From the Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201
Received for publication, October 29, 2002
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ABSTRACT |
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De novo pyrimidine biosynthesis is
activated in proliferating cells in response to an increased demand for
nucleotides needed for DNA synthesis. The pyrimidine biosynthetic
pathway in baby hamster kidney cells, synchronized by serum
deprivation, was found to be up-regulated 1.9-fold during S phase and
subsequently down-regulated as the cells progressed through the cycle.
The nucleotide pools were depleted by serum starvation and were not
replenished during the first round of cell division, suggesting that
the rate of utilization of the newly synthesized nucleotides closely
matched their rate of formation. The activation and subsequent
down-regulation of the pathway can be attributed to altered allosteric
regulation of the carbamoyl-phosphate synthetase activity of CAD
(carbamoyl-phosphate synthetase-aspartate
carbamoyltransferase-dihydroorotase), a multifunctional protein that
initiates mammalian pyrimidine biosynthesis. As the culture approached
S-phase there was an increased sensitivity to the allosteric
activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP
inhibition, changes that were reversed when cells emerged from S phase.
The allosteric regulation of CAD is known to be modulated by MAP kinase
(MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as
by autophosphorylation. CAD was found to be fully autophosphorylated in
the synchronized cells, but the level remained invariant throughout the
cycle. Although the MAPK activity increased early in G1,
the phosphorylation of the CAD MAPK site was delayed until just before
the onset of S phase, probably due to antagonistic phosphorylation by
PKA that persisted until late G1. Once activated,
pyrimidine biosynthesis remained elevated until rephosphorylation of
CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase.
Thus, the cell cycle-dependent regulation of pyrimidine
biosynthesis results from the sequential phosphorylation and
dephosphorylation of CAD under the control of two important signaling cascades.
The activity of the de novo pyrimidine biosynthetic
pathway closely parallels the growth rate of the cell and is highest
during periods of rapid proliferation (1-13). Cells growing in culture synthesize pyrimidines at a rapid rate during the exponential growth
phase, but the pathway is much less active when the cells enter the
stationary phase or have their growth arrested by serum deprivation
(14-18). In an earlier study, Mitchell and Hoogenraad (19) show that
pyrimidine biosynthesis is maximally activated during the S phase of
the cell cycle. Thus, the de novo biosynthetic pathway plays
a dominant role in providing precursors for the synthesis of DNA that
accompanies cell division. Furthermore, there is strong evidence (7,
11, 12) that the activation of the pathway is a prerequisite for the
proliferation of tumor and neoplastic cells.
The flux through the pathway is governed (20, 21) by the activity of
carbamoyl-phosphate synthetase II
(CPSase),1 a component of CAD
(Fig. 1), the multifunctional protein
(22-24) that initiates pyrimidine biosynthesis in mammalian cells.
CPSase catalyzes the rate-limiting step, the formation of carbamoyl
phosphate from bicarbonate, glutamine, and two ATP molecules. The
enzyme is subject to a complex network of control mechanisms that
include both allosteric regulation and phosphorylation. UTP, an end
product of the pathway, is a feedback inhibitor, whereas
5-phosphoribosyl-1-pyrophosphate (PRPP), a substrate for both the
parallel de novo purine biosynthetic pathway and a
subsequent step in pyrimidine biosynthesis, is an activator (13, 25,
26). Carrey et al. (28-31) made the important observation that purified CAD is phosphorylated at two sites (Fig. 1)
by cAMP-dependent protein kinase A (PKA). PKA
phosphorylation has no effect on the catalytic activity but decreases
UTP inhibition, a modification that would be expected to stimulate
pyrimidine biosynthesis. However, subsequent studies (32) showed that
PKA phosphorylation resulted in a decrease in the affinity for the activator, PRPP, an effect that would be expected to down-regulate the
pathway. Thus, PKA phosphorylation alone cannot account for the
activation of the pathway in rapidly growing cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural organization and regulation of
CAD. The first three steps in the de novo pyrimidine
biosynthetic pathway are catalyzed by the CPSase, aspartate
transcarbamoylase (ATC), and dihydroorotase
(DHO) domains of the multifunctional protein CAD, a complex
consisting of six copies of a 243-kDa polypeptide organized into
multiple domains and subdomains. The synthesis of carbamoyl phosphate
involves four partial reactions that are catalyzed by the concerted
action of the glutaminase domain (GLN) and two synthetase
subdomains (CPS.A and CPS.B). The CPSase activity of CAD controls the
flux through the pyrimidine pathway. The allosteric ligands, UTP, the
inhibitor, and PRPP, the activator, bind to the B3 regulatory and by
autophosphorylation (Autophos) subdomain of CPS.B. The
CPSase activity is also controlled by phosphorylation of the MAPK and
PKA1 sites. There is a second PKA target site (PKA2) unrelated to
regulation. The formation of carbamoyl aspartate and dihydroorotase is
catalyzed by the ATC and DHO domains, respectively.
MAP kinases (33) are ubiquitous components of the mitogen-activated cascade that result in cellular proliferation in response to growth factors. Stimulation of quiescent cells with epidermal growth factor results in the activation of the MAP kinase cascade and the phosphorylation of Thr-456 in the A1 subdomain (Fig. 1) of the CAD CPSase (34). MAP kinase phosphorylation both in vivo and in vitro has no effect on any of the catalytic activities of CAD but relieves UTP inhibition and stimulates PRPP activation of the CPSase. Both of these changes in allosteric regulation would be expected to increase the rate of pyrimidine biosynthesis.
We have recently shown (18) that the activation of pyrimidine
biosynthesis in exponentially growing BHK 165-23 cells is a consequence
of the MAP kinase-mediated phosphorylation of CAD and that its
subsequent down-regulation as the culture became confluent is
associated with dephosphorylation of the MAP kinase site and
phosphorylation by PKA. To more precisely define the mechanism of
growth-related activation and down-regulation of pyrimidine
biosynthesis, the activity of the pathway and the role of the diverse
control mechanisms was examined as BHK 165-23 cells traversed the cell cycle.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- BHK 165-23 (35), a baby hamster kidney cell line derived from BHK-21, was grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% dialyzed fetal bovine serum and 2 µg/ml gentamicin (Invitrogen). Two million cells were plated in T75 flasks containing 25 ml of media. The medium was changed every 2 days. Cells were counted using a hemocytometer, and viability was assessed by trypan blue staining.
Cell Synchronization-- BHK cells were synchronized by serum deprivation as described previously (36). Briefly, 10 T-75 flasks of exponentially growing cells (60-80% confluent) were serum-starved in DMEM, 0.1% fetal bovine serum for 24 h. The cells were then stimulated by the addition of 25 ml of DMEM supplemented with 10% fetal bovine serum and incubated at 37 °C. The cultures were harvested at 2-h intervals over a period of 20-24 h for pyrimidine biosynthesis assays and the preparation of cell extracts as previously described (18).
Pyrimidine Biosynthesis-- The rate of pyrimidine biosynthesis in live cells was assayed using a modification of the methods devised by Huisman et al. (1) and Simmonds and co-workers (37). Cells were harvested by trypsin treatment and suspended at a concentration of 2.5 × 106 cells/ml in 2.8 ml of DMEM, F-12 medium without bicarbonate. The assay was initiated by the addition of 0.33 ml of 49 mM 14C-labeled sodium bicarbonate (3.75 µCi/µmol). The final concentration of bicarbonate in the assay was 5.2 mM. After allowing the reaction to proceed with gentle shaking for 45 min at 37 °C the medium was removed, and the reaction was quenched by the addition of 0.375 ml of 6% cold trichloroacetic acid. The incorporation of 14C-labeled bicarbonate into UTP and CTP was immediately quantitated by HPLC. We found that pyrimidine biosynthesis proceeded linearly for at least 1 h.
HPLC Analysis of Nucleotides--
The nucleotides were analyzed
following the protocol of Dr. Melinda E. Wales,2 using a Waters Model
2695 HPLC system equipped with an IN/US systems -ram, Model 3 radioactivity flow detector. All reagents were ultrapure or HPLC grade.
The trichloroacetic acid supernatant was extracted twice with an equal
volume of freshly prepared 0.7 M
tri-N-octylamine/Freon solution (376 µl of 93% w/v
tri-N-octylamine (Sigma) diluted to a total volume of 5 ml
with freon (1,1,2-trichlorotrifluoroethane (Sigma)). The mixture was
vortexed for 1 min, placed on ice for 30 min, and then centrifuged for
1 min at 10,000 rpm. The aqueous phase (100 µl) was then immediately
injected onto a Whatman Partisil 10 SAX column (250 × 4.6 mm
(Phenomenex)) equilibrated with 5 mM ammonium phosphate
buffer at pH 2.7 (Buffer A). The nucleotides were eluted with a
discontinuous gradient of 5 mM
(NH4)3PO4, pH 2.7 (Buffer A), and
750 mM (NH4)3PO4, pH
3.8 (Buffer B), at a flow rate of 2 ml/min. Buffer B increased linearly
to 50% in 20 min and then from 50 to 100% over the next 10 min. The
absorbance at 254 and 280 nm and the radioactivity of the effluent were
monitored continuously. The peaks were integrated using the IN/US
systems software. The identity and total amount of each nucleotide were calculated from standard curves obtained using authentic standards. The
amount of the radiolabeled nucleotides were calculated from the
specific radioactivity of the 14C-labeled sodium bicarbonate.
Immunoblotting-- For immunoblotting the following antibodies were used at the previously described dilution (18) or as recommended by the manufacturer: phosphoserine (Z PS-1, Zymed Laboratories Inc.) rabbit polyclonal antibodies and phosphothreonine (Z PT-1, Zymed Laboratories Inc.) rabbit polyclonal antibodies; diphospho-Erks (anti-Thr-202/Tyr-204-phosphorylated p44/p42, Cell Signaling Technology) mouse monoclonal antibody; PKA (Santa Cruz) rabbit polyclonal antibody; affinity-purified goat anti-mouse IgG (H&L) antibody conjugated to horseradish peroxidase and affinity-purified goat anti-rabbit IgG (H&L) antibody conjugated to horseradish peroxidase (Cell Signaling Technology); rabbit polyclonal antibody against all Aequorea victoria green fluorescent protein variants (Living Colors A.v., Clontech); cyclin E (Upstate Biotechnology) rabbit polyclonal IgG; cyclin B1 (clone CB169, Upstate Biotechnology) mouse monoclonal IgG2b; cyclin A (Upstate Biotechnology) rabbit polyclonal IgG; purified mouse anti-human RB protein (Pharmingen). The rabbit polyclonal CAD serum was prepared as previously described (38). Immunoblotting and quantitation of the immunoblots were carried out as previously described (18).
Enzyme Assays-- The glutamine-dependent CPSase assay has been described (39). The 1-ml assay mixture contained 100 µg of protein, 100 mM Tris-HCl, pH 8.0, 100 mM KCl, 7.5% Me2SO, 2.5% glycerol, 1 mM dithiothreitol, 3.5 mM glutamine, 20.2 mM aspartate, 1.5 mM ATP, 3.5 mM MgCl2, and 5 mM sodium 14C-labeled bicarbonate (1.6 × 106 µCi/mol). The concentration of MgCl2 was adjusted so as to maintain a 2 mM excess over the sum of the concentration of ATP, UTP, and PRPP. Aspartate transcarbamoylase activity was assayed by the colorimetric method (40). The aspartate transcarbamoylase assay mixture contained 100 µg of protein, 5 mM carbamoyl phosphate, and 12 mM aspartate in a buffer consisting of 100 mM Tris-HCl, pH 8.0, 100 mM KCl, 7.5% Me2SO, 2.5% glycerol, and 1 mM dithiothreitol in a total volume of 1 ml. The MAP kinase (p44/p42 or Erk1/Erk2) activities were assayed using the New England Biolabs kit following the manufacturer's protocol. The phosphorylation of the MAP kinase substrate, glutathione S-transferase-Elk1 (2 µg), was followed by Western blotting using specific anti-phospho-Elk1 antibodies (18). Alternatively, Erk1/Erk2 were assayed using phospho-Erk1/2 antibodies that recognize only the activated kinases (Promega). The PKA activity in the cell extract (20-150 µg of protein) was assayed using the PepTag PKA assay kit (Promega), which relies on a change in the electrophoretic mobility of a fluorescent Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) derivative upon phosphorylation. Phosphorylation of the Kemptide could also be measured by electrophoresis on a 20% SDS-PAGE followed by immunoblotting using anti-phospho-serine antibodies.
CAD Phosphorylation-- Previous studies show (18, 41) that the extent of the phosphorylation of the MAP kinase site, Thr-456, could be quantitated by immunoblotting with phosphothreonine antibodies corrected for autophosphorylation. Similarly, the extent of phosphorylation of the CAD PKA sites (Ser-1406 and Ser-1859) could be quantitated using the phosphoserine antibodies. Autophosphorylation was measured by a previously described modification (41) of the "on membrane kinase assay" (42). The increase in phosphorylation of CAD, determined by quantitative immunoblotting with phosphothreonine antibodies, that occurred upon incubation of the protein with 0.5 mM ATP for 2 h was taken as a measure of the unmodified autophosphorylation site of CAD in cell extracts. Purified CAD incubated with and without ATP was used as a standard to determine the fractional autophosphorylation. Protein quantitation was performed by the Lowry method (43) and by scanning stained SDS-polyacrylamide gels calibrated with known amounts of bovine serum albumin as a standard using the software UNSCAN-IT (Silk Scientific Corp.).
Cloning of the Fluorescent Fusion Proteins--
A commercial PKA
clone (GeneStorm human PKA catalytic subunit from ResGen) was amplified
by PCR using the primers
5'-taaaaggtaccatggggaacgcggcgaccgccaagaaaggc-3', incorporating a
KpnI restriction site upstream of the first codon, and
5'-tttttgatcatcaaaattcaccaaattcttttgcac-3', incorporating a
BclI restriction site downstream of the coding sequence. The purified PCR product was cleaved with KpnI and
BclI (Invitrogen). The vector pCruz-GFP (Santa Cruz
Biotechnology) was digested using KpnI and BglII
and treated with thermosensitive alkaline phosphatase (Invitrogen). The
purified fragments were ligated and transformed into Escherichia
coli DH5 (Invitrogen). The plasmid encoded the PKA catalytic
subunit with the green fluorescent protein appended to the amino end.
The plasmid Npt7.5 Erk2 containing the murine Erk2 cDNA fragment, a
generous gift of Dr. Melanie Cobb (Dept. of Pharmacology, University of
Texas Southwestern Medical Center, Dallas, TX), was digested with
NcoI and filled in with Klenow (Invitrogen). The purified
fragment was digested with XbaI (New England Biolabs) and
repurified. The vector pECFP-C1 (Clontech) was
cleaved with BspE1, filled in with Klenow, digested with
XbaI, and dephosphorylated with thermosensitive alkaline
phosphatase. The vector and insert were ligated and transformed into
DH5
cells. The construct encodes MAPK (Erk2) with the enhanced cyan
fluorescent protein fused to the amino end. To construct the
glutaminase (GLN) CPS fusion protein, pCK-CAD10 (39) encoding the
full-length CAD cDNA in E. coli was cleaved with
BamHI (a unique site introduced at the 3' end of the coding
sequence) and BbsI for 2 h at 37 °C. The resulting
fragment encoding the entire GLN-CPSase domain (Fig. 1) was purified,
filled in with Klenow, and inserted into the pECFP-C1 vector
(Clontech) that had been linearized by cleavage with BspE1 and filled-in. The ligation products were transformed into
DH5
-subcloning efficiency bacteria, and recombinants were verified
by restriction digests and sequencing. The construct, pECFP-GLN-CPS,
encoded CAD GLN-CPS with the enhanced cyan fluorescent protein fused to
the amino end. For the construction of the full-length CAD fusion
protein, pCK-CAD10 was cleaved with XbaI. The 3750-bp fragment containing the end of the CPS.B domain and the remainder of
the CAD-coding sequences was inserted into pECFP-GLN-CPS that had been
cleaved with XbaI. XbaI cleaves pECFP-GLN-CPS in
the CPS.B domain and in the vector downstream of the insert. The
ligation products were transformed into DH5
. The construct,
pECFP-CAD, encoded the entire CAD protein with the enhanced cyan
fluorescent protein appended to the amino end. The fidelity of all
constructs was verified by DNA sequencing (Wayne State University DNA
Sequencing Facility).
Transfection and Fluorescence Microscopy--
The E. coli transformants were cultured overnight in Luria broth, 100 µg/ml kanamycin, and plasmids were purified using the Plasmid Maxi
kit (Qiagen). BHK cells were transfected with LipofectAMINE Plus
reagents (Invitrogen) using 2 µg of the DNA following the manufacturer's protocol. After 5 h, the transfection medium was replaced with DMEM complete medium. The expression of the recombinant proteins was monitored by fluorescence microscopy and Western blotting
using antibodies directed against, CAD, PKA, MAPK (p44/p42), and green
or cyan fluorescent protein. The recombinant proteins were fluorescent
and had the expected size on calibrated SDS gels. Fluorescence
microscopy was carried out using a Zeiss Axiophot Upright
Epifluorescent Digital Microscope (Karmanos Confocal Imaging Facility).
With the available filters, it was not possible to detect the cyan
chromophore at its maximum emission wavelength, but there is a
significant secondary maximum that allowed its visualization with the
green filters set. Photographs were obtained using a SPOT RT camera
(Diagnostic Instruments, Inc.) coupled to a Macintosh Personal Computer
with the SPOT software Version 3.0). The expression of cyclin A and
cyclin E in the transfectants was visualized by fluorescence microscopy
after immunostaining with cyclin-specific antibodies (44).
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RESULTS |
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Cell Synchronization--
Serum deprivation following well
established protocols (36) was used to synchronize cultures of
BHK165-23 cells. Replacing the complete media in exponentially growing
cultures with the same media supplemented with only 0.1% serum
resulted in the arrest of growth. Growth resumed 24 h later upon
stimulation with 10% fetal bovine serum, at which point the cells
began to traverse the cell cycle in synchrony. The periodic expression
of cyclins is commonly used to identify the different cell cycle phases
(45-47). In this study, cell cycle-dependent markers were
used to monitor the progression of the culture through the initial
phases of the cycle. Cyclin E expression, known to be confined to the
G1 phase, began to increase soon after serum stimulation,
peaked at 4 h, and then declined. The inactivation of the
retinoblastoma tumor suppressor protein by hyperphosphorylation is also
known to occur during G1 (46) and to remain phosphorylated
through mitosis. In the synchronized BHK cultures, retinoblastoma
phosphorylation preceded the expression of cyclin E, peaked at about
3 h, remained phosphorylated for 16 h, and then decreased.
Cyclin A expression, reported to begin at the G1/S boundary
and to reach the highest levels in late S phase (46, 47), peaked at
12 h in BHK cells, whereas cyclin B, another S phase marker,
peaked at about 13 h (Fig. 2). We
concluded that the midpoint of the S phase of the cycle occurred at
10-11 h after serum stimulation.
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Pyrimidine Biosynthesis--
The activity of the pyrimidine
biosynthetic pathway was assayed by measuring the incorporation of
14C-labeled bicarbonate into UTP and CTP. The basal level
of pyrimidine biosynthesis, measured during serum deprivation (Fig.
3, 0 h) was 1.0 pmol/min/106 cells. The level remained constant until just
before entry into the S phase of the cell cycle, at which point the
rate increased to 1.85 pmol/min/106 cells. The rate of
pyrimidine biosynthesis decreased as the cells emerged from S phase.
Thus, there was a strong correlation between the activity and the S
phase of the cell cycle. Radiolabeled bicarbonate is also a precursor
of purines, and there was a parallel increase in the incorporation of
14C-labeled bicarbonate into ATP from 1.6 to 3.0 pmol/min/106 cells in mid-S phase.
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The Intracellular Concentration of the Nucleotides-- The intracellular concentration of the nucleotides (Table I) was measured by HPLC as the cells traversed the cell cycle. The absorbance at 254 nm was monitored (Fig. 4), and the amount of each nucleotide was determined from the area of the corresponding peaks using standard curves. The concentration was calculated assuming 1 µl of intracellular fluid/106 packed cells (48). Although most of the nucleotides were well separated (Fig. 4), in the best cases CTP appeared as a small leading shoulder of the larger UTP peak. Because it proved impractical to reliably distinguish these species, the pyrimidine trinucleotide concentrations were expressed as a combined UTP and CTP.
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In serum-starved cells (t = 0 h), the concentration of UTP/CTP was 0.17 mM and remained reasonably constant for the duration of the cell cycle. This value was approximately half that observed in exponentially growing cultures of the same cells. A similar trend was observed for most of the other nucleotides. Notably, the ATP concentration in the synchronized cells was 5-fold lower than the value measured in the exponentially growing cells and did not significantly increase for 18 h post-serum stimulation. The observation that the nucleotide pools did not expand during the first round of cell division despite ongoing pyrimidine biosynthesis suggests that the newly synthesized nucleotides are utilized as rapidly as they are produced.
CAD Activity and Regulation--
The activity of CAD CPSase,
measured in the absence of allosteric ligands (Fig.
5A), increased ~2-fold from
1.2 nmol/min immediately after serum stimulation to 2.9 nmol/min at
22 h. We have shown previously (41) that when purified CAD is
incubated with ATP, the CPS domain undergoes autophosphorylation, a
phenomenon that is accompanied by a 2-fold increase in the
CPSase-specific enzymatic activity. The CAD autophosphorylation,
measured in synchronized cultures of BHK cells (Fig. 5A),
was found to be 90-95% complete and remained unchanged throughout the
cell cycle. Thus, the increase in CAD CPSase activity cannot be
attributed to an increase in autophosphorylation. Moreover, neither PKA
nor MAP kinase phosphorylation affects the CPSase activity in the
absence of allosteric ligands. Thus, it is likely that the approximate
2-fold increase in CPSase activity is simply a consequence of the
increase in the CAD concentration in the cell that must occur during
each turn of the cell cycle. This interpretation is supported by
assaying aspartate transcarbamoylase, an unregulated CAD activity,
which was also found to increase 2-fold by the end of the cell
cycle.
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These changes in CPSase activity cannot explain the activation of the pathway that occurred during the S phase of the cell cycle. For example, between 6 and 10 h, where pyrimidine biosynthesis was up-regulated by 85%, the CPSase activity increased only 5%. However, there were appreciable changes (Fig. 5B, Table II) in the allosteric properties of CPSase. As the cells approached S phase, the activation of the enzyme by 200 µM PRPP increased about 2-fold, from 1240 to 2524%. In serum-starved cells, UTP (0.5 mM) inhibition of CPSase activity was 40%, but as the cells approached S phase, the effect of UTP was completely abolished. Both of these changes in allosteric regulation, which would be expected to increase the rate of pyrimidine biosynthesis, were fully or partially reversed by 14 h post-serum stimulation. Thus, the changes in the sensitivity of CPSase to PRPP and UTP closely paralleled the S phase and the activation of the pyrimidine biosynthetic pathway.
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Kinase Activities and CAD Phosphorylation State--
The MAP
kinase activity (Fig. 6A) was
lowest in early G1 and increased 15-fold during the first
4 h after serum stimulation. The activation of MAP kinase
persisted for several hours and then began to decrease just before
entry into the S phase of the cell cycle. The levels remained at
~50% of the maximum value up to 20 h. Despite high levels of
MAP kinase activity during G1, the CAD phosphothreonine
remained unchanged until just before S phase and then increased 2-fold.
The phosphothreonine antibodies also detect phosphorylation of the
single CAD autophosphorylation site (Thr-1037), and this site was found
to be nearly fully phosphorylated throughout the cell cycle. Because
there was a background phosphorylation of 1 mol of phosphothreonine, a
2-fold increase can be attributed to the incorporation of 1 mol of
phosphate into the single CAD MAP kinase site (Thr-456). This
interpretation was reinforced by the observation that exposure of cells
10 h post-serum stimulation (Fig. 6C), at which point
the CAD MAP kinase site was 40% modified, to 20 µM
PD98059 or UO126, two MEK (MAP kinase kinase) inhibitors, for 30 min
resulted in a 35 and 40% reduction in phosphothreonine, respectively.
These inhibitors had no effect on CAD phosphoserine. Similar results
were obtained for cells harvested at other time points.
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The PKA activity (Fig. 5B) was highest immediately after serum stimulation and began to decrease to 50% of the maximum value over the next 6 h. The inactivation of PKA was accompanied by a simultaneous decline in CAD Ser phosphorylation. Exposure of cells 2.5 h after serum stimulation to the PKA inhibitor, H89 (Fig. 6C), reduced the CAD serine phosphorylation by another 25%, with no effect on CAD phosphothreonine. The levels remained low until 16 h, at which point both the PKA activity and the PKA phosphorylation of CAD began to increase once again.
Intracellular Localization of the Kinases and CAD--
For
phosphorylation to occur, the kinase and its substrate CAD must be
co-localized within the same intracellular compartment. Plasmids were
constructed encoding fusion proteins that had the green or
cyan fluorescent protein appended to the amino end of MAP kinase, the
PKA catalytic subunit, the GLN-CPS domain, and CAD. The constructs were
transiently transfected into BHK cells, and the intracellular location
was visualized (Fig. 7) by fluorescent microscopy at 12-h intervals after transfection. Previous studies (49)
show that the transfection procedure results in synchronization of the
subpopulation of cells that have taken up the plasmid. MAP kinase was
found to be primarily localized in the nucleus, although an appreciable
amount was cytoplasmic throughout the time course. By contrast, PKA was
initially localized in the nucleus but was subsequently translocated
into the cytoplasmic compartment. Interestingly, a significant fraction
of CAD, initially cytoplasmic, had migrated into the nucleus by 24 h. A similar distribution was observed for the GLN CPS domain,
indicating that the putative nuclear targeting signals are located
within this region of CAD. Immunofluorescence microscopy (not shown)
showed that the high levels of cyclin E were expressed at 0 and 12 h, whereas the levels were much lower in the 24-h cultures. These
results suggested that the 0- and 12-h samples represent
G0/G1 and that by 24 h, the transfected
subpopulation had entered the S phase of the cell cycle.
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DISCUSSION |
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The results reported here are in agreement with previous
studies of synchronously growing cells showing that the de
novo pyrimidine biosynthetic pathway is activated just before S
phase and subsequently down-regulated at the transition between S and
G2 (Fig. 8). We found that
the rate increased 1.9-fold from 1.0 pmol/min/106 cells in
G1 to 1.85 pmol/min/106 cells in mid-S phase.
These rates are in good agreement with the rate of pyrimidine
biosynthesis of 0.5 pmol/min/106 cells measured in
asynchronous cultures of the same cells during the first 24 h
after passage but are significantly lower than the rate of 4.2 pmol/min/106 cells measured in fully established,
exponentially growing cells (18). The lower rate of pyrimidine
biosynthesis after serum starvation can probably be attributed to the
low intracellular concentration of the CPSase substrate, ATP.
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The average values for the intracellular nucleotide triphosphates in mammalian cells, compiled by Traut in an exhaustive literature search (50), were found to be 567 ± 460, 278 ± 242, 3152 ± 1698, and 468 ± 224 µM for UTP, CTP, ATP, and GTP, respectively. The nucleotide triphosphate concentrations, measured by HPLC analysis of extracts of exponentially growing BHK cells (Table I), were for the most part within the expected range. It is noteworthy, that the nucleotide concentrations after stimulation of serum-deprived cells were appreciably lower that the values in exponentially growing cultures. Moreover, the nucleotide pools did not appreciably expand during the course of the cell cycle. Only modest increases were observed even after 20 h of post-serum stimulation (Table I). These results suggest that 1) the increased rate of pyrimidine biosynthesis nearly matches the rate of utilization of nucleotides for the synthesis of macromolecules during the S phase of the cell cycle, and 2) several cycles of cell division are apparently required before the nucleotide pools can be fully replenished after serum starvation. ATP, for example, was found to be 0.6 mM in serum-starved cells compared with a value of 2.9 mM obtained in fully established cultures. The low ATP concentration would be expected to result in an ~5-fold decrease3 in CPSase activity in serum-starved cells, in reasonable agreement with the 7-fold decrease in the flux through the pyrimidine pathway compared with the rate in exponential cultures.
As described above, the increase in pyrimidine biosynthesis during the S-phase of the cell cycle cannot be attributed to an increase in the CPSase activity measured in the absence of allosteric effectors or to autophosphorylation of CAD. The CAD autophosphorylation site was nearly completely phosphorylated in vivo, representing a constant phosphothreonine background throughout the cell cycle. However, previous studies show (18) that the CPSase activity of CAD, and consequently the rate of pyrimidine biosynthesis, is controlled by PKA- and MAP kinase-mediated phosphorylation of the CAD CPSase domain.
The activity of the pyrimidine biosynthetic pathway was up-regulated as the cells entered the S phase, reached a maximum at 11 h, and then decreased as the cells exited the S phase. The increase in the pyrimidine biosynthetic activity during S phase closely paralleled the response of CPSase to allosteric effectors. During S phase, UTP inhibition was abolished, and PRPP activation increased 2-fold. This altered response to allosteric effectors could readily account for the 1.9-fold activation of the pathway.
Similar changes in allosteric regulation have been observed in asynchronous cultures in response to MAP kinase phosphorylation (18). In synchronized BHK cells, the MAP kinase activity increased rapidly and peaked at 5 h but remained elevated for the duration of the cycle. However, the phosphorylation of the MAP kinase site of CAD did not correlate with the highest MAP kinase activities, and no significant phosphorylation of this site could be detected for 7 h. The resistance of CAD to MAP kinase phosphorylation can probably be attributed to PKA phosphorylation since we have found4 that prior phosphorylation of CAD by PKA blocks phosphorylation by MAP kinase. CAD serine phosphorylation closely paralleled the activity of PKA, highest initially after serum stimulation and decreasing during the next 7 h. A decrease in PKA activity has been previously shown to be a prerequisite for entry into S phase (44). These results suggest that the activating MAP kinase phosphorylation of CAD is delayed until the PKA site is dephosphorylated. Another factor that could potentially influence the phosphorylation of CAD may be its intracellular location relative to the kinases. MAP kinase was found by fluorescence microscopy to be distributed throughout the cell but was concentrated in the nucleus during G1 and S. In contrast, PKA was initially localized in the nucleus but is translocated into the cytosol late in G1. CAD was cytosolic during G1 and migrated into the nucleus, where there were high levels of MAP kinase and low levels of PKA during S phase. Experiments are currently under way to follow up on these interesting observations.
The down-regulation of the pathway occurred as cells emerged from the S phase at about 14-15 h post-serum stimulation. At this point, the MAP kinase activity remained elevated, and the CAD MAP kinase site began to dephosphorylate. Moreover, at 15 h the phosphorylation of CAD by PKA began to increase once again. PKA phosphorylation is known to reduce the sensitivity of CAD to PRPP activation (32). Accordingly, a 1.7-fold decrease in the response to PRPP was observed (Fig. 5) as the cells exited the S-phase that coincided with the down-regulation of the pyrimidine pathway.
In summary, we conclude that the cell cycle-dependent up-
and down-regulation of the rate of pyrimidine biosynthesis can be attributed to the sequential, concerted action of the PKA and MAP
kinase signaling cascades. The phosphorylation and dephosphorylation of
the regulatory sites on the CPSase domain of CAD alter the response to
UTP and PRPP, allosteric effectors that control the flux through the
pyrimidine pathway.
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ACKNOWLEDGEMENTS |
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We thank Drs. Ibrahim Almansour and Dr. David Evans for helpful discussions, Tracey Schultz for expert technical assistance, Dr. Melinda E. Wales at Texas A&M University for generous advice on the HPLC analysis of nucleotides, and Dr. Melanie Cobb, University of Texas Southwestern Medical Center, for the kind gift of the Erk2 clone.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant GM/CA60371 and a seed grant from the Karmanos Cancer Center (American Cancer Society Research Grant IRG-85-003-14).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1016; Fax:
313-577-2765; E-mail: hguy@cmb.biosci.wayne.edu.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M211078200
2 M. E. Wales, personal communication.
3 The CPSase activity in the cell was calculated at the ATP concentrations of 0.4 and 2.9 mM using the Hill equation assuming a Km of 3.4 mM and a Hill coefficient of 1.3, values measured (41) for purified CAD in the absence of allosteric effectors. Because the kinetic parameters are dependent on the concentration of effectors, the calculation is only approximate.
4 D. H. Kotsis, M. Sigoillot, H. I. Guy, and D. Evans, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CPSase, carbamoyl-phosphate synthetase; CPS, the CAD domain that catalyzes carbamoyl phosphate synthesis from NH3, bicarbonate, and ATP; CAD, the multifunctional protein that has CPSase, aspartate transcarbamoylase, and dihydroorotase activities; Erk1 and Erk2, extracellular signal-regulated kinases 1 and 2; MAPK, mitogen-activated protein (MAP) kinase; MEK, MAP kinase kinase, the kinase in the MAP kinase cascade that phosphorylates and activates MAP kinase; PKA, cAMP-dependent protein kinase A; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium; BHK cells, baby hamster kidney cells; PRPP, 5-phosphoribosyl-1-pyrophosphate; ATC, the CAD domain that catalyzes the synthesis of carbamoyl aspartate; DHO, the CAD domain that catalyzes the formation of dihydroorotate; GLN, the CAD domain that hydrolyzes glutamine and transfers the ammonia to the CPS domain.
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