From the
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.
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.
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
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
-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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.