Uridine Phosphorylase Association with Vimentin

INTRACELLULAR DISTRIBUTION AND LOCALIZATION*

Rosalind L. Russell, Deliang Cao, Dekai Zhang, Robert E. Handschumacher, and Giuseppe PizzornoDagger

From the Department of Internal Medicine, Section of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut 06520

Received for publication, September 18, 2000, and in revised form, November 21, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pM and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ability of cells to maintain a constant supply of pyrimidine and purine nucleotides is dependent on both de novo synthetic and salvage pathways. The relative importance of either the de novo or the salvage pathway in the maintenance of nucleotide pools is variable and dependent on the cell or tissue type (reviewed in Refs. 1, 2). Uridine phosphorylase (UPase)1 is an important enzyme in the pyrimidine salvage pathway and catalyzes the reversible phosphorolysis of uridine to uracil (3-5). This enzyme is present in most human cells and tissues analyzed, and it is frequently elevated in tumors (4, 5). Enzymatic activity may also be induced in different cell lines by cytokines such as tumor necrosis factor-alpha , interleukin-1alpha , and interferon-alpha and -gamma as well as vitamin D3 (6-8). UPase has also been shown to be important in the activation and catabolism of fluoropyrimidines (9, 10), and the modulation of its enzymatic activity may affect the therapeutic efficacy of these chemotherapeutic agents (11, 14).

UPase also plays an important role in the homeostatic regulation of both intracellular and plasma uridine concentrations (11-14). Uridine plasma concentration is under very stringent regulation (15, 16) mostly as a function of liver metabolic control (17), intracellular UPase enzymatic activity (11-14), and cellular transport by both facilitated diffusion and Na+-dependent active transport mechanisms (18-25). Uridine is critical in the synthesis of RNA and biological membranes through the formation of pyrimidine-lipid and pyrimidine-sugar conjugates (reviewed in Ref. 1), and it has been associated with the regulation of a number of biological processes (1).

Whereas there is evidence that uridine and its nucleotides are associated with different biological processes (reviewed in Refs. 1 and 2) the precise mechanisms that allow uridine to modulate these processes are not well defined. Uridine has been shown to cause increased vascular resistance (25), hyperpolarize amphibian and rat ganglia (26, 27), potentiate dopaminergic transmission, and reduce anxiety in animal models (28, 29), as well as potentiate barbiturate effects and induce sleep in rats (30, 31). Uridine perfusion has been shown to maintain brain metabolism during ischemia (32, 33) and to rapidly restore myocardial ATP and UDPG (34) following myocardial ischemia.

Characterization of UPase intracellular localization and association with other proteins may provide some insight into the mechanisms that control uridine metabolism in cells. In this study, we characterize the cellular distribution and the associated enzymatic activity of UPase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Primary antibodies against UPase were prepared at Yale (rabbit anti-UPase polyclonal antibody to human recombinant UPase) (35), or purchased from Sigma Chemical Co. (St. Louis, MO) (mouse anti-Vimentin Clone V9). Secondary antibodies, horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit (fluorescein isothiocyanate-conjugated and Texas red-conjugated donkey anti-rabbit) or (fluorescein isothiocyanate- or Texas red-conjugated sheep anti-mouse) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Antibodies for vimentin (V9 or monoclonal antibody clone 13.2) or polyclonal goat anti-vimentin were purchased from Sigma (St. Louis, MO). Immunoaffinity support for antibody immobilization and antibody purification was purchased from Pierce. Affi-Gel-10-activated agarose and protein assay reagent were purchased from Bio-Rad (Hercules, CA). 5'-Amino benzylacyclouridine and benzylacyclouridine (BAU) were a generous gift from Dr. S. Chu at Brown University (Providence, RI).

Tissue Culture-- Cells (NIH 3T3 and colon 26) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Sigma) and were maintained in a humidified atmosphere containing 5% CO2 in air. NIH 3T3 cells were purchased from ATCC, and colon 26 cells were derived from the solid tumor (grown in vivo) using standard procedures. Colon 26 cells were originally obtained from Southern Research Institute (Birmingham, AL) and grown subcutaneously in BALB/C mice.

Affinity Chromatography-- The BAU affinity column was prepared by coupling amino-BAU to an Affi-Gel-10-agarose matrix as previously described (35). Antibody affinity columns were prepared using a cyanoborohydride coupling procedure (Pierce) following manufacturer guidelines. Vimentin antibody was purchased from Sigma (goat anti-vimentin and clone V9 mouse anti-vimentin were both used to prepare affinity columns).

In all affinity chromatographic procedures, cells (colon 26 or NIH 3T3) or solid tumors (colon 26) were lysed in 50 mM Tris-HCl, pH 7.4 (containing 2 mM dithiothreitol), using a Dounce homogenization apparatus or tissue homogenizer, respectively. Cell lysates were prepared at 4 °C, and the supernatant from the 30,000 × g centrifugation was applied to the column. Following sample application, the column was washed with ~10 volumes of buffer or until the column effluent contained no detectable protein (Bio-Rad Coomassie Brilliant Blue G-250 dye reagent). Sample elution was accomplished using 4-8 column volumes of 0.1 M glycine (antibody affinity column) or 20 mM uridine (BAU affinity column). The eluted proteins were concentrated using Ultrafree-4 centrifugal filters (Millipore) and analyzed by SDS-polyacrylamide gel electrophoresis and Western blot techniques.

Gel Filtration Chromatography-- Cell lysates from colon 26 cells grown as a monolayer in 150-cm2 dishes were prepared in 20 mM Tris-HCl, 137 mM NaCl pH 7.4 (TBS). Cytosolic fractions were centrifuged at 100,000 × g for 1 h and applied to a Sephacryl-s300 column (Amersham Pharmacia Biotech) that had been calibrated using known molecular weight markers (Amersham Pharmacia Biotech). The mobile phase was 20 mM Tris-HCl, 137 mM NaCl with a flow rate of 1 ml/min. Protein elution of standards was monitored in fractions using the Bio-Rad Coomassie Blue G-250 dye reagent.

In Vitro Protein Binding Assay-- The ability of UPase and vimentin to form a stoichiometric complex was evaluated using purified recombinant vimentin (cytoskeleton) directly applied to nitrocellulose membranes. Known quantities of recombinant vimentin (431-3.4 pM) or similar protein concentrations of BSA as a negative control was applied to nitrocellulose membranes and allowed to air dry. Membranes were blocked for 1 h at room temperature in 5% nonfat milk. Purified UPase was directly coupled to horseradish peroxidase (Pierce) and used to probe the nitrocellulose membranes containing BSA and vimentin. The concentration of UPase bound to vimentin was calculated from a standard curve containing 5-0.15 µg of horseradish peroxidase-UPase directly applied to nitrocellulose and exposed to ECL (Amersham Pharmacia Biotech) at the same time as the vimentin membrane.

Construction of Prokaryotic Expression Vector and Preparation of Recombinant UPase Protein-- Large quantities of human UPase recombinant protein were prepared using the pQE expression system (Qiagen, Santa Clarita, CA). Briefly, human UPase cDNA was released from pMal/Hup with EcoRI and HindII (35) and inserted into pBluescript KS II vector (Stratagene, La Jolla, CA) generating a pBlue/Hup construct. Then, human UPase cDNA was released by BamHI and HindIII and inserted into a pQE 30 vector, generating a prokaryotic expression vector, pQE/Hup, that produces the full-length human UPase recombinant protein. After the construct was confirmed by restriction enzyme digestions, a single M15 transformant was incubated overnight in LB broth with 100 µg/ml ampicillin and 50 µg/ml kanamycin. The overnight culture was diluted 1:100 in 1 liter of LB broth and grown until A600 reached 0.5. The growth was induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h at 37 °C. Bacteria were pelleted by centrifugation for 15 min, 7000 × g at 4 °C, and resuspended in 30 ml lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10 mM beta -mercaptoethanol, 0.5% Triton X-100, 10% glycerol, and 20 mM imidazole, pH 8.0). The bacterial suspensions were incubated on ice for 30 min with 1 mg/ml lysozyme, followed by sonication at 300 watts for 6 × 10 s with 10-s intervals, and the lysate was clarified by centrifuging at 10,000 × g for 20 min at 4 °C (Sorvall, Newton, CT). Protein binding was performed at 4 °C for 2 h by addition of 2 ml of nickel-nitrilotriacetic acid resin into the supernatant. The resin-protein mix was then loaded onto the column and washed with wash buffer (same as lysis buffer except for imidazole increased to 40 mM). UPase protein tagged with 6× histidine remained on the column and was eluted by imidazole step-gradient buffer (50 mM NaH2PO4, 500 mM NaCl, 10% glycerol, plus imidazole at 120, 180, 240, and 300 mM). The presence of UPase in the fractions was examined by SDS-polyacrylamide gel electrophoresis/Coomassie Blue staining and confirmed by enzyme activity assay.

Enzyme Assay-- UPase enzyme activity was measured by uridine conversion to uracil, using TLC chromatographic separation as described previously (35). Briefly, cell lysates were prepared using 50 mM Tris-HCl. The cell pellet remaining following disruption by Dounce homogenization was sequentially extracted using 1% Triton X-100 in 50 mM Tris-HCl, and the supernatant following the 30,000 × g centrifugation was analyzed for enzyme activity. Finally, the pellet remaining after Triton X-100 solubilization was extracted using radioimmune precipitation (RIPA) buffer (1% Triton X-100, 0.5% deoxycholic acid, and 0.1% SDS), and the supernatant was analyzed for enzyme activity. Enzyme activity was measured as the percent conversion of [3H]uridine to [3H]uracil (scintillation counting) following separation on silica TLC plates (Kieselgel 60, Merck), using an 85:15:5 mixture of chloroform and methanol to acetic acid, respectively. The effect of detergents on UPase enzymatic activity was evaluated using purified recombinant UPase to which the detergents were added to the final concentrations used in the extraction methods. No significant alteration in activity was noted in the presence of the detergents used.

Immunofluorescence Techniques-- For whole cell immunofluorescence analyses, cells were grown to 50-70% confluence on glass cell culture slides. After a brief wash with PBS, cell monolayers were fixed with 3.8% paraformaldehyde in PBS for 10 min at 4 °C. Fixed cells were washed briefly in PBS (5 min) and permeabilized using 0.1% Triton X-100 in PBS (10 min), and nonspecific binding was blocked using 3% BSA or serum of the same specificity as the secondary antibody when available (10 min). Incubation with the primary antibody was performed for 1 h at room temperature. After washing the excess unbound antibody (two times 5 min with PBS), sample was exposed for 1 h to secondary antibody at room temperature. The excess secondary antibody was washed with PBS, and the slides were mounted in fluorescent antibody-compatible medium (Molecular Probes, Eugene, OR). Photographs were taken using a Ziess Axiophot microscope and camera apparatus. All experiments included a negative nonspecific serum (preimmune serum) control to ensure specificity of the observed fluorescence. In the case of dual antibody detection, reagent compatibility was determined using normal or preimmune serum and secondary antibodies as negative controls singly and in combination.

Cytoskeleton immunofluorescence was performed as described previously (36). Cells grown to 70% confluence on glass slide covers were rinsed briefly in PBS, and then soluble proteins were extracted for 3-5 min at room temperature using cytoskeleton buffer (100 mM PIPES, pH 6.9, 1 mM MgCl2, 1 mM EGTA containing 4% polyethylene glycol and 1% Triton X-100). Following extraction, cell cytoskeletons were rinsed three times with cytoskeleton stabilizing buffer without detergent. The immunofluorescence analysis was performed as described above.

MALDI-MS-- The unknown protein band was excised from a Coomassie Blue-stained gel and digested overnight using trypsin as described (37). The peptides were subsequently analyzed using MALDI-MS and the Profound peptide data base at the Howard Hughes Medical Institute Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University (38, 39).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Uridine phosphorylase was purified from NIH 3T3 and colon 26 cells using an affinity column coupled to the high affinity uridine phosphorylase inhibitor, 5'-amino-benzylacyclouridine. For colon 26 cells, solid tumor homogenate was affixed to the BAU column. The Coomassie Blue-stained blot of UPase eluted using 20 mM uridine is shown in Fig. 1 (lane 1). The one-step purification procedure results in the isolation of UPase and the copurification of a 58-kDa species. The identification of the lower band as UPase using Western blot analysis is shown in Fig. 1 (lane 2). No cross reactivity of the UPase antibody with the upper band was noted. The lower band at 34 kDa in Fig. 1, lane 1, represents purified UPase, and the upper band (~58 kDa) was identified as vimentin using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) of the tryptic digest (Fig. 2). The tryptic digest of the Coomassie Blue-stained 58-kDa band excised from acrylamide gel covered 57% of the protein using a mass tolerance of ±0.2 atomic mass unit for monoisotopic and ±0.5 atomic mass unit for observed average masses.


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Fig. 1.   UPase was purified from colon 26 cells using BAU affinity column as described under "Experimental Procedures." Lane 1 is the Coomassie Blue-stained polyvinylidene difluoride membrane; lane 2 is the Western blot of the same polyvinylidene difluoride membrane using the UPase antibody.


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Fig. 2.   The tryptic digest of the upper band shown in Fig. 1 was identified as vimentin using MALDI-MS. The internal standards were 100 fmol of bradykinin, which has a protonated, monoisotopic mass of 1060.57, and adrenocorticotropic hormone clip, which has a protonated, monoisotopic mass of 2465.2.

We analyzed NIH 3T3 cells to evaluate whether the copurification of UPase with vimentin was a phenomenon specific to colon 26 tumor cells, which contain highly elevated levels of UPase. UPase was purified from NIH 3T3 and colon 26 cell lines using the BAU column as described, and the results of the Western blots are shown in Fig. 3 (A and B), respectively. In both cases, vimentin was identified in the BAU eluate using Clone V9 monoclonal antibody (Sigma) as shown in Figs. 3A, lane 2, and 3B, lane 2.


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Fig. 3.   UPase was purified from NIH 3T3 (A) and colon 26 cells (B) using a BAU affinity column. Lane 1 (A and B) are the Western blots of the UPase isolated from the affinity column for NIH 3T3 and colon 26, respectively. Lane 2 (A and B) are the same Western blots re-analyzed for vimentin using clone V9 monoclonal antibody as described.

We, and others (35, 40) have previously shown that the majority of the UPase in cells exists in a soluble form that is readily extractable using near-physiologic buffers (40). This is in contrast to what is known about the intermediate filament vimentin, which is one of the most insoluble proteins known. In fact, it has been shown that less than 1% of the total vimentin exists in cells as soluble tetramers (41). The fact that UPase and vimentin are copurified in cell/tumor extracts using physiologic buffers suggests that a fraction of UPase exists in combination with this soluble pool of vimentin. To confirm the association of UPase with vimentin in the cytosol, we performed gel filtration chromatography using a Sephacryl-s300 column to separate the UPase monomer from that which is associated with vimentin. As shown in Fig. 4A, UPase elutes from the column as two distinct peaks. The high molecular mass species elutes at ~400-500 kDa, and the low molecular mass peak elutes as a broad peak with a mean value of about 34 kDa, suggesting that it exists predominantly as a monomer in the cytoplasm. The shoulder on the low molecular mass peak may indicate that UPase exists in both monomeric and dimeric forms or is associated with another protein, under these experimental conditions. Vimentin coelutes from the Sephacryl-s300 column in the same fractions containing the high molecular mass UPase, again suggesting they exist in the cytosolic pool as a complex. The Western blot shown in Fig. 4B shows the UPase and vimentin present in each fraction depicted in Fig. 4A.


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Fig. 4.   A, all lysates were prepared as described under "Experimental Procedures" and applied to a 27- × 1.5-cm2 Sephacryl-s300 column. Fractions were collected at 0.5-min intervals and analyzed using Western blot for UPase and vimentin. The solid circles indicate the molecular mass calibration standards (thyroglobin, 634 kDa; ferritin, 440 kDa; catalase, 199 kDa; aldolase, 177 kDa; bovine serum albumin, 67 kDa; carbonic anhydrase, 29 kDa; and cytochrome c, 12.9 kDa). The open circles represent the densitometric analysis of UPase isolated from each fraction, and the solid triangles represent the densitometric analysis of the vimentin isolated from each fraction as determined by Western blot of 5% of the effluent. B, the Western blot of vimentin (upper row) and UPase (lower row) present in each of the fractions 15-30 as shown in A.

Confirmation of the association of UPase with the soluble pool of vimentin was also confirmed using a combination of gel filtration chromatography and immune precipitation (data not shown).

The stoichiometry of the UPase-vimentin complex, was estimated using a slot blot binding assay to measure direct protein-protein interactions. Purified recombinant human UPase was directly coupled to horseradish peroxidase and used to probe known concentrations of purified recombinant vimentin (inset, Fig. 5, lane A) or BSA (inset, Fig. 5, lane B) and the Scatchard transformation is shown in Fig. 5. Using this technique, the stoichiometry of UPase binding to vimentin is calculated at ~1:2 and the Kd of the complex is 120 pM.


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Fig. 5.   Human recombinant UPase (5 µg/ml) directly conjugated to horseradish peroxidase was used to probe a nitrocellulose membrane to which purified recombinant vimentin (inset, lane A) or BSA (inset, lane B) was affixed. Vimentin and BSA concentrations of 25, 12.5, 6.25, 3.2, 1.7, and 0.85 µg are shown in lanes 1-8, respectively. ECL was used to develop the blots, and the optical density of the resulting film was converted to picomole values of UPase using a standard curve containing known quantities of UPase as described. Data transformation was performed, and the Scatchard analysis is shown.

We wanted to determine whether UPase could be found in combination with the polymeric form of vimentin, so NIH 3T3 cells and colon 26 cells were sequentially extracted using Tris-buffered saline, followed by 1% Triton X-100, and finally RIPA buffer. The detergent extracts were utilized to solubilize cytoskeleton (including vimentin) and associated proteins while maintaining optimum enzyme activity (Table I) and UPase-vimentin association (Fig. 6). Extracts from each of these conditions were affinity-purified using anti-vimentin chromatography. The results from these experiments demonstrate that UPase and vimentin exist together in a complex in each of these fractions. Fig. 6B shows a Western blot of UPase isolated from colon 26 cell lysates purified using anti-vimentin affinity column and indicates the presence of a UPase-vimentin complex in each of these fractions. Although the extraction of vimentin from both the Tris and Triton X-100 fractions are similar (Fig. 6A, lanes 1 and 2), less vimentin was retained on the affinity column in the RIPA solubilized fraction, probably a function of lower antibody efficiency in the presence of detergents contained in the RIPA buffer. The predominant form of UPase exists in the Tris-soluble fraction in combination with the soluble tetrameric vimentin. Further solubilization of the cell pellet with Triton X-100 liberates a smaller percentage of UPase (~15-30% based on enzymatic activity shown in Table I) suggesting less UPase is associated with the polymeric membrane-associated form of vimentin liberated using this technique. Finally, there is a very small percentage of UPase released by the final RIPA solubilization of the pellet, which is between 10-20% based on enzymatic activity (Table I) and is difficult to visualize on the Western blot shown in Fig. 6. This additional pool of UPase is also associated with the polymeric form of vimentin. Taken together, both the Triton X-100 and RIPA buffer extracted UPase represent between 25-50% additional UPase enzymatic activity found in association with the polymeric and membrane-associated vimentin pool.

                              
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Table I
Fraction of total UPase associated with the soluble and detergent insoluble cytoskeleton
Cells grown to 70% confluence were trypsinized and extracted sequentially using 50 mM Tris-HCl, 1% Triton X-100 and RIPA buffer as described under "Experimental Procedures." Enzymatic activity was measured as a fraction of the total in three independent extractions and the mean of these data are shown with the standard error of the mean in parentheses.


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Fig. 6.   Colon 26 tumor homogenates were prepared by sequentially extracting the cell pellet in 50 mM Tris-HCl (lane 1), followed by a 1% Triton X-100 extraction of the pellet (lane 2), and a final extraction of the residual pellet using RIPA buffer (lane 3). The lysates were chromatographed on an anti-vimentin column and eluted using 0.1 M glycine, pH 3.0. The upper panel (A) is the Coomassie Blue-stained Western blot of each of these extraction protocols eluted from a vimentin affinity column. B is a Western blot for the UPase present in each of the fractions.

Western blots of NIH 3T3 (lanes 1-3) and colon 26 cells (lanes 4-6) sequentially extracted without detergent (lanes 1 and 4), using 1% Triton X-100 (lanes 2 and 5) and RIPA buffer (lanes 3 and 6) are shown in Fig. 7. The distribution pattern of vimentin in these fractions is shown in the upper panel (A), and the distribution of UPase in the same fractions is shown in the lower panel (B). Although UPase and vimentin copurify in all three fractions as determined by affinity chromatography (Fig. 6), there is an inverse relationship between the relative abundance of each protein in these fractions. Although the majority of UPase exists in the Tris buffer-soluble fraction (lanes 1 and 4) for NIH 3T3 and colon 26 cells, respectively, less than 1% of the total vimentin has been shown to exist in this pool (41) (Fig. 7A, lanes 1 and 4). Although 99% of the vimentin exists in the polymeric form and is extractable using detergents (Fig. 7A, lanes 3 and 6), less than 20% of the total UPase is present in this fraction (RIPA) based on enzyme activity (Table I).


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Fig. 7.   NIH 3T3 cell homogenates were sequentially extracted using 50 mM Tris HCl (lane 1), followed by 1% Triton X-100 (lane 2), and finally RIPA buffer (lane 3). Equal protein was loaded for each extraction and analyzed by Western blot for UPase (B) and vimentin (A). Colon 26 cell homogenates were sequentially extracted using 50 mM Tris HCl (lane 4), followed by 1% Triton X-100 (lane 5) and finally RIPA buffer (lane 6). Equal protein was loaded for each extraction and analyzed by Western blot for UPase (B) and vimentin (A).

Further evidence for the association of UPase with the polymeric form of vimentin was demonstrated using NIH 3T3 cells from which soluble proteins were extracted in the presence of cytoskeleton stabilizing agents (36). We extracted NIH 3T3 cells grown on glass slides with cytoskeleton-stabilizing buffer containing 1% Triton X-100 and 4% polyethylene glycol as described (36) before fixing and processing them for immunofluorescent microscopy. Subcellular localization of UPase in NIH 3T3 demonstrated a distinctly filamentous pattern (Fig. 8A). The staining for UPase was particularly intense in the perinuclear area of the cell and extends outward toward the cell periphery in a filamentous network, which is identical to the intermediate filament vimentin as shown in Fig. 8B. Regions of the cells where vimentin and UPase colocalize are shown in Fig. 8C and appear yellow.


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Fig. 8.   NIH 3T3 cells were grown overnight on glass cover slides and processed for immunofluorescent antibody detection as described, using cytoskeleton-stabilizing buffers to maintain microtubule integrity. In A, UPase staining is shown as red fluorescence. Cells were double-labeled for vimentin, as shown in B, and appear green. Areas where UPase and vimentin are colocalized appear yellow using double filters (C). NIH 3T3 cells were grown overnight on glass cover slides and on the second day, 0.125 nM nocodazole was added for 24 h (D-F). UPase staining following nocodazole is shown in D, vimentin staining is shown in E, and areas of colocalization are shown in F and appear yellow.

Because the intermediate filament network is found in close association with the microtubule system, we used nocodazole to depolymerize the microtubules as a means of dissociating the microtubule and intermediate filament networks (42, 43). This treatment is characteristically associated with a perinuclear collapse of the intermediate filament network away from the cell periphery. In Fig. 8 (D-F), changes in the UPase staining that result from treating NIH 3T3 cells with nocodazole are shown. The UPase is no longer associated with the filamentous network, which extends toward the cell membrane, but is surrounding the nucleus in a dense network (Fig. 8D) that is coincidental with the vimentin intermediate filament as shown in Fig. 8E. Areas of colocalization are shown in Fig. 8F and appear yellow.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that UPase and the intermediate filament protein vimentin are colocalized using immunofluorescent antibody techniques, affinity chromatography, gel filtration, and immunoprecipitation. We have further shown these proteins interact in vitro using binding assays. The difficulty is in determining the physiological relevance of this observation. The role of uridine phosphorylase in the salvage pathway of pyrimidine nucleoside biosynthesis does not readily translate into a role for this enzyme in association with the cytoskeleton. Additional difficulty in interpreting these data lies with the inability to clearly establish a functional role for the intermediate filament vimentin. Although a number of theories have been proposed for the function of this network, the data are not conclusive. Cellular processes as diverse as differentiation, motility, signal transduction, cell division, cytoskeletal stability, and vesicular trafficking have been associated with alterations in the dynamics of the intermediate filaments (reviews in Refs. 44-48, and references within). Deletion of the vimentin protein in mice had no detrimental characteristics, and the mice apparently developed and reproduced normally (49). It has recently been shown that vimentin null mice exhibit neurological defects and impaired motor coordination (50). Further in vitro analyses of fibroblasts isolated from wild type and vimentin null mouse embryos show that vimentin null cells exhibit reduced mechanical stability, decreased growth factor-directed and random motility, and reduced capacity to cause contraction of collagen fibrils (51), a process necessary in wound healing.

In recent years, a number of proteins have been shown to be associated with the vimentin intermediate filament scaffold, including p53 (52), protein kinase C (53), Yes and cGMP kinase (54, 55), glycolytic enzymes pyruvate kinase, creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase (56-58), and nucleoside diphosphate kinase (NDPK) (56, 59) as well as the cross-linking proteins plectin, IFAP-300, and filamin, which link intermediate filaments to other cytoskeletal elements and membranes (59-63). It is particularly interesting to note the number of proteins involved in signal transduction and energy metabolism that have been associated with vimentin. Although the phenotype of the vimentin knockout mice is not evident under "normal" conditions, the recent observations of reduced mechanical strength and the cellular response to motility stimulating growth factors in fibroblasts isolated from these animals supports a role for the vimentin three-dimensional network in the coordination of these responses.

The proposed role for nucleoside diphosphate kinase (NDPK, nm23) in nucleotide channeling (59), production of most cellular non-ATP nucleoside triphosphates (64), and copurification with vimentin and enzymes involved in ATP formation/regeneration (56), together with our observation of UPase colocalization with this same cellular machinery, suggests that such observations are biologically relevant. If vimentin serves a largely structural role in cellular homeostasis, the localization of the vimentin-associated proteins within the milieu of the cell may represent a mechanism for "docking" these proteins to the cytoskeleton scaffold. In this case, proteins associated with glycolytic processes and signal transduction may be bound to vimentin as a mechanism of sequestration of enzymatic activity or signal transduction. If the vimentin network serves as a functional scaffold that directs mRNA and vesicular trafficking, as proposed (reviewed in Ref. 45), the association of glycolytic, UPase, NDPK, and signal transduction molecules with this filamentous network may be under dynamic control. In the case of UPase, it may be relevant that a large fraction of this enzyme is associated with the soluble pool of vimentin. Because soluble vimentin represents the fraction of this protein that is added to existing filaments in response to changing cell dynamics, it seems relevant that this form of the filament is associated with UPase. Particularly, if newly synthesized vimentin targets areas of mRNA translation, having a pyrimidine degradation enzyme in close proximity to areas of high mRNA translation would seem reasonable.

It is possible that the UPase complex with vimentin represents the biologically active form of this enzyme. It has been shown by Vita et al. (65) that in Escherichia coli B., enzymatically active UPase exists as a tetramer. From our observations in colon 26 cells, the majority of soluble UPase (55-60%) exists as a monomer and the remaining UPase is found in association with the soluble vimentin tetramer, possibly in a 1:2 stoichiometry as suggested by in vitro binding assays. It is possible that the biologically active species of UPase is the UPase:vimentin multimer. Alternatively, it is possible that in mammalian cells UPase could exist predominantly either as a monomer or an easily dissociated tetramer not detected with our techniques.

The association of proteins with the cytoskeleton may serve different functions, including activation or inactivation (reviewed in Ref. 66) of enzymatic activity, localization of a particular enzymatic activity to multiprotein complexes (67-69), or sequestration of proteins from the soluble or nuclear pool (52, 69). Whether any or all these possibilities are true for UPase sequestered to the vimentin filaments remains to be proven. In vitro enzymatic analyses of the detergent-extractable pool of UPase demonstrated that this source of enzyme retains enzymatic activity. It is difficult to say whether this is true in the intact cell where it exists in an insoluble form. The UPase found in association with the polymeric vimentin network may represent a pool of enzyme able to mobilize that is only active when liberated from its three-dimensional network, possibly regulated by variations in the intracellular concentrations of uridine. The association with the insoluble vimentin network could also represent a way of localizing enzymatic activity to a particular area within the cell.

It is possible that the interaction between UPase and vimentin is a function of nonspecific interactions between two relatively hydrophobic molecules or is mediated by an intermediate element. Based on the variety of different biochemical analyses that demonstrate the colocalization of these proteins, this seems unlikely. Experiments are in progress to further analyze the specificity and nature of the interactions between these two proteins.

    ACKNOWLEDGEMENTS

We thank Dr. Michael Kashgarian, Professor of Pathology and Molecular Cell and Developmental Biology at Yale University School of Medicine, for his generous assistance and the use of the Fluorescence Microscopy facilities.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Dept. of Internal Medicine, Section of Oncology, Yale University School of Medicine, 333 Cedar, SHM I 220, New Haven, CT 06520. Tel.: 203-785-4549; Fax: 203-785-7670; E-mail: Giuseppe.Pizzorno@yale.edu.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M008512200

    ABBREVIATIONS

The abbreviations used are: UPase, uridine phosphorylase; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; BAU, benzylacyclouridine; NDPK, nucleoside diphosphate kinase; RIPA buffer, radioimmune precipitation buffer; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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