(Received for publication, January 27, 1997)
From the Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201-0509
Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.
Thymidylate synthase (TS, EC 2.1.1.45)1 is a unique enzyme in nature by virtue of the fact that one of the substrates in the reaction, CH2H4PteGlu serves to reductively methylate the second substrate, dUMP, to yield dTMP and H2PteGlu. Because dTMP plays an essential role in the synthesis of DNA, the enzyme has been a chemotherapeutic target since its discovery about 40 years ago (1). The DNA sequences of some 30 different species of TS have been clarified (2) establishing it as one of the most phylogenetically conserved proteins known. X-ray crystal structures for the Lactobacillus casei (3), Escherichia coli (4, 5), and human TSs (6) have been utilized to define the mechanism by which the substrates interact with the enzyme to form product and the nature of the inhibition affected by substrate analogues, as well as to aid in the rational design of potential chemotherapeutic agents (7).
While much is known about the physical and enzymic properties of TS, less is known about how the enzyme is regulated within the cell, its structural location, and its potential interaction with other proteins. Recent studies indicate that this enzyme may be regulated at both the transcriptional (8) and translational levels of synthesis (9), while earlier studies suggested that TS forms multienzyme complexes involved in DNA synthesis both in T-even phage-infected E. coli (10, 11) and eukaryotic cells (12, 13). These findings are compounded further now by the enzyme's apparent association with the nucleolus of the cell and its state of phosphorylation, as will be described in this paper. In addition we will provide evidence that TS may be located also in the mitochondria of cells as has been described recently for a bifunctional dihydrofolate reductase·TS plant enzyme (14) and a dUTPase in HeLa cells (15).
Attempts to establish the intracellular location of TS have been inconclusive since depending on the technique and cells used, TS has been shown to be associated either with the nucleus (16, 17) or the cytoplasm (18, 19). Fluorescent antibody studies by us some 10 years ago using human TS antibody2 indicated that the enzyme was present mainly within the nucleus of the HeLa cell, but could not be confirmed with H35 hepatoma cells. More recently by using a similar technique with yeast the enzyme was shown to be localized to the nuclear periphery (20). To more clearly define the enzyme's location within a eukaryote, we will demonstrate, using immunogold electron microscopy and autoradiography, the nuclear and nucleolar presence of TS in a rat hepatoma (H35) cell line.
The fixation of cells and the labeling procedures used for the localization of the TS antigen after first treating the cells with a purified anti-TS rabbit antibody then with an IgG secondary antibody (goat anti-rabbit antibody conjugated with 5 nm colloidal gold, Janssen Life Sciences, Piscataway, NJ) were essentially as described in the instructions provided by Janssen and by Hechemy et al. (21). The TS antigen was purified to homogeneity (22) and 1 mg injected as an emulsion with Freund's complete adjuvant several times along the midline region of a rabbit's spine. After 2 weeks antibody production was boosted with another 0.5 mg of TS in saline that was injected into the rabbit's hind legs intramuscularly and neck pad. After two more weeks 50 ml of blood was removed and allowed to clot. The TS antibody (IgG) was purified from the rabbit serum by CM-Affi-Gel blue chromatography (Bio-Rad) according to the manufacturer's instructions.
Quantitative Evaluation of ImmunogoldGold particles were counted on electron micrographs (final magnification × 45,000). Cell areas were determined by overlaying each micrograph with a calibrated lattice of known size, and underlying areas were determined for each cell compartment. The density of gold label is defined as the number of gold particles encountered per square micron of surface counted.
Electron Microscopy AutoradiographyH35S (FdUrd-sensitive) and H35R (FdUrd-resistant) cells (6 × 104 in 12 ml of Swim's medium containing 5% human serum 10% fetal bovine serum) were grown for 43 h in 100-mm plates in a CO2 incubator, after which 8 ml of medium was removed from each plate. Folinic acid was added to a final concentration of 100 µM to the remaining 4 ml, while [6-3H]FdUrd (22 Ci/mmol), DuPont NEN) was added to a final concentration of 0.26 µM. The plates were incubated for an additional 4 h, washed three times with media and incubated for 4 h with fresh Swim's media. At the end of this period, the cells were fixed with 2 ml of Trump's fixative for 15 min and washed two additional times with 5 ml of fixative. The plates were then incubated overnight at 40 °C and the cells gently scraped off the Petri dishes and centrifuged, followed by rinsing in 0.1 M sodium cacodylate buffer, pH 7.4. After a secondary fixation for 30 min at room temperature in a solution of 1% osmium tetroxide, 0.1 M cacodylate buffer, pH 7.4, the pellets were embedded in agar, dehydrated in a graded series of ethanol, and embedded in Epon/Oraldite. Sections of 0.1 µm were collected on carbon-coated grids, and autoradiographic emulsions were prepared essentially as described by Caro and van Tubergen (23). Grids were post-stained with uranyl acetate and Reynold's lead citrate (24) and examined in a Zeiss 910 transmission electron microscope at 100 kv.
Mitochondrial Isolation and Ternary Complex FormationH35S
and H35R cells were grown in Swim's medium to about 90% confluence in
one to three roller bottles and harvested with 0.05% trypsin, 0.02%
versene. The cells were added to an equal volume of Swim's medium and
centrifuged for 5 min at 1000 rpm followed by washing with 20 ml of
phosphate-buffered saline. The centrifuged cells were resuspended in 4 ml of isolation buffer (230 mM mannitol, 70 mM
sucrose, 0.5 mM EGTA, 0.5% bovine serum albumin, and 10 mM Hepes, pH 7.4). The cells were then homogenized and
centrifuged in a 15-ml plastic centrifuge tube at 1000 × g for 10 min at 4 °C using a Sorvall GLC-2B centrifuge.
The supernatant fraction after transferring to another tube was
recentrifuged at 20,000 × g in a Sorvall RC-2B
centrifuge for 15 min. The pellet was resuspended in 5 ml of isolation
buffer and recentrifuged. Alternatively, mitochondria were purified by
isopycnic centrifugation as described by Rickwood et al.
(25). In either case, the final mitochondrial pellet was suspended in 1 ml of STE (100 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl, pH 7.4), and centrifuged in an Eppendorf centrifuge at 14,000 rpm for 10 min at 4 °C. The pellet was
resuspended in 150 µl of STE and sonicated for 7 s in an
ethanol/ice bath and centrifuged as above. Alternatively, the
mitochondria were suspended in cold water and after lysis the
membranous fraction was centrifuged, as above, in an Eppendorf
centrifuge. Ternary complexes containing [32P]FdUMP were
obtained by a slight modification of the procedure of Maley et
al. (26) as follows: to 8 µl of H35R cell extract, mitochondrial
supernatant fraction, or purified recombinant rat TS (27) was added 1 µl of 50 mM MgCl2, 3 µl of a 1.6 mM (R,S)-CH2H4PteGlu solution (26), 3 µl of 0.1 M cytidine 5-monophosphate,
and 5 µl of [32P]FdUMP (1.1 × 106
dpm). After incubating at room temperature for 30 min the reactions were stopped with an equal volume of SDS-loading buffer and boiled for
3 min. Half of the sample was subjected to 12.5% SDS-PAGE for 1 h
at 180 volts. The gel was dried and exposed to Kodak X-Omat x-ray film
for 15-60 min at room temperature.
H35R rat hepatoma cells were grown in Swim's medium as
above on 60-mm tissue culture plates for 24 h prior to labeling.
The cells, which were at 80% confluence at this time, were washed 3 × with 2 ml of phosphate-free Dulbecco's modified Eagle's
medium (DMEM) and were then incubated with 2 ml of phosphate-free DMEM, at 37 °C in a CO2 incubator for 1 h. The media were
replaced with 1.5 ml of phosphate-free DMEM containing 1% fetal bovine
serum (1.5 ml/plate) and 120 µCi of 32Pi,
which was incubated in a 37 °C CO2 incubator for 4 h. The plates were washed four times with 2 ml of phosphate-buffered saline and the cells lysed in 0.5 ml of a modified RIPA (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS,
1% Triton X-100, 2 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM NaF, 0.1 mM Na3VO4) on ice with gentle
rocking for 30 min. The lysates were transferred to 1.5-ml Eppendorf
tubes, which were centrifuged at 14,000 rpm for 10 min at 4 °C in an
Eppendorf 5415 centrifuge. The supernatant fractions were transferred
to fresh Eppendorf tubes, to which were added 25 µl of protein
A-Sepharose (10% slurry in modified RIPA), incubated for 1 h, and
centrifuged for 10 min at 14,000 rpm at 4 °C. To the supernatant
fractions transferred to fresh tubes was added 10 µl of rabbit
anti-rat TS serum, and after incubation on ice for 1 h, 25 µl of
protein A-Sepharose beads was added to each tube, which were shaken for 1 h. The beads were centrifuged and washed four times with 200 µl of modified RIPA, then resuspended in 20 µl of SDS-loading buffer containing 2-mercaptoethanol and boiled for 5 min. Each supernatant fraction (10 µl) was loaded onto a 12.5% SDS-PAGE gel,
and after 1 h at 175 volts, the gel was stained with Coomassie Blue, followed by destaining and drying onto Whatman No. 3MM paper. The
dried gel was exposed to Kodak X-Omat film for 1-3 days at 70 °C
using an enhancing screen.
H35R cells
were grown for 24 h as described above. The medium was removed,
and the plates were rinsed with a buffer solution containing 0.25 M sucrose, 50 mM Tris-Cl, pH 7.4, and 5 mM MgSO4. The cells from six plates were
scraped off with the above buffer, but containing 2 mM
phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 mM NaF, and 0.1 mM sodium
vanadate, and sonicated for 10 s in an ethanol/ice bath. Aliquots
of the sonicates (0.28 ml) were added to each Eppendorf tube with 20 µCi of [-32P]ATP. The incubations, conducted at room
temperature, were terminated at various times by the addition of 1 µmol of cold ATP, which so diluted the [
-32P]ATP to
effectively end the measurable incorporation of labeled phosphate into
the protein acceptors. The cellular debris was centrifuged for 10 min
at 14,000 rpm in an Eppendorf centrifuge at 4 °C. The supernatant
fractions were transferred to fresh Eppendorf tubes, where they were
treated with 7 µl of rabbit anti-rat TS serum for 1 h on ice,
followed by 15 µl of protein A-Sepharose (Pharmacia Biotech Inc.) for
1 h on ice.
Earlier studies by us using fluorescent guinea pig antibody to HeLa cell TS, as well as immunogold electron microscopy, strongly implicated the association of TS with the nucleus.2 However, attempts to repeat these studies by exposing H35S cells to rat TS fluorescent antibody were inconclusive, mainly due to the difference in membrane permeability of the two cell lines. Since we had available to us much larger quantities of rat TS (22) than HeLa TS for the preparation of antibody, we explored the use of immunogold secondary antibody (goat anti-rabbit IgG) fixation to rabbit anti-rat TS antibody following treatment of H35S cells with the latter.
Fig. 1A reveals that little or no gold
particles could be detected when the cells were exposed to nonspecific
IgG. In contrast, when the H35S cells were treated with purified
anti-TS IgG, a major share of the immunogold particles were found to be
localized to the nucleus (Fig. 1B) with most of the
particles being associated with the nucleolus (Fig. 1C). The
nuclear distribution of the particles could be partially blocked if the
antibody was first treated with H35 TS, verifying the
specificity of the reaction (Fig. 2). That the nuclear
(nucleolar) association of the TS antibody was not a random event was
verified by measuring a statistically significant number of immunogold
particles in a large number of areas inside, as well as outside of the
cell (Fig. 2). However, because of the uniqueness of these findings and
the possibility that the results of Fig. 1 were a consequence of the
technology employed and did not truly represent what was observed, a
more direct approach was sought, that of autoradiography.
Nuclear-Nucleolar Location of TS via Autoradiography
To
unambiguously confirm the immunogold results a biochemical approach was
taken, that of exposing the cells to [6-3H]FdUrd and
leucovorin, which in the case of the former compound will be converted
to [6-3H]FdUMP once in the cell, while the latter is
metabolized to 5,10-methylenetetrahydrofolylpolyglutamate. Both
compounds then combine with TS to form a tight ternary complex, which
is the basis, in most cases, of the chemotherapeutic response to
fluorouracil or FdUrd (28). Although leucovorin was added to make
certain the folate pool was not limiting, it was found in most
instances that the concentration of this pool was adequate. After
thorough washing of the cells to remove unbound
[6-3H]FdUrd, it was hoped that the location of the TS
complex within the cell would be revealed on autoradiography.
As shown in Fig. 3A, most of the labeled
ternary complex resided in the nucleus of the H35S cells and, similar
to the immunogold electron micrographs of Fig. 1, appears to be
localized primarily to the nucleolar region. Since the possibility
existed that the nucleolar presence of the label was due to the
breakdown of FdUrd to fluorouracil and its subsequent incorporation
into nucleolar RNA, this experiment was repeated in the presence of a
100-fold excess of unlabeled uracil or deoxyuridine. No dilution of the
radioactivity was apparent, indicating that incorporation into RNA was
not the source of the label within the cell (data not shown). In
addition, SDS-PAGE of H35S and H35R cells treated as described in Fig.
3, followed by autoradiography, revealed that all of the radioactivity
was associated with the FdUMP·TS ternary complex with none of the label at the origin, which would be indicative of RNA (data not presented).
In an attempt to increase the immunogold particles and labeled ternary complexes within the cell, an FdUrd-resistant cell line (H35R) that contained 50-100-fold greater levels of TS than the FdUrd-sensitive line (H35S) (22) was employed. In contrast to the findings with the H35S cell line (Fig. 3A), the H35R cells revealed that although some radioactivity was in the nucleus, most of the ternary complex was spread throughout the cytoplasm (Fig. 3B). It is of interest to note that the morphology of the two cell lines differed in that the H35R cells are larger than the H35S cells. It should be emphasized though that the level of TS found in dividing cells in culture and in tissues, both normal and neoplastic, is more like that found in H35S than H35R, that is quite low. As in the case of the immunogold studies, these findings were reproduced in numerous autoradiographs where it was possible to easily distinguish between H35S and H35R cells.
Mitochondrial Presence of Thymidylate SynthaseAs a
consequence of recent findings suggesting the presence of TS in plant
mitochondria as a dihydrofolate reductase·TS bifunctional complex
(14) and the presence of a dUTPase in rat liver mitochondria, we
extensively purified mitochondria from H35S and H35R cells by the same
procedure used for dUTPase (15) and found considerable TS activity in
the latter relative to the former using a sensitive tritium release
assay (29). To confirm the assay, we incubated mitochondrial extracts
with [32P] FdUMP and
CH2H4PteGlu, and as shown in Fig.
4, mitochondria do indeed contain TS as evidenced by
their ability to form a ternary complex. To demonstrate that the enzyme
was not associated with the mitochondrial membranes, lysis of the
mitochondria in water followed by centrifugation to remove the
membranous fraction revealed TS to be present mainly in the supernatant
fraction. As in the case of the dUTPase (15), TS appears to be
imported, since evidence for a sequence in mitochondrial DNA, which
hybridizes to the rat TS cDNA could not be detected, although it is
possible that the mitochondrial TS sequence, if it does exist, is
sufficiently different from the genomic DNA that it does not hybridize
to mitochondrial TS DNA. However, based on the high degree of
evolutionary homology of the various TSs described to date (2), this
would appear to be unlikely.
Evidence That Thymidylate Synthase Is Phosphorylated
In view
of the fact that many proteins, in particularly those associated with
the regulation of cell division are phosphorylated (30), we examined
this possibility for TS. H35R cells were incubated with
32Pi, and the TS present within the cells was
extracted with antibody specific for rat TS using protein A. As shown
in the SDS-PAGE autoradiogram (Fig. 5), the TS extracted
from the cells was labeled with 32Pi (Fig. 5,
lane 2), but no label was present in this region when nonspecific serum was used (Fig. 5, lane 1). In the presence
of increasing amounts of okadaic acid, an inhibitor of
phosphoserine/phosphothreonine phosphatases, the amount of label
associated with TS appeared to increase (Fig. 5, lanes 3 and
4), while the addition of staurosporine, a protein kinase
inhibitor, blocked TS labeling completely (Fig. 5, lane
5).
Phosphorylation of Thymidylate Synthase in Vitro
To verify
the results in Fig. 5, we determined if the apparent phosphorylation of
TS that was occurring in situ could be measured in cellular
extracts. It is apparent from the results in Fig. 6 that
a kinase is present in the extracts that transferred 32P
from [-32P]ATP to a specific site on the TS present in
the extracts. For the purpose of measuring potential kinase activity,
we used H35R cells rather than H35S cells, since a much greater
quantity of TS is present in extracts from these cells, which did not
require the addition of TS as a substrate. To reduce the background and to basically terminate the transfer of 32P, a large excess
of cold ATP was added at the indicated times in Fig. 6, which appeared
to have the desired effect of ending measurable incorporation of
radioactivity into protein. It is clear from the results in Fig. 6 that
the transfer is time-dependent. Preliminary results
indicate that at least one serine in the enzyme is phosphorylated.
Numerous proteins, particularly those associated with cell division, have been found in the past decade to be associated with the nucleus. Even before these more recent developments, TS was reported to be present in the nucleus of eukaryotic cells as part of a "replitase" complex consisting of enzymes involved mainly in providing substrates for DNA synthesis (17), although the cytoplasmic location of TS has also been reported (18, 19). We have shown in this paper by both immunogold labeling and autoradiography that not only is TS associated with the nucleus of H35S cells, it is located predominantly in the nucleolus. It was hoped that cells selected for their resistance to FdUrd (H35R), as a result of a 50-100-fold increase in TS, would show an even greater nuclear content of TS. However, much to our surprise, when these cells were examined by autoradiography to locate TS by means of ternary complex formation, most of the TS was found in the cytoplasm, although some was still in the nucleus. It should be emphasized that the presence of such high concentrations of TS in H35R cells is not encountered normally, since TS, even in actively dividing cells, can be detected only by a sensitive tritium release assay (29), whereas the enzyme can be measured easily in H35R cell extracts using a spectrophotometric assay (26). This massive level of TS in the cytoplasm of H35R cells is also associated with an altered morphology in that these cells appear to be considerably larger than the H35S cells. It is not known at this time whether the morphologic differences are associated with an apparent impaired transport of TS to the nucleus. Even so, it is not obvious how TS is directed to the nucleus, since it does not possess a typical nuclear localization signal (31), although there are three sites in the protein sequence of human, mouse, and rat TS (27) with three to four basic residues within a seven- to eight-amino acid peptide sequence that could serve this purpose. Alternatively, the enzyme might be chaperoned into the nucleus by a protein similar to that described recently for the Id family of helix-loop-helix proteins (32). It is of interest to note that dUTPase, like TS, although associated with the nucleus, does not contain a characteristic nuclear localization signal either (15, 33).
Other properties that dUTPase and TS share relate to their presence in mitochondria and their state of phosphorylation. It is possible that the mitochondrial location of TS is due to the nonspecific adherence of TS to the surface of the mitochondria, but the extensive washing procedure employed would appear to mitigate against this possibility, as well as our recent finding that the enzyme can be released on hypotonic lysis of the mitochondria.3 Preliminary results reveal that the H35 TS is labeled on a serine, as has been found also for dUTPase (33). Because of these common properties of TS and dUTPase, it would not be unreasonable to assume that the two enzymes reside in the same regions of the cell, possibly in the proposed replitase complex (12). As shown in Fig. 6, extracts of H35 cells contain a kinase (5) that phosphorylates TS. More recently, to test TS's susceptibility to phosphorylation, we have found such kinases as protein kinase C and calmodulin kinase to phosphorylate recombinant rat TS quite well, and with a fair degree of specificity, as several other proteins including E. coli TS, were not phosphorylated.3
A function for the phosphorylation of TS, if any, is not known, although based on similar studies, it has been shown that phosphorylation can affect the stability, the activity, and even the location of a protein (34). As shown recently in the case of MADR2, its nuclear accumulation and signaling depend on it being phosphorylated (35). This does not appear to be an isolated case as more and more instances are being reported on the influence of protein phosphorylation on the cellular location of various proteins (34, 36, 37), particularly those associated with cell division. It is interesting to note that only the nuclear form of dUTPase is also phosphorylated (33). Whether a similar case can be made for TS remains to be determined. In a similar vein, questions can be raised regarding the function of TS in the nucleus of the cell and even more so the purpose of its nucleolar presence. Since TS has been shown to interact with its own mRNA to regulate its translation (9), as well as that of c-myc RNA (38), it is possible that what is observed in the autoradiographic patterns (Fig. 3A) is an association of TS with its mRNA or pre-mRNA or possibly its association with a replitase complex (17). As far as the mitochondrial location of TS, mitochondrial DNA must be maintained, and since TS is involved in providing a substrate for the synthesis and repair of DNA, this could very well be its function. In any event it would appear from the data presented here that the functional role of TS may be more complex than merely serving as a provider of dTMP. A preliminary account of these findings has been presented (39).
We express our appreciation to Judith Valentino for her excellent assistance in preparing this manuscript and to Zenia Nimec for her assistance in some of these experiments. We also thank Dr. Carmen Mannella of our institution for his helpful suggestions on the lysis of mitochondria.