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• W2102259689 abstract "The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway. The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway. CTP synthetase is an essential enzyme in all organisms. The essential nature of this enzyme emanates from the fact that the product of its reaction CTP is required for the synthesis of nucleic acids and membrane phospholipids (1Stryer L. Biochemistry. Fourth Ed. W. H. Freeman and Company, New York1995Google Scholar). The enzyme catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (2Liberman I. J. Biol. Chem. 1956; 222: 765-775Google Scholar, 3Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Google Scholar). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (3Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Google Scholar, 4Levitzki A. Koshland Jr., D.E. Biochemistry. 1972; 11: 241-246Google Scholar, 5Bearne S.L. Hekmat O. Macdonnell J.E. Biochem. J. 2001; 356: 223-232Google Scholar, 6Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Google Scholar). In eukaryotic cells, regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (6Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Google Scholar, 7Aronow B. Ullman B. J. Biol. Chem. 1987; 262: 5106-5112Google Scholar, 8Robert de Saint Vincent B. Buttin G. Biochim. Biophys. Acta. 1980; 610: 352-359Google Scholar, 9Meuth M. L'Heureux-Huard N. Trudel M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6505-6509Google Scholar, 10Ozier-Kalogeropoulos O. Fasiolo F. Adeline M.-T. Collin J. Lacroute F. Mol. Gen. Genet. 1991; 231: 7-16Google Scholar, 11Ozier-Kalogeropoulos O. Adeline M.-T. Yang W.-L. Carman G.M. Lacroute F. Mol. Gen. Genet. 1994; 242: 431-439Google Scholar, 12Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Google Scholar) and in the synthesis of membrane phospholipids (12Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Google Scholar, 13Hatch G.M. McClarty G. J. Biol. Chem. 1996; 271: 25810-25816Google Scholar, 14McDonough V.M. Buxeda R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Google Scholar). The importance of understanding the regulation of CTP synthetase is further emphasized by the fact that unregulated levels of CTP synthetase activity is a common property of various human cancers (15van den Berg A.A. van Lenthe H. Busch S. de Korte D. Roos D. van Kuilenburg A.B.P. van Gennip A.H. Eur. J. Biochem. 1993; 216: 161-167Google Scholar, 16van den Berg A.A. van Lenthe H. Kipp J.B. de Korte D. Van Kuilenburg A.B. van Gennip A.H. Eur. J. Cancer. 1995; 31A: 108-112Google Scholar, 17Verschuur A.C. van Gennip A.H. Muller E.J. Voute P.A. Van Kuilenburg A.B. Adv. Exp. Med. Biol. 1998; 431: 667-671Google Scholar, 18Kizaki H. Williams J.C. Morris H.P. Weber G. Cancer Res. 1980; 40: 3921-3927Google Scholar, 19Weber G. Lui M.S. Takeda E. Denton J.E. Life Sci. 1980; 27: 793-799Google Scholar, 20Weber G. Olah E. Lui M.S. Tzeng D. Adv. Enzyme Regul. 1979; 17: 1-21Google Scholar, 21Verschuur A.C. van Gennip A.H. Brinkman J. Voute P.A. Van Kuilenburg A.B. Adv. Exp. Med. Biol. 2000; 486: 319-325Google Scholar, 22Verschuur A.C. Brinkman J. van Gennip A.H. Leen R. Vet R.J. Evers L.M. Voute P.A. Van Kuilenburg A.B. Leuk. Res. 2001; 25: 891-900Google Scholar).We utilize the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and the impact of this regulation on phospholipid synthesis (Fig. 1). In yeast, CTP synthetase is encoded by the URA7 (10Ozier-Kalogeropoulos O. Fasiolo F. Adeline M.-T. Collin J. Lacroute F. Mol. Gen. Genet. 1991; 231: 7-16Google Scholar) and URA8 (11Ozier-Kalogeropoulos O. Adeline M.-T. Yang W.-L. Carman G.M. Lacroute F. Mol. Gen. Genet. 1994; 242: 431-439Google Scholar) genes. The yeast CTP synthetases (10Ozier-Kalogeropoulos O. Fasiolo F. Adeline M.-T. Collin J. Lacroute F. Mol. Gen. Genet. 1991; 231: 7-16Google Scholar, 11Ozier-Kalogeropoulos O. Adeline M.-T. Yang W.-L. Carman G.M. Lacroute F. Mol. Gen. Genet. 1994; 242: 431-439Google Scholar) contain a conserved glutamine amide transfer domain common to CTP synthetases from other organisms (24Yamauchi M. Yamauchi N. Meuth M. EMBO J. 1990; 9: 2095-2099Google Scholar, 25Weng M. Makaroff C.A. Zalkin H. J. Biol. Chem. 1986; 261: 5568-5574Google Scholar, 26Tipples G. McClarty G. J. Biol. Chem. 1995; 270: 7908-7914Google Scholar, 27Trach K. Chapman J.W. Piggot P. Lecoq D. Hoch J.A. J. Bacteriol. 1988; 170: 4194-4208Google Scholar, 28Van Kuilenburg A.B. Meinsma R. Vreken P. Waterham H.R. van Gennip A.H. Adv. Exp. Med. Biol. 2000; 486: 257-261Google Scholar, 29Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Google Scholar, 30Van Kuilenburg A.B. Meinsma R. Vreken P. Waterham H.R. van Gennip A.H. Biochim. Biophys. Acta. 2000; 1492: 548-552Google Scholar, 31Hendriks E.F. O'Sullivan W.J. Stewart T.S. Biochim. Biophys. Acta. 1998; 1399: 213-218Google Scholar, 32Mahony T.J. Miller D.J. FEMS Microbiol. Lett. 1998; 165: 153-157Google Scholar, 33Willemoes M. J. Biol. Chem. 2003; 278: 9407-9411Google Scholar). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (34Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Google Scholar) and is responsible for the majority of the CTP synthesized in vivo (11Ozier-Kalogeropoulos O. Adeline M.-T. Yang W.-L. Carman G.M. Lacroute F. Mol. Gen. Genet. 1994; 242: 431-439Google Scholar). Like CTP synthetase from mammalian cells (35Van Kuilenburg A.B. Elzinga L. van Gennip A.H. Adv. Exp. Med. Biol. 1998; 431: 255-258Google Scholar), the yeast enzymes are allosterically regulated by their substrates and product CTP (6Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Google Scholar, 34Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Google Scholar).The S. cerevisiae URA7-encoded CTP synthetase is also regulated by phosphorylation. In vivo, CTP synthetase is phosphorylated on multiple serine residues (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar). In vitro studies have shown that CTP synthetase is a substrate for protein kinase A (37Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Google Scholar) and for protein kinase C (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar, 38Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Google Scholar). In S. cerevisiae, protein kinase A is the principal mediator of signals transmitted through the Ras-cAMP pathway (39Broach J.R. Deschenes R.J. Adv. Cancer Res. 1990; 54: 79-139Google Scholar, 40Thevelein J.M. Yeast. 1994; 10: 1753-1790Google Scholar) whereas protein kinase C is required for the cell cycle (41Nishizuka Y. Nature. 1984; 308: 693-698Google Scholar, 42Nishizuka Y. Science. 1992; 258: 607-614Google Scholar, 43Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Google Scholar, 44Levin D.E. Fields F.O. Kunisawa R. Bishop J.M. Thorner J. Cell. 1990; 62: 213-224Google Scholar, 45Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Google Scholar) and plays a role maintaining cell wall integrity (46Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Google Scholar). Independently, the phosphorylation of CTP synthetase by protein kinase A (37Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Google Scholar) and by protein kinase C (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar, 38Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Google Scholar) results in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover and decreases enzyme sensitivity to CTP product inhibition.In this work, we addressed the question of whether the phosphorylation of CTP synthetase by protein kinase A affects the phosphorylation by protein kinase C. Amino acid residue Ser424 has been identified as the target site for protein kinase A phosphorylation in CTP synthetase (47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). Therefore, we utilized a S424A mutant CTP synthetase enzyme for our studies. The S424A mutant enzyme is not phosphorylated in response to the activation of protein kinase A in vivo, and the mutant enzyme is not phosphorylated and stimulated by protein kinase A in vitro (47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). This mutant enzyme exhibits lower catalytic activity and greater sensitivity to CTP product inhibition when compared with the wild type enzyme (47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). These properties are consistent with the effects that protein kinase A phosphorylation has on the activity of wild type CTP synthetase (37Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Google Scholar, 47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). We showed here that the S424A mutation reduced the ability of CTP synthetase to be a substrate for protein kinase C. An explanation for this effect was that Ser424 was also a target site for protein kinase C phosphorylation. We also showed that cells bearing the S424A mutant CTP synthetase enzyme exhibited a decrease in the synthesis of the membrane phospholipid PC 1The abbreviations used are: PC, phosphatidylcholine; PVDF, polyvinylidene difluoride. 1The abbreviations used are: PC, phosphatidylcholine; PVDF, polyvinylidene difluoride. via the CDP-choline pathway.EXPERIMENTAL PROCEDURESMaterials—All chemicals were reagent grade. Growth medium supplies were purchased from Difco Laboratories. Nucleotides, l-glutamine, phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, pepstatin, histone, casein, choline, phosphocholine, CDP-choline, and bovine serum albumin were purchased from Sigma. PVDF paper was from Amersham Biosciences. Protein kinase C (rat brain) and protein kinase A catalytic subunit (bovine heart) were purchased from Promega. Protein assay reagent, electrophoresis reagents, immunochemical reagents, and protein molecular mass markers were purchased from Bio-Rad. Phosphocellulose filters were purchased from Pierce. Radiochemicals were purchased from PerkinElmer Life Sciences. Scintillation counting supplies and acrylamide solutions were from National Diagnostics. Phospholipids were from Avanti Polar Lipids. Silica Gel 60 thin-layer chromatography plates and cellulose thin-layer glass plates were purchased from EM Science. The peptide SLGRKDSHSA was synthesized and purified commercially by BioSynthesis, Inc.Strain and Growth Conditions—The wild type URA7 and mutant URA7S424A alleles coding for CTP synthetase were expressed from multicopy (pTP1 and pTP2, respectively) and single copy (pTP3 and pTP4, respectively) plasmids in the ura7 ura8 double mutant strain OK8 (47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). Methods for growth and analysis of yeast were performed as described previously (48Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 49Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast cultures were grown in complete synthetic medium minus inositol (50Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Google Scholar) containing 2% glucose at 30 °C. Yeast cell numbers were determined by microscopic examination with a hemacytometer or spectrophotometrically at an absorbance of 600 nm.Purification of Wild Type and S424A Mutant CTP Synthetases— Cells expressing the wild type and S424A mutant CTP synthetases from multicopy plasmids were used for enzyme purification. The enzymes were purified by the method of Yang et al. (6Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Google Scholar) with the following modifications. The Sephacryl 300 HR chromatography step was replaced by dialysis and the Superose 6 chromatography step was replaced by Mono Q chromatography. This procedure resulted in the isolation of nearly homogeneous enzyme preparations as evidenced by SDS-PAGE.Enzyme Assays and Protein Determination—CTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 m–1 cm–1, respectively) by following the increase in absorbance at 291 nm on a recording spectrophotometer (3Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Google Scholar). The standard reaction mixture contained 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 10 mm 2-mercaptoethanol, 2 mm l-glutamine, 0.1 mm GTP, 2 mm ATP, 2 mm UTP, and an appropriate dilution of enzyme protein in a total volume of 0.1 ml. Enzyme assays were performed in triplicate with an average standard deviation of ± 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 product/min. Protein concentration was estimated by the method of Bradford (51Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as the standard.Electrophoresis and Immunoblotting—SDS-PAGE (52Laemmli U.K. Nature (London). 1970; 227: 680-685Google Scholar) was performed with 10% slab gels. Molecular mass standards for electrophoresis 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. Immunoblot assays were performed with IgG anti-URA7-encoded (6Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Google Scholar) CTP synthetase antibodies as described previously (53Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Google Scholar). The density of the URA7-encoded CTP synthetase bands on immunoblots was quantified by scanning densitometry. Immunoblot signals were in the linear range of detectability.Phosphorylation Reactions—Phosphorylation reactions were routinely measured for 10 min at 30 °C in a total volume of 40 μl. The indicated concentrations of purified wild type and S424A mutant CTP synthetase enzymes or synthetic peptide were phosphorylated with the indicated concentrations of protein kinase C in a reaction mixture that contained 50 mm Tris-HCl buffer (pH 8.0), 10 mm MgCl2, 10 mm 2-mercaptoethanol, 0.375 mm EDTA, 0.375 mm EGTA, 1.7 mm CaCl2, 20 μm diacylglycerol, 50 μm phosphatidylserine, and 50 μm [γ-32P]ATP (5,000 cpm/pmol). CTP synthetase was phosphorylated with protein kinase A (0.2 unit/ml) in a reaction mixture that contained 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, and 50 μm [γ-32P]ATP (5,000 cpm/pmol). Samples containing 32P-labeled CTP synthetase were treated with an equal volume of 4× Laemmli sample buffer (52Laemmli U.K. Nature (London). 1970; 227: 680-685Google Scholar) followed by SDS-PAGE, transfer to PVDF paper, and visualized by phosphorimaging. The extent of phosphorylation was analyzed using ImageQuant software. Phosphorylation signals were in the linear range of detectability. Reactions containing the synthetic peptide were terminated by spotting an aliquot of the reaction mixture onto phosphocellulose filters. The filters were washed with 75 mm phosphoric acid and subjected to scintillation counting. Phosphorylation reactions were performed in triplicate.Tryptic Digestion and Two-dimensional Peptide Mapping—Pieces of PVDF paper containing 32P-labeled CTP synthetase were subjected to digestion with l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin and two-dimensional peptide mapping analysis as described by MacDonald and Kent (54MacDonald J.I.S. Kent C. J. Biol. Chem. 1994; 269: 10529-10537Google Scholar). Electrophoresis (1% ammonium bicarbonate buffer at 1000 volts for 20 min) and ascending chromatography (n-butyl alcohol/glacial acetic acid/pyridine/water, 10:3:12:15) were performed on cellulose thin-layer glass plates. Dried plates were then subjected to phosphorimaging analysis.Analysis of Phospholipids—Phospholipids were labeled with 32Pi and [methyl-3H]choline as described previously (14McDonough V.M. Buxeda R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Google Scholar, 55Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Google Scholar, 56Homann M.J. Poole M.A. Gaynor P.M. Ho C.-T. Carman G.M. J. Bacteriol. 1987; 169: 533-539Google Scholar). Phospholipids were extracted from cells by the method of Bligh and Dyer (57Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) as described previously (58Morlock K.R. Lin Y.-P. Carman G.M. J. Bacteriol. 1988; 170: 3561-3566Google Scholar). Phospholipids were separated by two-dimensional thin-layer chromatography using silica gel 60 thin-layer chromatography plates. The solvent systems for dimensions one and two were chloroform/methanol/glacial acetic acid (65:25:10, v/v) and chloroform/methanol/88% formic acid (65:25:10, v/v), respectively (59Esko J.D. Raetz C.R.H. J. Biol. Chem. 1980; 255: 4474-4480Google Scholar). 32P-Labeled phospholipids were visualized by phosphorimaging analysis. The positions of the labeled lipids on chromatography plates were compared with standard phospholipids after exposure to iodine vapor. The amount of each labeled phospholipid was determined by liquid scintillation counting of the corresponding spots on the chromatograms.Analysis of CDP-choline Pathway Intermediates—Labeling of the CDP-choline pathway intermediates with [methyl-3H]choline was performed as described by McDonough et al. (14McDonough V.M. Buxeda R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Google Scholar). Choline, phosphocholine, and CDP-choline were isolated from whole cells following lipid extraction (57Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). The aqueous phase was neutralized and dried in vacuo, and the residue was dissolved in deionized water. Samples were centrifuged for 3 min at 12,000 × g to remove insoluble material. The CDP-choline pathway intermediates were separated by thin-layer chromatography with silica gel 60 plates using the solvent system methanol/0.5% sodium chloride/ammonia (50:50:1) (60Teegarden D. Taparowsky E.J. Kent K. J. Biol. Chem. 1990; 265: 6042-6047Google Scholar). The intermediates were detected on chromatograms by fluorography using EN3HANCE and compared with standards. Liquid scintillation counting was used to quantify the amounts of the intermediates.Data Analyses—Kinetic data were analyzed according to the Michaelis-Menten equation using the EZ-FIT enzyme kinetic model-fitting program (61Perrella F. Anal. Biochem. 1988; 174: 437-447Google Scholar). Statistical analyses were performed with SigmaPlot 5.0 software.RESULTSEffect of the S424A Mutation on the Phosphorylation and Stimulation of CTP Synthetase by Protein Kinase C—The URA7-encoded CTP synthetase is phosphorylated by protein kinase A (37Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Google Scholar) and by protein kinase C (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar, 38Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Google Scholar). Residue Ser424 in the enzyme has been identified as the protein kinase A target site (47Park T.-S. Ostrander D.B. Pappas A. Carman G.M. Biochemistry. 1999; 38: 8839-8848Google Scholar). We questioned whether phosphorylation at Ser424 affects phosphorylation of the enzyme by protein kinase C. Accordingly, we analyzed the phosphorylation of the S424A mutant CTP synthetase by protein kinase C. Protein kinase C was incubated with [γ-32P]ATP and various concentrations of the purified S424A mutant and wild type CTP synthetase enzymes. After the phosphorylation reactions, samples were subjected to SDS-PAGE and transferred to PVDF paper, followed by phosphorimaging analysis. Protein kinase C activity was dependent on the concentration of both the wild type and mutant forms of CTP synthetase (Fig. 2). The S424A mutation caused a decrease in enzyme phosphorylation at each CTP synthetase concentration (Fig. 2). The apparent Km value for the S424A mutant enzyme (50 μg/ml) was 20-fold higher than that of the wild type enzyme (2.5 μg/ml). The effect of the S424A mutation on the dependence of protein kinase C activity on ATP concentration was also examined (Fig. 3). The extent of CTP synthetase phosphorylation at each ATP concentration was reduced for the S424A mutant enzyme when compared with the wild type enzyme. The apparent Km value for the mutant enzyme (25 μm) was 2-fold higher when compared with the wild type enzyme (Km = 12.5 μm). The phosphorylation reactions using the S424A mutant CTP synthetase as a substrate were performed for different time intervals and with various concentrations of protein kinase C. The phosphorylation of the S424A mutant CTP synthetase by protein kinase C was time-dependent (Fig. 4) and dose-dependent (Fig. 5), but the rate and extent of phosphorylation was reduced by about 50% when compared with the wild type enzyme.Fig. 2Effect of the S424A mutation on the dependence of protein kinase C activity on the concentration of CTP synthetase. The indicated concentrations of the purified wild type (WT) and S424A mutant CTP synthetases were incubated with protein kinase C and [γ-32P]ATP for 10 min. Following the incubations, samples were subjected to SDS-PAGE and transferred to PVDF paper. The phosphorylated proteins were subjected to phosphorimaging analysis. Panel A, portions of the images showing the phosphorylation of CTP synthetase are shown. The arrow in the figures denotes the position of CTP synthetase. The position and amounts of the wild type and S424A mutant CTP synthetases on the PVDF papers were confirmed by immunoblot analysis. The data shown are representative of two independent experiments. Panel B, the relative amounts of phosphate incorporated into CTP synthetase were quantified using ImageQuant software where the maximum phosphorylation of the wild type CTP synthetase protein was set as 100. The figure shows the double reciprocal plot of the data.View Large Image Figure ViewerDownload (PPT)Fig. 3Effect of the S424A mutation on the dependence of protein kinase C activity on the concentration of ATP. Purified wild type (WT) and S424A mutant CTP synthetases (0.8 μg each) were incubated with protein kinase C and the indicated concentrations of [γ-32P]ATP for 10 min. The phosphorylated samples were analyzed as described in the legend to Fig. 2. The data shown are representative of two independent experiments. The figure shows the double reciprocal plot of the data.View Large Image Figure ViewerDownload (PPT)Fig. 4Effect of the S424A mutation on the time-dependent phosphorylation of CTP synthetase by protein kinase C. Purified wild type (WT) and S424A mutant CTP synthetases (0.8 μg each) were incubated with protein kinase C and [γ-32P]ATP for the indicated time intervals. The phosphorylated samples were analyzed as described in the legend to Fig. 2. The data shown are representative of two independent experiments.View Large Image Figure ViewerDownload (PPT)Fig. 5Effect of the S424A mutation on the dose-dependent phosphorylation of CTP synthetase by protein kinase C. Purified wild type (WT) and S424A mutant CTP synthetases (0.8 μg each) were incubated with [γ-32P]ATP and the indicated concentrations of protein kinase C for 10 min. The phosphorylated samples were analyzed as described in the legend to Fig. 2. The data shown are representative of two independent experiments.View Large Image Figure ViewerDownload (PPT)The effect of the S424A mutation on the stimulation of CTP synthetase activity by protein kinase C was examined. The purified S424A mutant and wild type CTP synthetase enzymes were phosphorylated with various concentrations of protein kinase C for 10 min. Following the phosphorylation reactions, samples were assayed for CTP synthetase activity using subsaturating concentrations of UTP and ATP. These assay conditions were used to accentuate the effect of phosphorylation on the stimulation of CTP synthetase activity (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar, 38Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Google Scholar). As described previously (36Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Google Scholar, 38Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Google Scholar), protein kinase C phosphorylation of the wild type enzyme resulted in a dose-dependent stimulation of CTP synthetase activity (Fig. 6). The activity of the S424A mutant enzyme was also stimulated by protein kinase C phosphorylation; however, the extent of stimulation was much reduced (Fig. 6). At the highest protein kinase C concentration, the stimulation of CTP synthetase activity was reduced by 80%.Fig. 6Effect of the S424A mutation on the stimulation of CTP synthetase activity by protein kinase C" @default.
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• W2102259689 date "2003-06-01" @default.
• W2102259689 modified "2023-10-17" @default.
• W2102259689 title "Phosphorylation of Saccharomyces cerevisiae CTP Synthetase at Ser424 by Protein Kinases A and C Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway" @default.
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• W2102259689 doi "https://doi.org/10.1074/jbc.m303337200" @default.
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