichiometry of the reaction given the number of phosphopeptides, which resulted from the proteolysis of the enzyme phosphorylated in vitro and in vivo. Indeed, the alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold inactivation of activity and an increase in the stoichiometry of protein kinase C phosphorylation from 0.4 to 2.2 mol of phosphate/mol of enzyme.

Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro. However, CTP synthetase was primarily phosphorylated on serine residues in vivo. The number of phosphopeptides derived from CTP synthetase phosphorylated by protein kinase C in vitro was more than those derived from the enzyme phosphorylated in vivo. These additional phosphorylation sites may not be physiologically relevant. On the other hand, most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from the enzyme phosphorylated by protein kinase C in vitro. Thus, the common phosphopeptides derived from the enzyme labeled in vivo and in vitro raised the suggestion that CTP synthetase was a substrate for protein kinase C in vivo.

Protein kinase C is essential for progression of S. cerevisiae cell cycle (12) and plays a role in cell wall formation (37). In mammalian cells, protein kinase C is a transducer of lipid second messengers and is the receptor for phorbol ester and other tumor promoters(9, 10, 11) . Protein kinase C activity plays a central role in the regulation of a host of cellular functions through its activation by growth factors, hormones, and other agonists(38, 39, 40) . These functions include cell growth and proliferation(38, 39, 40) . The synthesis of CTP is essential for the synthesis of RNA, DNA, and membrane phospholipids(4, 5) . Thus, the phosphorylation and activation of CTP synthetase by protein kinase C may represent a mechanism by which lipid signal transduction pathways are linked to CTP synthesis needed for cell growth and proliferation.

In summary, we have shown that CTP synthetase from S. cerevisiae was phosphorylated and activated by protein kinase C. Our studies provided evidence that CTP synthetase was phosphorylated by protein kinase C in vivo. Future work will be directed toward defining the mechanism of CTP synthetase activation by protein kinase C and the role of this phosphorylation on cellular growth and metabolism in S. cerevisiae.


FOOTNOTES

*
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-10581-1-95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 908-932-9663; Fax: 908-932-6776; E-mail: george@a1.caft1vax.rutgers.edu.

The abbreviations used are: TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PS, phosphatidylserine; DG, diacylglycerol.


ACKNOWLEDGEMENTS

We thank Carlos Gonzalez and Charles Martin for providing assistance with PhosphorImager analysis. We also acknowledge Virginia McDonough for many helpful discussions during the course of this work.


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