Uridine Phosphorylase Association with Vimentin
INTRACELLULAR DISTRIBUTION AND LOCALIZATION*
Rosalind L.
Russell,
Deliang
Cao,
Dekai
Zhang,
Robert E.
Handschumacher, and
Giuseppe
Pizzorno
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
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ABSTRACT |
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.
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INTRODUCTION |
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-
, interleukin-1
,
and interferon-
and -
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.
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EXPERIMENTAL PROCEDURES |
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-
-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
-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).
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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DISCUSSION |
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.
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.
 |
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