From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901
Received for publication, February 10, 2003
, and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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The yeast CTP synthetase is regulated by genetic and biochemical mechanisms. Like many enzymes involved in macro-molecular 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 Glu161 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). Ser424 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 Ser36, Ser330, Ser354, and Ser454 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 alterations 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
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, and Growth ConditionsThe 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 µg/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.
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DNA Manipulations, Amplification of DNA by PCR, and DNA SequencingPlasmid 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 PlasmidsThe codons for Ser36, Ser330, Ser354, and Ser454 in the URA7-encoded CTP synthetase were changed to alanine codons by site-directed mutagenesis. The URA7S36A (primers: 5'-CCCTCGGTTTAAAGGTTACcgCcATTAAAATTGACCCTTATATGA-3' and 5'-TCATATAAGGGTCAATTTTAATgGcgGTAACCTTTAAACCGAGGG-3'), URA7S330A (primers: 5'-GCATTGGAACATTCAgCaATGAAGTGTCGTCGTAAG-3' and 5'-CTTACGACGACACTTCATtGcTGAATGTTCCAATGC-3'), URA7S354A (primers: 5'-GGAACCTGAAGCACAAGAAgcCAACAAAACTAAATTTCATG-3' and 5'-CATGAAATTTAGTTTTGTTGgcTTCTTGTGCTTCAGGTTCC-3'), and URA7S454A (primers: 5'-GGAAACCATGGGGGGCgCAATGAGATTAGGTTT-3' and 5'-AAACCTAATCTCATTGcGCCCCCCATGGTTTCC-3') 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 URA7-encoding 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 URA7- and URA8-encoded enzymes.
In Vivo Labeling of CTP SynthetaseCells 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
32Pi (0.3 mCi/ml) and U-14C-labeled
L amino acids (5 µCi/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 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).
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 SynthetasesCells 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 x g for 5 min. The cytosol was obtained by centrifugation at 100,000 x 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 x 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 x 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 x 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 (01.5 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 80 °C in buffer B with the glycerol concentration raised to 25%. The enzyme preparations were stable for at least 6 months when stored at 80 °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 CuCl2 (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 CThe 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 ImmunoblottingStandard 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 SynthetaseThe 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 CPhosphorylation of CTP synthetase was performed in a total
volume of 40 µl at 30 °C. CTP synthetase (1 µg) was incubated for 15
min with 50 mM Tris-HCl (pH 8.0), 50 µM
[-32P]ATP (4 µCi/nmol), 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
0.1 nmol/min/ml protein kinase C
(35). At the end of the
phosphorylation reactions, samples were treated with an equal volume of
2x 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 [-32P]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 µl 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 µl 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 DeterminationCTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M1 cm1, 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 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 0.2 ml. Enzyme assays were performed in triplicate with an average S.D. 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 (55) using bovine serum albumin as the standard.
Analysis of CTPCells 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 PhospholipidsPhospholipids were labeled with 32Pi and [methyl-3H]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 DataKinetic data were analyzed with the EZ-FIT enzyme kinetic model-fitting program (62). Statistical analyses were performed with SigmaPlot software.
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RESULTS |
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Construction and Characterization of CTP Synthetase S36A, S330A, S354A,
and S454A MutantsMutagenesis of Ser36,
Ser330, Ser354, and Ser454 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.
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Cells expressing the wild type and mutant CTP synthetase alleles on the single-copy plasmid were labeled with 32Pi to detect phosphorylated CTP synthetase and with U-14C-labeled L-amino acids to normalize for the amount of CTP synthetase isolated. The ratio of counts/min of 32P incorporated into CTP synthetase to the counts of 14C 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 Purification of the S36A, S330A, S354A, and S454A Mutant CTP SynthetasesThe wild type and the S36A, S330A, S354A, and S454A mutant CTP synthetase enzymes were purified to examine the effects of the mutations on the properties of the enzyme. The purification scheme developed by Yang et al. (5) was modified to obtain partially purified preparations of the enzyme in a relatively short amount of time. A 50-fold overexpression of the URA7 gene on the multicopy plasmid facilitated the purification of the enzymes. The purification scheme included ammonium sulfate fractionation of the cytosol 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 (1727%) and degree of purification (2026-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 2.32.5 units/mg (5, 36).
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Effects of the S36A, S330A, S354A, and S454A Mutations on the Phosphorylation of CTP Synthetase by Protein Kinase C in VitroThe 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 32P-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 Ser330 when compared with the other peptide substrates.
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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 derived from the wild type CTP synthetase were c and d. Ofthe 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, Ser330 should be contained in phosphopeptide d.
Effects of the S36A, S330A, S354A, and S454A Mutations on the Enzymic Properties of CTP SynthetaseA 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 Vmax 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 Vmax 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 Km value for ATP (Table III). The mutations did not affect the Hill number (n = 1.4) for UTP.
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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 IC50 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 SynthetaseIn 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 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.
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Effects of the S36A, S330A, S354A, and S454A Mutations in CTP Synthetase on the Cellular Concentration of CTPWe 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).
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Effects of the S36A, S330A, S354A, and S454A Mutations in CTP
Synthetase on Phospholipid CompositionCTP is essential for the
synthesis of PC, the major membrane phospholipid in S. cerevisiae
(64,
65,
66,
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,
65,
66,
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
phosphatidylserine
phosphatidylethanolamine
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,
70,
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
32Pi and [methyl-3H]choline. The
32Pi 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 µM, a concentration too low to affect
the rate of synthesis of PC by the CDP-choline pathway
(73).
The 32P-labeling experiments showed that the S36A, S330A, S354A, and S454A mutations did not have any major effects on phospholipid composition. The 3H-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 3H incorporated into PC to the cpm of 32P incorporated into PC. This allowed us to determine whether the mutations affected the pathways by which cells synthesized PC (17, 30). The 3H/32P ratio decreased in cells bearing the S36A, S354A, and S454A mutations by 35, 20, and 19%, respectively, whereas the 3H/32P 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.
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DISCUSSION |
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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 Ser36,
Ser330, Ser354, and Ser454 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
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 Ser330 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 Vmax 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 Vmax of the reaction. These data raised the suggestion that the phosphorylation of Ser330 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 Ser36 and Ser354 on the nucleotide-dependent oligomerization of CTP synthetase. This analysis indicated that phosphorylation of Ser330 and Ser454 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 Ser36, Ser330, Ser354, and Ser454 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, S330A, S354A, and S454A mutant CTP synthetase enzymes were within the range of the Km 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 Km 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 CDP-diacylglycerol 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, 65, 66, 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.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Food Science, Rutgers
University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext.
217); Fax: 732-932-6776; E-mail:
carman{at}aesop.rutgers.edu.
1 The abbreviations used are: PC, phosphatidylcholine; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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ACKNOWLEDGMENTS |
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REFERENCES |
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