Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

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), 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, EC2.1.1.45) 1The abbreviations used are: TS, thymidylate synthase; dUMP, 2′-deoxyuridine 5′-monophosphate; FdUMP, 5-fluoro-2′-deoxyuridine 5′-monophosphate; dTMP, 2′-deoxythymidine 5′-monophosphate; FdUrd, 5-fluoro-2′-deoxyuridine; CH2H4PteGlu, 5,10-methylene tetrahydrofolate; H2PteGlu, dihydrofolate; dUTPase, deoxyuridine triphosphatase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation buffer. 1The abbreviations used are: TS, thymidylate synthase; dUMP, 2′-deoxyuridine 5′-monophosphate; FdUMP, 5-fluoro-2′-deoxyuridine 5′-monophosphate; dTMP, 2′-deoxythymidine 5′-monophosphate; FdUrd, 5-fluoro-2′-deoxyuridine; CH2H4PteGlu, 5,10-methylene tetrahydrofolate; H2PteGlu, dihydrofolate; dUTPase, deoxyuridine triphosphatase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation buffer. 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 (1Friedkin M. Kornberg A. McElroy W.D. Glass B. The Chemical Basis of Heredity. Johns Hopkins Press, Baltimore, MD1957: 609-614Google Scholar). The DNA sequences of some 30 different species of TS have been clarified (2Carreras C.W. Santi D.V. Annu. Rev. Biochem. 1995; 64: 721-762Crossref PubMed Scopus (653) Google Scholar) establishing it as one of the most phylogenetically conserved proteins known. X-ray crystal structures for theLactobacillus casei (3Hardy L.W. Finer-Moore J. Montfort W.R. Jones M.O. Santi D.V. Stroud R. Science. 1987; 235: 448-455Crossref PubMed Scopus (293) Google Scholar), Escherichia coli (4Montfort W.R. Perry K.M. Fauman E.B. Finer-Moore J.S. Maley G.F. Hardy L. Maley F. Stroud R.M. Biochemistry. 1990; 29: 6964-6977Crossref PubMed Scopus (260) Google Scholar, 5Matthews D.A. Villafranca J.E. Janson C.A. Smith W.W. Welsh K. Freer S. J. Mol. Biol. 1990; 214: 937-948Crossref PubMed Scopus (144) Google Scholar), and human TSs (6Schiffer C.A. Clifton I.J. Davisson V.J. Santi D.V. Stroud R.M. Biochemistry. 1995; 34: 16279-16287Crossref PubMed Scopus (111) Google Scholar) 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 (7Reich S.H. Fuhry M.A.M. Nguyen D. Pino M.J. Welsh K.M. Webber S. Janson C.A. Jordan S.R. Matthews D.A. Smith W.W. Bartlett C.A. Booth C.L.J. Herrmann S.M. Howland E.F. Morse C.A. Ward R.W. White J. J. Med. Chem. 1992; 35: 847-858Crossref PubMed Scopus (57) Google Scholar). 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 (8Horie N. Nozawa R. Takeishi K. Biochem. Biophys. Res. Commun. 1992; 185: 127-133Crossref PubMed Scopus (14) Google Scholar) and translational levels of synthesis (9Chu E. Koeller D.M. Casey J.L. Drake J.C. Chabner B.A. Elwood P.C. Zinn S. Allegra C.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8977-8981Crossref PubMed Scopus (325) Google Scholar), while earlier studies suggested that TS forms multienzyme complexes involved in DNA synthesis both in T-even phage-infected E. coli (10Reddy G.P.V. Singh A. Stafford M.E. Mathews C.K. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3152-3156Crossref PubMed Scopus (37) Google Scholar,11Wheeler L. Wang Y. Mathews C.K. J. Biol. Chem. 1992; 267: 7664-7670Abstract Full Text PDF PubMed Google Scholar) and eukaryotic cells (12Noguchi H. Reddy G.P.V. Pardee A.B. Cell. 1983; 32: 443-451Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 13Ayusawa D. Shimizu K. Koyama H. Takeishi K. Seno T. J. Biol. Chem. 1983; 258: 48-57Abstract Full Text PDF PubMed Google Scholar). 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 (14Neuburger M. Réberllé F. Jourdain A. Nakamura S. Douce R. J. Biol. Chem. 1996; 271: 9466-9472Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and a dUTPase in HeLa cells (15Ladner R.D. McNulty D.E. Carr S.A. Roberts G.D. Caradonna S.J. J. Biol. Chem. 1996; 271: 7745-7751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). 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 (16Brown S.S. Neal G.E. Williams D.C. Biochem. J. 1965; 97: 34C-36CCrossref PubMed Scopus (23) Google Scholar, 17Reddy G.P.V. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3312-3316Crossref PubMed Scopus (285) Google Scholar) or the cytoplasm (18Kucera R. Paulus H. Exp. Cell Res. 1986; 167: 417-428Crossref PubMed Scopus (21) Google Scholar, 19Johnston P.G. Liang C.-M. Henry S. Chabner B.A. Allegra C.J. Cancer Res. 1991; 51: 6668-6676PubMed Google Scholar). Fluorescent antibody studies by us some 10 years ago using human TS antibody 2W. A. Samsonoff, G. Maley, and F. Maley, unpublished observations. 2W. A. Samsonoff, G. Maley, and F. Maley, unpublished observations. 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 (20Poon P.-P. Storms R.K. J. Biol. Chem. 1994; 269: 8341-8347Abstract Full Text PDF PubMed Google Scholar). 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. (21Hechemy K. Fox J. Samsonoff W.A. Anacker R. Silverman D. Eisemann C. Green I.S. J. Clin. Microbiol. 1989; 27: 377-384Crossref PubMed Google Scholar). The TS antigen was purified to homogeneity (22Rhee M.S. Balinksa M. Bunni M. Priest D.G. Maley G.F. Maley F. Galivan J. Cancer Res. 1990; 50: 3979-3984PubMed Google Scholar) 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. Gold 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. H35S (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 for in in a after which ml of medium was removed from each was to a of to the while was to a of The were for an times with and for with Swim's the of this the cells were with 2 ml of for and two times with 5 ml of The were then at 40 and the cells the and by in After a secondary fixation for 30 at in a of the were in in a of and in of were on and were essentially as described by and J. Cell Biol. PubMed Scopus Google Scholar). were with and J. Cell Biol. PubMed Scopus Google Scholar) and in a electron at H35S and H35R cells were in Swim's medium to about in one to and with The cells were to an of Swim's medium and for 5 at by with ml of The cells were in ml of 0.5 bovine serum and 10 The cells were then and in a at for 10 at using a The after to another was at × in a for The was in 5 ml of and mitochondria were purified by as described by et D. D. Press, D. Scholar). In either case, the mitochondrial was in 1 ml of 10 50 and in an at for 10 at The was in of and for in an and as the mitochondria were in and after the was as in an complexes containing [32P]FdUMP were by a of the of Maley et al. F. L. Biochemistry. 1995; 34: PubMed Scopus Google Scholar) as to of H35R cell mitochondrial or purified rat TS J. Maley G.F. Maley F. Biophys. 1995; PubMed Scopus Google Scholar) was 1 of 50 of a F. L. Biochemistry. 1995; 34: PubMed Scopus Google Scholar), of and 5 of [32P]FdUMP × After at for 30 the were with an of and for of the was to for 1 at The gel was and to for at H35R rat hepatoma cells were in Swim's medium as on for to The cells, which were at at this were × with 2 ml of Dulbecco's modified Eagle's medium and were then with 2 ml of DMEM, at in a for 1 The were with ml of containing fetal bovine serum and of which was in a for The were times with 2 ml of saline and the cells in 0.5 ml of a modified 2 10 10 10 on with for 30 The were to which were at for 10 at in an The were to to which were of in modified for 1 and for 10 at at To the to was 10 of rabbit TS and after on for 1 of was to each which were for 1 The were and times with of modified RIPA, then in of containing and for 5 was a and after 1 at the gel was with by and paper. The gel was to for at using an H35R cells were for as described The medium was and the were with a containing 50 and 5 The cells from were with the but containing 2 10 10 10 and and for 10 in an of the were to each with of The at were at times by the addition of 1 of which the to the of into the The was for 10 at in an at The were to were with of rabbit TS serum for 1 on by of for 1 on studies by us using antibody to HeLa cell TS, as well as immunogold electron the association of TS with the However, to these studies by H35S cells to rat TS antibody were mainly to the in of the two cell we to us much of rat TS (22Rhee M.S. Balinksa M. Bunni M. Priest D.G. Maley G.F. Maley F. Galivan J. Cancer Res. 1990; 50: 3979-3984PubMed Google Scholar) than HeLa TS for the of we the use of immunogold secondary antibody (goat anti-rabbit fixation to rabbit TS antibody of H35S cells with the 1 that or gold particles could be the cells were to In the H35S cells were with purified anti-TS a of the immunogold particles were found to be localized to the nucleus 1 with most of the particles associated with the nucleolus 1 The nuclear of the particles could be the antibody was first with H35 TS, the of the the nuclear association of the TS antibody was not a was by a number of immunogold particles in a number of areas as well as of the cell However, of the of these findings and the that the of 1 were a of the and not was a more was that of of immunogold particles in H35S cells, which is by the in The density of gold particles in each cell was determined as described and of cells was counted in each of with the by the To the immunogold a was that of the cells to and which in the of the will be to in the cell, while the latter is to then with TS to form a tight ternary which is the in most of the chemotherapeutic to or B. M. Santi D.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was to the was not it was found in most that the of this was After of the cells to it was that the location of the TS complex within the cell be on autoradiography. shown in most of the ternary complex in the nucleus of the H35S cells similar to the immunogold electron micrographs of appears to be localized primarily to the nucleolar the that the nucleolar presence of the label was to the of to and its into nucleolar this was in the presence of a of or of the was that into was not the of the label within the cell not In addition, of H35S and H35R cells as described in by autoradiography, that of the was associated with the ternary complex with of the label at the which be of not In an to the immunogold particles and ternary complexes within the cell, an cell that 50–100-fold greater levels of TS than the (22Rhee M.S. Balinksa M. Bunni M. Priest D.G. Maley G.F. Maley F. Galivan J. Cancer Res. 1990; 50: 3979-3984PubMed Google Scholar) was In to the findings with the H35S cell the H35R cells that although some was in the nucleus, most of the ternary complex was the cytoplasm is of to that the of the two cell in that the H35R cells are than the H35S cells. be that the of TS found in cells in and in both and is more that found in H35S than that is in the of the immunogold these findings were in it was to H35S and H35R cells. a of findings the presence of TS in plant mitochondria as a dihydrofolate reductase·TS bifunctional complex (14Neuburger M. Réberllé F. Jourdain A. Nakamura S. Douce R. J. Biol. Chem. 1996; 271: 9466-9472Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and the presence of a dUTPase in rat we purified mitochondria from H35S and H35R cells by the used for dUTPase (15Ladner R.D. McNulty D.E. Carr S.A. Roberts G.D. Caradonna S.J. J. Biol. Chem. 1996; 271: 7745-7751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and found TS activity in the latter relative to the using a D. Biochemistry. PubMed Scopus Google Scholar). To the we mitochondrial extracts with and CH2H4PteGlu, and as shown in mitochondria TS as by to form a ternary To that the enzyme was not associated with the mitochondrial of the mitochondria in by to the TS to be present mainly in the in the of the dUTPase (15Ladner R.D. McNulty D.E. Carr S.A. Roberts G.D. Caradonna S.J. J. Biol. Chem. 1996; 271: 7745-7751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), TS appears to be since evidence for a in mitochondrial DNA, which to the rat TS could not be although it is that the mitochondrial TS it is different from the DNA that it not to mitochondrial TS However, on the of of the TSs described to (2Carreras C.W. Santi D.V. Annu. Rev. Biochem. 1995; 64: 721-762Crossref PubMed Scopus (653) Google Scholar), this to be In of the fact that in associated with the of cell division are Cell. 1995; Full Text PDF PubMed Scopus Google Scholar), we this for H35R cells were and the TS present within the cells was with antibody for rat TS using A. shown in the the TS from the cells was with but label was present in this region serum was used In the presence of amounts of an of the of label associated with TS to while the addition of a TS labeling To the in we determined the apparent of TS that was in could be in is apparent from the in that a is present in the extracts that from to a on the TS present in the the of potential we used H35R cells than H35S cells, since a much greater of TS is present in extracts from these cells, which not the addition of TS as a To the and to the of a of was at the indicated times in which to have the of of into is from the in that the is indicate that at one in the enzyme is associated with cell have been found in the to be associated with the these more TS was to be present in the nucleus of eukaryotic cells as of a complex of involved mainly in substrates for DNA synthesis G.P.V. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3312-3316Crossref PubMed Scopus (285) Google Scholar), although the location of TS has also been (18Kucera R. Paulus H. Exp. Cell Res. 1986; 167: 417-428Crossref PubMed Scopus (21) Google Scholar, 19Johnston P.G. Liang C.-M. Henry S. Chabner B.A. Allegra C.J. Cancer Res. 1991; 51: 6668-6676PubMed Google Scholar). have shown in this by both immunogold labeling and autoradiography that not is TS associated with the nucleus of H35S cells, it is located in the was that cells for to as a of a 50–100-fold in TS, an greater nuclear of However, much to these cells were by autoradiography to TS by of ternary complex most of the TS was found in the although some was still in the be that the presence of of TS in H35R cells is not encountered since TS, in cells, be by a D. Biochemistry. PubMed Scopus Google Scholar), the enzyme be in H35R cell extracts using a F. L. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). of TS in the cytoplasm of H35R cells is also associated with an in that these cells to be than the H35S cells. is not known at this the are associated with an apparent of TS to the it is not how TS is to the nucleus, since it not a nuclear localization signal L. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), although are in the of and rat TS J. Maley G.F. Maley F. Biophys. 1995; PubMed Scopus Google Scholar) with to within a to that could this the enzyme be into the nucleus by a similar to that described recently for the of proteins R.W. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is of to that dUTPase, TS, although associated with the nucleus, not a nuclear localization signal either (15Ladner R.D. McNulty D.E. Carr S.A. Roberts G.D. Caradonna S.J. J. Biol. Chem. 1996; 271: 7745-7751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, R.D. Carr S.A. M.J. McNulty D.E. Caradonna S.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). properties that dUTPase and TS to presence in mitochondria and state of is that the mitochondrial location of TS is to the of TS to the surface of the but the to this as well as that the enzyme be on of the that the H35 TS is on a as has been found also for dUTPase R.D. Carr S.A. M.J. McNulty D.E. Caradonna S.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Because of these properties of TS and dUTPase, it not be to that the two in the of the cell, in the complex (12Noguchi H. Reddy G.P.V. Pardee A.B. Cell. 1983; 32: 443-451Abstract Full Text PDF PubMed Scopus (123) Google Scholar). shown in extracts of H35 cells a D.A. Villafranca J.E. Janson C.A. Smith W.W. Welsh K. Freer S. J. Mol. Biol. 1990; 214: 937-948Crossref PubMed Scopus (144) Google Scholar) that More to to phosphorylation, we have found as and to rat TS and with a of as several other proteins E. coli TS, were not and F. Maley, unpublished for the of TS, is not although on similar it has been shown that the the and the location of a D.A. Biochem. J. 1995; PubMed Scopus Google Scholar). shown recently in the of its nuclear and on it M. S. R. L. J.L. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). not to be an as more and more are on the of on the location of proteins D.A. Biochem. J. 1995; PubMed Scopus Google Scholar, K. J. Exp. Cell. Res. PubMed Scopus Google J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), associated with cell is to that the nuclear form of dUTPase is also R.D. Carr S.A. M.J. McNulty D.E. Caradonna S.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a similar be for TS remains to be In a similar be the of TS in the nucleus of the cell and more the of its nucleolar TS has been shown to interact with its to its (9Chu E. Koeller D.M. Casey J.L. Drake J.C. Chabner B.A. Elwood P.C. Zinn S. Allegra C.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8977-8981Crossref PubMed Scopus (325) Google Scholar), as well as that of E. T. Jones D.M. S.M. Maley G.F. Maley F. S. Allegra C.J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar), it is that is in the is an association of TS with its or or its association with a complex G.P.V. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3312-3316Crossref PubMed Scopus (285) Google Scholar). as the mitochondrial location of TS, mitochondrial DNA be and since TS is involved in a substrate for the synthesis and of DNA, this could well be its In it from the that the role of TS may be more complex than as a of of these findings has been J. Samsonoff W.A. M. B. Galivan J. Maley G. Maley F. Proc. Cancer Res. 1996; Scholar). to for in this and to for in some of these also of for on the of