(Received for publication, January 19, 1996; and in revised form, February 22, 1996)
From the
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is
an allosterically regulated enzyme in the yeast Saccharomyces
cerevisiae. In this work we examined the regulation of CTP
synthetase activity by S. cerevisiae protein kinase C (Pkc1p)
phosphorylation. The results of labeling experiments with S.
cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated
by Pkc1p in vivo. In vitro, Pkc1p phosphorylated
purified CTP synthetase on serine and threonine residues, which
resulted in the activation (3-fold) of enzyme activity. The mechanism
of this activation involved an increase in the apparent V of the reaction and an increase in the
enzyme's affinity for ATP. In vitro phosphorylated CTP
synthetase also exhibited a decrease in its positive cooperative
kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP
synthetase did not have a significant effect on the kinetic properties
of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP
synthetase resulted in a decrease in the enzyme's sensitivity to
product inhibition by CTP. Phosphorylation did not affect the mechanism
by which CTP inhibits CTP synthetase activity.
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme that is essential for the growth and metabolism of cells. The product of its reaction, CTP, is required for the synthesis of RNA, DNA, phospholipids, and sialoglycoproteins(1) . CTP synthetase catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (2, 3) . GTP activates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate(3, 4) . Genes encoding for CTP synthetase have been isolated from Escherichia coli(5) , Chlamydia trachomatis(6) , Bacillus subtilis(7) , Saccharomyces cerevisiae(8, 9) , and human cells(10) . The deduced amino acid sequences of the cloned CTP synthetase genes have a relatively high degree of identity, including a conserved glutamine amide transfer domain characteristic of glutamine amidotransferases(5, 6, 7, 8, 9, 10) .
CTP synthetases have been purified to apparent homogeneity from E. coli(3, 11) , S. cerevisiae(12, 13) , and rat liver cells(14) . Purified CTP synthetases exist as dimers that oligomerize to tetramers in the presence of their substrates UTP and ATP(3, 12, 13, 14, 15) . CTP synthetases from E. coli(3, 4, 16, 17, 18, 19) and S. cerevisiae(12, 13) exhibit positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP. The positive cooperative kinetics toward UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme(3, 15) .
A characteristic common to the pure CTP synthetases is the inhibition of their activities by the product CTP(3, 12, 13, 14) . CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP(12, 13, 14) . Regulation of CTP synthetase activity by CTP inhibition plays an important role in vivo. For example, mutant mammalian cell lines with CTP synthetase activity insensitive to CTP inhibition exhibit abnormally high intracellular levels of CTP and dCTP(20, 21) , resistance to nucleotide analog drugs used in cancer chemotherapy(22, 23, 24, 25) , and an increased rate of spontaneous mutations (23, 25, 26) . In addition, elevated CTP synthetase activity is a common property of leukemic cells (27) and rapidly growing tumors found in liver(28) , colon(29) , and lung(30) .
In S. cerevisiae, CTP synthetase is encoded by the URA7 and URA8 genes(8, 9) . Comparison of the nucleotide and deduced amino acid sequences of the open reading frames of the URA7 and URA8 genes show 70 and 78% identity, respectively(8, 9) . Biochemical characterization of the purified enzymes (12, 13) and phenotypic analysis of ura7 and ura8 mutants (9) have shown that the two CTP synthetases are not functionally identical. Our studies on the pure URA7-encoded enzyme have revealed that CTP synthetase is also regulated by protein kinase C phosphorylation(31) . Protein kinase C is a transducer of lipid second messengers (32, 33, 34) and plays a central role in the regulation of a host of cellular functions including cell growth and proliferation(35, 36, 37) . Rat brain protein kinase C phosphorylates the yeast CTP synthetase in vitro on serine and threonine residues, which results in an activation of CTP synthetase activity(31) . In this study we demonstrated that CTP synthetase was a substrate for the S. cerevisiae protein kinase C (Pkc1p). Our data also demonstrated that this phosphorylation regulated CTP synthetase activity by changing the kinetic properties of the enzyme and its sensitivity to inhibition by CTP.
Figure 1:
Effect of Pkc1p expression on the
phosphorylation of CTP synthetase. Wild-type cells, cells defective in
the expression of Pkc1p (pkc1::LEU2), and cells
that overexpress Pkc1p (YEp352 [PKC1]) were labeled
with
P
and
[
C(U)]L-amino acids. CTP synthetase was
immunoprecipitated from cells using anti-CTP synthetase antibodies. CTP
synthetase was dissociated from the enzyme-antibody complex, and the
amount of the label incorporated into CTP synthetase was determined by
scintillation counting. The values are reported as the cpm of
P incorporated into CTP synthetase relative to the cpm of
C incorporated into CTP synthetase. The data are from an
average of two independent growth studies.
We examined if
purified CTP synthetase was a substrate for Pkc1p in vitro.
These studies were performed with a purified preparation of a ZZ-tagged
Pkc1p(45) . The ZZ-tag facilitates the purification of Pkc1p
but does not alter the biochemical properties of the
enzyme(41, 45) . We examined the phosphorylation of
CTP synthetase with Pkc1p under the conditions used to phosphorylate
the enzyme with rat brain protein kinase C(31) . Pkc1p
catalyzed the incorporation of the -phosphate of
P-labeled ATP into CTP synthetase (Fig. 2A, lane 2). The omission of the protein
kinase C cofactors calcium, diacylglycerol, and phosphatidylserine from
the phosphorylation reaction resulted in a 70% decrease in the
phosphorylation of CTP synthetase (Fig. 2A, lane
4). (
)CTP synthetase was phosphorylated on serine and
threonine residues (Fig. 2B), and the stoichiometry of
phosphorylation was 0.4 mol of phosphate/mol enzyme.
Figure 2:
Phosphorylation of CTP synthetase by
Pkc1p and phosphoamino acid analysis of the P-labeled
enzyme. A, CTP synthetase (0.8 µg) was incubated with
Pkc1p and [
-
P]ATP for 10 min in the
presence (lane 2) and the absence (lane 4) of protein
kinase C cofactors (1.7 mM calcium, 20 µM diacylglycerol, and 50 µM phosphatidylserine).
Following the incubations, samples were subjected to SDS-polyacrylamide
gel electrophoresis, immunoblot analysis, and autoradiography. The
position of phosphorylated CTP synthetase was confirmed by immunoblot
analysis and is indicated by the arrow. Lane 1 shows the
position of
C-labeled bovine serum albumin (66.2 kDa)
standard. Lane 3 did not contain CTP synthetase. B,
CTP synthetase (0.8 µg) was phosphorylated with Pkc1p using
[
-
P]ATP and subjected to SDS-polyacrylamide
gel electrophoresis. An SDS-polyacrylamide gel slice containing
P-labeled CTP synthetase was 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.
The effect of phosphorylation on CTP synthetase activity was measured. Phosphorylation of the enzyme resulted in a dose-dependent activation (3-fold) of CTP synthetase activity (Fig. 3). Maximum activation of activity occurred when the enzyme was measured with subsaturating concentrations of UTP and ATP (Fig. 3). When CTP synthetase activity was measured with saturating concentrations of UTP and ATP, Pkc1p did not have a significant effect on CTP synthetase activity (Fig. 3). Overall, these results were similar to those reported for rat brain protein kinase C phosphorylation of yeast CTP synthetase(31) . This indicated that the yeast Pkc1p and the rat brain protein kinase C phosphorylated and activated yeast CTP synthetase in a similar manner.
Figure 3:
Activation of CTP synthetase activity by
Pkc1p. CTP synthetase was incubated with the indicated amounts (U = nmol/min) of Pkc1p for 10 min. Following the incubations,
samples were diluted 5-fold, and CTP synthetase activity was measured
as described in the text using subsaturating () concentrations of
ATP (0.5 mM) and UTP (0.2 mM) and saturating (
)
concentrations of ATP (2 mM) and UTP (2 mM). The
concentrations of glutamine, GTP, and MgCl
were maintained
at 2, 0.1, and 10 mM,
respectively.
Figure 4:
Effect of phosphorylation on the kinetics
of CTP synthetase activity with respect to UTP. Phosphorylated ()
and native (
) CTP synthetase activities were measured as a
function of the concentration of UTP using 0.5 (A) and 2
mM ATP (B). The concentrations of glutamine, GTP, and
MgCl
were maintained at 2, 0.1, and 10 mM,
respectively.
We next
examined the effect of phosphorylation on the dependence of CTP
synthetase activity on ATP using subsaturating and saturating
concentrations of UTP. At the subsaturating UTP concentration, the
phosphorylation of CTP synthetase altered the apparent K of the reaction and the positive cooperative kinetic behavior of
the enzyme (Fig. 5A and Table 1). Under these
conditions, the apparent K
value for ATP of the
phosphorylated enzyme was 2.5-fold lower than the apparent K
value for the native enzyme. Phosphorylated CTP
synthetase exhibited a decrease in its positive cooperative kinetic
behavior toward ATP when compared with the native enzyme (n = 1.4 and 2.5, respectively). At a saturating UTP
concentration, phosphorylation of CTP synthetase resulted in a 5.3-fold
reduction in its apparent K
value for ATP and a
loss of its positive cooperative kinetic behavior with respect to ATP (Fig. 5B and Table 1). Phosphorylation of CTP
synthetase resulted in a modest increase in the apparent V
of the reaction when activity was measured
with subsaturating or saturating concentrations of UTP ( Fig. 5and Table 1).
Figure 5:
Effect of phosphorylation on the kinetics
of CTP synthetase activity with respect to ATP. Phosphorylated ()
and native (
) CTP synthetase activities were measured as a
function of the concentration of ATP using 0.1 (A) and 2
mM UTP (B). The concentrations of glutamine, GTP, and
MgCl
were maintained at 2, 0.1, and 10 mM,
respectively.
Figure 6:
Effect of phosphorylation on the kinetics
of CTP synthetase activity with respect to glutamine. Phosphorylated
() and native (
) CTP synthetase activities were measured as
a function of the concentration of glutamine with 0 (A) and
0.1 mM GTP (B). The concentrations of UTP, ATP, and
MgCl
were maintained at 2, 2, and 10 mM,
respectively.
Figure 7:
Effect of phosphorylation on the
activation of CTP synthetase activity by GTP. Phosphorylated ()
and native (
) CTP synthetase activities were measured in the
absence and the presence of the indicated concentrations of GTP. The
concentrations of UTP, ATP, glutamine, and MgCl
were
maintained at 2, 2, 2, and 10 mM,
respectively.
Figure 8:
Effect of phosphorylation on the
inhibition of CTP synthetase activity by CTP. Phosphorylated ()
and native (
) CTP synthetase activities were measured with 0.1
mM UTP in the absence and the presence of the indicated
concentrations of CTP using 0.5 (A) and 1 mM ATP (B). The concentrations of glutamine, GTP, and MgCl
were maintained at 2, 0.1, and 10 mM,
respectively.
As described previously(12) , the presence
of CTP in the assay for the native enzyme caused an increase in the
positive cooperativity toward UTP, an increase in the apparent K for UTP, and a decrease in the apparent V
(Fig. 9). We questioned what effect
phosphorylation had on the dependence of CTP synthetase activity on UTP
in the presence of CTP. In these experiments the concentration of ATP
was held constant at 1 mM, and the other substrates in the
reaction were saturating. The apparent V
value
of the phosphorylated enzyme measured in the presence of CTP was
greater than the apparent V
value of the native
enzyme measured in the presence of CTP ( Fig. 9and Table 1). However, enzyme phosphorylation did not have a
significant effect on the cooperative kinetic behavior of the enzyme
toward UTP nor the apparent K
value for UTP ( Fig. 9and Table 1).
Figure 9:
Effect of CTP on the kinetics of
phosphorylated CTP synthetase activity with respect to UTP.
Phosphorylated () and native (
,
) CTP synthetase
activities were measured as a function of the concentration of UTP in
the presence of 0.3 mM CTP as indicated. The concentrations of
ATP, glutamine, GTP, and MgCl
were maintained at 1, 2, 0.1,
and 10 mM, respectively.
Phosphorylation/dephosphorylation is a major mechanism by which the activity of an enzyme is regulated(51, 52) . The aim of this work was to examine the regulation of yeast CTP synthetase activity by protein kinase C. We demonstrated that purified CTP synthetase was a substrate for yeast Pkc1p and that Pkc1p mediated the phosphorylation of the enzyme in vivo. This does not, however, demonstrate that CTP synthetase is a substrate for Pkc1p in vivo. An alternate explanation could be that Pkc1p modulates the activity of another kinase that is responsible for phosphorylating CTP synthetase. We do know on the basis of our labeling experiments with pkc1 mutant cells that another kinase phosphorylated the enzyme in vivo.
Phosphorylation of the
enzyme in vitro resulted in the activation of CTP synthetase
activity. This regulation of activity involved changes in the kinetic
properties of the enzyme. Pkc1p phosphorylation of CTP synthetase
activated the enzyme by increasing the apparent V of the reaction. The change in the apparent V
was most evident when the kinetics of the enzyme toward UTP was
measured using a subsaturating concentration of ATP. The phosphorylated
enzyme also showed a greater affinity for ATP when compared with the
native enzyme. This was reflected in the apparent K
values for ATP. Another striking effect of Pkc1p phosphorylation
of CTP synthetase was the decrease in the positive cooperativity toward
UTP and ATP. This was most evident when activity was measured with
respect to ATP.
Pkc1p phosphorylation of CTP synthetase did not have
a significant effect on the kinetic properties of the enzyme with
respect to glutamine and GTP. This indicated that phosphorylation did
not affect the reaction involving the formation of the glutaminyl
enzyme intermediate nor the role GTP plays as an enzyme
activator(3, 4) . Phosphorylated CTP synthetase was
less sensitive to inhibition by CTP when activity was measured with a
subsaturating concentration of ATP. However, enzyme phosphorylation did
not affect the mechanism by which CTP inhibited CTP synthetase
activity. The cooperative kinetic behavior toward UTP and the apparent K value for UTP were not significantly affected by
Pkc1p phosphorylation of the enzyme. The only kinetic parameter
affected by phosphorylation was the apparent V
.
CTP synthetase is one of several allosteric enzymes whose kinetic properties are changed by phosphorylation. For example, phosphorylation changes the kinetic behavior of glycogen phosphorylase (53, 54) and phosphofructokinase (55) with respect to their substrates, activators, and inhibitors. Moreover, these changes in kinetic behavior play a role in the regulation of glycogen phosphorylase and phosphofructokinase activities in vivo(1) .
How could the phosphorylation of CTP synthetase by Pkc1p affect activity in vivo? The steady-state cellular concentrations of UTP (0.75 mM) and ATP (2.3 mM) are saturating for CTP synthetase activity(8, 12) . Under these conditions Pkc1p phosphorylation of CTP synthetase would not be expected to affect activity in vivo. At the same time, enzyme phosphorylation would not affect the regulation of CTP synthetase activity by CTP inhibition. On the other hand, if the cellular concentrations of UTP and ATP were to decrease to subsaturating concentrations for the enzyme, Pkc1p phosphorylation would affect CTP synthetase activity. For example, the cellular concentration of ATP can vary between 0.57 to 2.3 mM depending on growth conditions(56) . These concentrations fall within the range of the subsaturating to saturating ATP concentrations for CTP synthetase activity(12) . It was the subsaturating ATP concentrations that had the major effect on the kinetic properties of the phosphorylated enzyme. In addition, CTP synthetase activity was less sensitive to product inhibition by CTP when ATP levels were subsaturating. The activation of CTP synthetase activity by Pkc1p phosphorylation may be a mechanism by which the cell regulates CTP synthesis when cellular ATP levels are limiting. At the present time it is not known what role phosphorylation plays in the function of CTP synthetase under different growth conditions. Future studies will address this question.
Our interest in CTP synthetase originates from the role CTP plays in the regulation of the pathways by which phosphatidylcholine is synthesized(57) . Phosphatidylcholine is the essential end product of phospholipid synthesis and the major phospholipid found in S. cerevisiae(58, 59) . CTP is required for the synthesis of phosphatidylcholine through the CDP-choline- and CDP-diacylglycerol-based pathways(58, 59) . In mammalian cells, phosphatidylcholine plays a major role in lipid signal transduction pathways(60) . The diacylglycerol derived from the receptor-mediated hydrolysis of phosphatidylcholine is responsible for the sustained activation of protein kinase C(60) . The phosphorylation and activation of CTP synthetase by Pkc1p in yeast may represent a mechanism by which lipid signal transduction pathways are coordinately regulated to CTP synthesis and cell growth.