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Originally published In Press as doi:10.1074/jbc.M607230200 on January 7, 2007

J. Biol. Chem., Vol. 282, Issue 10, 7512-7521, March 9, 2007
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The Coxsackie and Adenovirus Receptor Binds Microtubules and Plays a Role in Cell Migration*

Patrick T. Fok{ddagger}§12, Kuo-Cheng Huang{ddagger}13, Paul C. Holland{ddagger}, and Josephine Nalbantoglu{ddagger}§4

From the {ddagger}Montreal Neurological Institute and Departments of Neurology & Neurosurgery and §Experimental Medicine, McGill University, Montreal, Quebec H3A 2B4, Canada

Received for publication, July 31, 2006 , and in revised form, November 29, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, inhibits cell growth of a variety of tumors. The cytoplasmic domain of CAR has been implicated in decreased invasion and intracerebral growth of human U87 glioma cells. Using affinity binding, we identified tubulin as an interaction partner for the cytoplasmic domain of CAR. The interaction was specific; CAR and tubulin co-immunoprecipitated in cells expressing endogenous CAR and partially co-localized in situ. The binding of CAR to tubulin heterodimers and to microtubules was direct, with dissociation constants of ~1 µM for tubulin and ~32 nM for in vitro assembled microtubules. Whereas CAR-expressing U87 glioma cells had decreased migration in a chemotactic assay in Boyden chambers as compared with control cells, an effect that depended on the presence of the cytoplasmic domain of CAR, the difference was abrogated at low, non-cytotoxic doses of the taxane paclitaxel, a microtubule-stabilizing agent. These results indicate that CAR may affect cell migration through its interaction with microtubules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CAR5 (Coxsackie and adenovirus receptor) is a 46-kDa plasma membrane glycoprotein (1, 2), which constitutes the primary site of attachment for all serotypes of Coxsackie B virus and for many adenovirus serotypes (3, 4). CAR belongs to a newly recognized subclass of the immunoglobulin superfamily of transmembrane proteins termed the CTX family (5). Members of the CTX subclass of immunoglobulin superfamily proteins are homologous type I transmembrane proteins with an extracellular moiety consisting of one variable (V-type) and one constant (C2-type) immunoglobulin (Ig) domain, a single transmembrane domain, and a cytoplasmic tail. The founding member of the CTX subfamily (cortical thymocyte marker in Xenopus) was discovered in 1996 (6). Other CTX subfamily members include A33 antigen (7), the junctional adhesion molecules JAM-A, -B, and -C (8), the endothelial cell-selective adhesion molecule ESAM (9), the brain- and testis-specific immunoglobulin superfamily protein (BT-immunoglobulin superfamily) (10) and CAR-like membrane protein (11). Members of the CTX subclass are typically cell-cell adhesion molecules (9, 12) and, in adult tissues, are predominantly localized to cell-cell contacts between epithelial and endothelial cells. CAR is a component of tight junctions in polarized epithelial cells (13, 14). It binds homotypically (15) but its heterotypic interaction with the junctional adhesion molecule homolog JAML has also been shown to mediate transendothelial migration of neutrophils (16).

The V-type Ig domain of CAR is sufficient for adenovirus binding (17, 18) and the transmembrane and cytoplasmic domains of CAR are not required in adenovirus infection (19). Nevertheless, the cytoplasmic domain of CAR has several features of potential functional importance for the molecule. The cytoplasmic domain of CAR is highly conserved between species and constitutes nearly one-third of the entire molecule. The membrane-proximal cysteines 259 and 260 provide a palmitoylation motif required for stable plasma membrane expression of CAR (20). Several regions are involved in basolateral targeting in epithelial cells (21). Furthermore, both full-length isoforms of CAR contain PDZ domain binding sequences; these isoforms are identical over much of their length, including the transmembrane and most of the cytoplasmic domain, but, through alternative splicing, differ at their C termini (CAR-SIV versus CAR-TVV). Accordingly CAR has been shown to bind the PDZ domain-containing proteins Ligand-of-Numb Protein-X (22, 23), MAGI-1b and PICK1 (24), ZO-1 (13), and MUPP-1 (25).

Whereas their normal tissue counterparts express readily detectable levels of CAR, low or absent expression of CAR is seen in many primary tumor tissue and cell lines (26-29). Forced expression of CAR results in inhibition of tumor cell growth in human prostate cancer (30), bladder cancer (31), and glioma (32) cell lines. We have recently shown that rates of glioma cell invasion and tumor growth are reduced by forced expression of full-length CAR but not by expression of cytoplasmic domain-deleted CAR, indicating the C-terminal cytoplasmic domain of CAR is required for inhibition of glioma cell invasion and tumor growth (33). To understand how the cytoplasmic domain of CAR may mediate these effects, we performed affinity binding assays and identified tubulin as a protein that interacts with CAR. Our results indicate that the binding of CAR to tubulin, and more specifically to microtubules, may have functional importance in cell migration.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Culture—U87CAR and U87LNCX cell lines have been described and characterized previously (33). Briefly, they were generated from the parental human glioma U87-MG cell line by infection with a retroviral vector expressing either full-length CAR (m1 isoform (34)) or an empty retroviral vector, respectively. They both consist of pooled populations of clones. The U87CAR cells express CAR at levels equivalent to that observed in lysates prepared from postnatal day 6 mouse brain (33). The U87CAR781 cell line has also been described and characterized previously (33). The pooled clones were generated through retroviral infection with a truncated CAR construct containing amino acids 1-260. The human bladder 253J cell line was obtained from Dr. Hsieh (MD Anderson, Houston, TX), and expresses robust levels of endogenous CAR (31). The additional CAR-expressing cell lines, human embryonic kidney 293A and human cervical HeLa cells were obtained from the American Type Culture Collection (Rock-ville, MD). Cells were maintained (unless stated otherwise) at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 2 mML-glutamine, 10% heat inactivated fetal bovine serum, and an antibiotic mixture (final concentration of 30 µg/ml gentamicin, 100 units of penicillin/ml, and 100 µg of streptomycin/ml) (Invitrogen).

Fusion Proteins—To create the GST-CAR fusion protein the intracellular domain (nucleotides 778-1098, encoding amino acids 259-365) of CAR (GenBankTM accession Y07593 [GenBank] ) was cloned into pGEX-3X plasmid (Amersham Biosciences). BL21 bacteria (Promega, Madison, WI) were transformed with pGEX-3X-CAR (778-1098). GST and GST-CAR fusion proteins were produced by overnight growth, followed by a 4-h induction with 1 and 0.5 mM of isopropyl-beta-D-thiogalactopyranoside (Fisher Scientific, Ottawa, ON, Canada), respectively. Bacterial cells were sonicated in phosphate-buffered saline (PBS) with protease inhibitor mixture (Roche, Mississauga, ON, Canada). Cleared bacterial lysate was incubated with gluthathione-Sepharose beads (Amersham Biosciences) for 5 min at room temperature and washed three times with PBS supplemented with protease inhibitors (Roche). Aliquots of the washed beads were run on SDS-PAGE and stained with Coomassie Blue to verify fusion protein integrity.

The preparation of a His-tagged fusion protein expressing amino acids 259-339 of the cytoplasmic domain of CAR has been described previously (35). This fusion protein contains the domains that are common to the two full-length CAR isoforms (m1 and m2 (34)) but lacks the distal most 26 amino acids of the m1 isoform, including the PDZ binding residues (SIV).

GST Pulldown Assay—A 70-80% confluent 15- or 10-cm2 plate of U87CAR or 293A cells, respectively, was used for affinity binding to GST-CAR. Cell lysates (20 mM HEPES, pH 8, 150 mM NaCl, 1% Triton X-100, protease tablet mixture (Roche)) were pre-cleared by incubating with unbound gluthathione-Sepharose beads, followed by GST-bound glutathione-Sepharose beads (50 µl, 2 h each at 4 °C under rotation) to remove any nonspecific binding. The pre-cleared lysate was then incubated with 50 µl of GST-CAR-bound glutathione-Sepharose beads for 2 h at 4°C. The beads were washed three times with 500 µl of lysis buffer. Interacting proteins were eluted by boiling with 2x Laemmli SDS-PAGE loading buffer containing 5% beta-mercaptoethanol (Bio-Rad). Samples were electrophoresed on an 11% SDS-polyacrylamide gel followed by silver staining for mass spectroscopic analysis, or transblotting for Western blot analysis.

Proteomic Analysis—Following SDS-PAGE, silver staining was performed using a protocol that was compatible with subsequent mass spectrometry. Gels were fixed in 10% acetic acid, 50% methanol overnight and washed twice in water for 30 min each. Sensitization was performed for 1 min with 0.02% thiosulfate followed by two rinses of 20 s with water. Gels were stained in 0.2125% silver nitrate, 0.0259% formaldehyde and washed twice for 15 s in water. Development occurred by treatment with 3% potassium, 0.00925% formaldehyde, 0.00125% thiosulfate for 2 min. The reaction was stopped with 3% Tris, 2% acetic acid for 1 min and rinsed twice for 10 min in water. Gels were kept in storage solution of 2% acetic acid and bands were selected for further mass spectroscopy processing. Gel slices were excised and digested with trypsin (6 ng/µl)for 5 h on a MassPrep robotic work station (Micromass, Manchester, United Kingdom). Peptides were extracted in a final volume of 45 µl of 0.5% formic acid (v/v) and 9% acetonitrile (v/v). Tryptic peptides were analyzed on an LC-QToF (liquid chromatography quadrupole time-of-flight) mass spectrometer (Micro-Mass). Briefly, the sample was applied to a 10 x 75-cm Pico Frit column containing BioBasic C18 packing. Peptides were eluted from the column using a gradient of 10-95% acetonitrile (v/v) containing 0.1% formic acid (v/v) throughout at a flow rate of 200 nl/min. Eluted peptides were electrosprayed as they exited the column and doubly or triple charged ions were selected for passage into a collision cell. Fragmentation was induced by collision with argon gas and data collected in 1-s scans for up to 5 s. Peaklists of MS/MS data were prepared using Masslynx software (MicroMass) and submitted to Mascot (Matrix Science, Boston, MA) for identification by analysis against the National Center for Biotechnology Information (NCBI) non-redundant data base.

SDS-PAGE and Western Blot—SDS-PAGE and Western blots were performed as previously described (33). For Coomassie Blue staining, protein polyacrylamide gels were incubated for 1 h at room temperature with 0.1% Coomassie Brilliant Blue R-250 stain (Bio-Rad) in 45% methanol, 10% acetic acid and destained overnight in 40% methanol, 10% acetic acid.

Immunoprecipitation Analysis—Cells grown in a 10-cm2 dish were washed twice in PBS, then scraped into 1 ml of immunoprecipitation lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, protease inhibitor mixture (1 tablet, Roche)) and incubated on ice for 30 min. The sample was then centrifuged at 3,600 x gmax for 15 min at 4 °C. The supernatant was kept and spun again at 17,500 x gmax in a microcentrifuge at 4 °C for 30 min. The insoluble pellet (which contained the majority of microtubules) was discarded and the soluble supernatant was then incubated overnight at 4 °C with 5 µg of antipan beta-tubulin mouse monoclonal antibody (Clone Tub 2.1, Sigma) pre-coupled to protein G-Sepharose. Beads were then washed three times with immunoprecipitation buffer and bound protein eluted with 2x SDS-PAGE buffer for Western blot analysis. Blots were incubated with a polyclonal anti-CAR antibody (RP291), which reacts against the cytoplasmic domain of CAR m1 isoform (23) (kind gift of Dr. Kerstin Sollerbrant (Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, Stockholm, Sweden)).

Indirect Immunofluorescence—Cells were grown in 8-well chamber slides (Nunc, Rochester, NY). Cells were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 5 min with 0.1% Triton X-100 in PBS, and blocked for 30 min with 1% milk, PBS, washing once with PBS between each step. Antibodies RP291 (anti-CAR) and anti-tubulin (Clone Tub 2.1, Sigma) were diluted 1:1000 and 1:2000, respectively, in blocking buffer and incubated overnight at 4 °C. Primary antibody was washed off three times and Alexa 555 goat anti-rabbit and Alexa 488 goat anti-mouse diluted at 1:250 (Invitrogen) were applied for 1 h at room temperature. After three washes with PBS, the chambers were removed and the coverslip applied. Controls consisted of chambers processed similarly but in the absence of primary antibody. Slides were visualized under an oil immersion objective (x63) on a Leica DMIRE2 (Richmond Hill, ON, Canada) wide-field fluorescence microscope. The same exposure time was used for all samples. The images were processed using Openlab (Lexington, MA) software. In some experiments, images were collected using a laser scanning confocal microscope (LSM 510, Carl Zeiss MicroImaging Inc.) equipped with argon (488 nm) and krypton (568 nm) lasers and a Plan Apochromat x100/1.4 oil immersion objective lens.

For the nocodazole treatments, cells were plated at a density of 10,000 cells/cm2 on coverslips in 24-well tissue culture plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotic mixtures for 2 days. The culture medium was aspirated and replaced with culture medium of the same composition supplemented with 4 µM nocodazole (Sigma) for 5 min at 37 °C in 5% CO2. Cells were then rinsed once with PBS and then with PEM buffer (80 mM PIPES, 5 mM EGTA, 1 mM MgCl2, pH 6.8) after which cells were incubated with PEM containing 0.5% (w/v) Nonidet P-40 and 0.3% glutaraldehyde for 10 min for simultaneous fixation of microtubules and extraction of unpolymerized tubulin heterodimers. After fixation, cells were rinsed with PBS and further permeabilized by incubation with 0.5% Triton X-100 in PBS for 10 min at room temperature. The cells were washed with PBS and then incubated with 0.1 M glycine in PBS for 20 min to quench uncross-linked glutaraldehyde. The cells were then washed once with PBS, blocked in blocking solutions, stained for beta-tubulin and visualized as outlined above. All microphotographs were taken at the same exposure time and magnification.

Cytotoxicity Assays—Cells were seeded in 96-well plates at 5000 cells/well. The next day, cells were treated with paclitaxel (Sigma) at 2:3 serial dilutions of 15.8 nM in triplicate. After 72 h incubation, cells were fixed and survival calculated with the sulforhodamine B cytotoxicity assay, as previously described (36).

Tubulin Binding Affinity Measurements—Purified bovine brain tubulin (Cytoskeleton Inc., Denver, CO) at 10 µM dimer concentration in binding buffer (80 mM PIPES, pH 6.9, 0.5 mM EGTA, 2 mM MgCl2, 1% Triton X-100, 1 mM GTP, 10% glycerol) was placed on ice for 30 min to ensure complete depolymerization. The tubulin sample was then centrifuged for 10 min at 10,000 x g to remove insoluble aggregates and then diluted into 250-µl aliquots at different final concentrations from 50 nM to 3.7 µM and added to a fixed amount (10 µl) of GST fusion protein slurry (50%) for 4 h at 4 °C after which the samples were spun down and washed twice with ice-cold binding buffer. Tubulin bound to the beads was released by SDS-PAGE loading buffer and analyzed by Western blot analysis. The amount of tubulin bound to the GST fusion protein beads was quantitated against a tubulin standard curve by densitometry using Gene-Tool software (Syngene, Frederick, MD). A dissociation curve was constructed and the dissociation constant (Kd) calculated using Prism software (Graph Pad, San Diego, CA).

Microtubule (MT) Preparation—Pre-formed MTs used for microtubule-binding protein spin down assays, microtubule binding affinity measurements, and electron microscopy were prepared using a microtubule binding protein spin-down assay kit (Cytoskeleton Inc.) immediately before use as per the manufacturer's instructions. Briefly, 2.5 µl of cushion buffer (80 mM PIPES, pH 7, 1 mM MgCl2, 1mM EGTA, 50% glycerol) was added to 20-µl aliquot of 5 mg/ml tubulin protein in general tubulin buffer (80 mM PIPES, pH 7, 1 mM MgCl2, 1mM EGTA) and incubated at 37 °C. After 20 min of incubation, the MTs were diluted with 200 µl of general tubulin buffer plus 2 µl of 2 mM paclitaxel for stabilization and the prepared MTs were used at room temperature.

Microtubule-binding Protein Spin-down Assay—The ability of fusion protein constructs and test proteins to bind to microtubules was assessed using a microtubule-binding protein spindown assay kit (Cytoskeleton Inc.) according to the manufacturer's instructions. Briefly, 20 µl of Taxol-stabilized MT were added to 30 µl of purified MAP2, bovine serum albumin (BSA), or purified GST-CAR fusion protein or His6-tagged CAR fusion protein at room temperature. After 30 min of incubation, the samples were placed on top of a 100-µl Taxol (20 µM)-supplemented cushion buffer and centrifuged at 100,000 x g at room temperature for 40 min in a Beckman Airfuge (Mississauga, ON, Canada). After centrifugation, the supernatants were carefully removed by pipetting off 50 µl from the top. A volume of 12 µl of 5x SDS-PAGE loading buffer was then added for SDS-PAGE analysis. The cushion buffer was then gently removed from the ultracentrifugation tubes and the microtubule pellets collected by washing the bottom of the tube with 1x SDS gel loading buffer and loaded on gels for SDS-PAGE analysis. For peptide competition assays, an excess of preformed microtubules were preincubated with varying concentrations of peptides for 30 min at room temperature. The CAR peptide (amino acids 340-365) was VAAPNLSRMGAVPVMIPAQSKDGSIV (Mr 2609; pI 8.9). The control peptide was CSSRGRNTPGKPMREDTMKLH (Mr 2401; pI 10). After this preincubation, GST-CAR proteins were added into the mixture and incubated for 30 min as usual before the spindown.

Microtubule Binding Affinity Measurements—Purified GST-CAR protein was eluted from glutathione beads using freshly prepared 10 mM reduced glutathione in 50 mM Tris-HCl buffer (pH 8.0). This was then dialyzed for 24 h in general tubulin buffer after which the protein concentration was quantified using the BCA protein assay reagent concentrate as per manufacturer's instructions (Bio-Rad). The protein sample was diluted into 45-µl aliquots of concentrations ranging from 5.63 µM to 0.115 µM in general tubulin buffer. A volume of 5 µl of MT prepared as above was then mixed with each of the diluted GST-CAR protein samples and incubated for 1 h at room temperature at which time the samples were spun down at 100,000 x g at room temperature for 40 min on top of a 100 µl of Taxol-supplemented cushion buffer as in the MT-binding protein spin-down assay described above. The MT pellets were then collected and analyzed by Western blot analysis for the presence of GST-CAR protein. The amount of GST-CAR protein that had bound and spun-down with the MTs was quantitated by comparing the intensity of the bands against standard curves using the Syngene computer software. Constant MT quantity in the pellets of all of the samples was also monitored using Western blot analysis. A dissociation curve was constructed and the Kd calculated using Prism software (Graph Pad).


Figure 1
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  		FIGURE 1.
Identification of tubulin as a CAR-interacting protein by mass spectrometry. The peptides that were isolated after MS/MS analysis are boxed. The Mowse score is also given (significance set at >32). These hits were considered highly significant because of the Mowse score, the number of peptides matched as well as the sequence coverage obtained for both {alpha}- and beta-tubulin. In beta-tubulin, some peptides occurred twice, with different lengths as indicated (LAVNMVPFPRLHFFMPGFAPLTSR).

 
Electron Microscopy of Microtubules—For electron microscopic analysis of fusion protein binding to MTs, fusion proteins (GST or GST-CAR) at 5.63 µM were incubated with MTs as in the microtubule binding affinity measurement described above. After incubation, the samples were applied to a carbon-supported copper 300 mesh electron microscope grid (Canemco Inc., Lakefield, Quebec, Canada), air dried, and counterstained with lead citrate and uranyl acetate for examination with a JEOL (St-Hubert, QC, Canada) 100 CX transmission electron microscope. The magnification used was x5000.

Cell Migration Assays—Glioma migration was assessed using a Boyden transwell chemotactic assay adapted from those described in Refs. 37 and 38. The transwell chamber consisted of upper chambers with 8-µm polycarbonate filter inserts (Corning, Acton, MA) placed in 24-well tissue culture plates (Nunc). Serum-starved glioma cells (5000 cells), suspended in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum and 1 mg/ml BSA, were seeded into the top compartment of each chamber. In some experiments, U87CAR cells were infected at a multiplicity of 50 with control adenovirus recombinant expressing blue fluorescent protein (AdVBFP) or antisense full-length CAR (AdVAS) (both generated with the AdEasy system (QBiogene, Montreal, QC, Canada). The bottom well contained U251 glioma conditioned medium to act as directional chemoattractant. In selected experiments, the bottom well was supplemented with 0.5 nM paclitaxel (Sigma). After 16 h of incubation, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, washed twice with PBS, and stained with Hoechst dye (Molecular Probes, Burlington, ON, Canada) diluted 1:10,000 in PBS for 30 min in the dark. Chambers were washed twice with PBS and non-migrated cells were then gently removed from the upper side of the filter insert with a cottontipped applicator. The trans-migrated cells remaining on the lower side of the filter insert were then quantified using a Leica wide-field fluorescence microscope with an objective of x40. For each filter insert, at least 5 random fields were quantified. Each assay was run in triplicate. Analysis of variance statistical analysis was performed using Prism software (Graph Pad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of Tubulin with the Cytoplasmic Domain of CAR—To identify proteins that bind the intracellular domain of CAR (amino acids 259-365, m1 isoform (34)), we performed affinity assays using a GST-CAR fusion protein with cell lysates prepared from U87 glioma cells stably expressing the full-length CAR (U87CAR (33)). The lysates were sequentially applied to glutathione-Sepharose and GST-Sepharose to remove nonspecific binding, followed by application to GST-CAR-Sepharose. After elution and electrophoretic separation of the GST-CAR-binding proteins, proteomic analysis was performed on individual bands that were enriched in the GST-CAR pull-downs as described under "Experimental Procedures." Highly significant hits were seen with multiple peptides of beta- and {alpha}-tubulin identified by mass spectrometry as shown in Fig. 1.


Figure 2
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  		FIGURE 2.
CAR interaction with tubulin. A and B, cell lysates (lane 1) from U87CAR (A) and 293A (B) cells were sequentially applied to and eluted from glutathione-Sepharose (lane 2), GST-Sepharose (lane 3), and GST-CAR-Sepharose (lane 4), followed by immunoblotting of the eluted material with an anti-tubulin antibody. Only GST-CAR bound to tubulin. C and D, soluble cell lysates (enriched in tubulin heterodimers after pelleting of microtubules) were immunoprecipitated (IP) with a monoclonal antibody against tubulin, followed with immunoblotting with a polyclonal antibody against the C terminus of CAR (RP291). CAR signal was detected only in the anti-tubulin lane and not in the control IgG lane.

 
To validate the results of the proteomic analysis, we verified that the GST-CAR fusion protein could indeed pull down tubulin from U87CAR cell lysates. As shown is Fig. 2A, tubulin was detected only in the samples that had been incubated with the GST-CAR fusion protein (lane 4) and was absent in samples incubated with either glutathione-Sepharose or GST-Sepharose (lanes 2 and 3, respectively). Similar results were obtained with cell lysates prepared from human embryonic kidney 293A cells that express only endogenous CAR (Fig. 2B).


Figure 3
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  		FIGURE 3.
Partial co-localization of CAR with microtubules in U87CAR cells. A, cells were double stained for beta-tubulin and CAR as described under "Experimental Procedures." Partial co-localization is seen at the cell membrane in the merged image. B, double stained cells were also examined by confocal microscopy. Superimposed, merged pictures are shown in the right panel, with yellow indicating co-localization. A similar extent of co-localization was observed in all U87CAR cells that were visualized (over 50 cells).

 
In Vivo Interaction of CAR with Tubulin—To confirm the interaction of CAR with tubulin, we immunoprecipitated tubulin from U87CAR cells with an anti-tubulin monoclonal antibody and analyzed for the presence of CAR by Western blotting (Fig. 2C). A portion of the total cellular CAR was present in these immunocomplexes. Moreover, immunoprecipitation of tubulin from human HeLa cells that express endogenous CAR (2) revealed that tubulin and CAR also have the ability to associate in these cells (Fig. 2D). Similar co-immunoprecipitation results were obtained in additional cell lines that express endogenous CAR such as the human bladder cancer cell line 253J (31) (data not shown).


Figure 4
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  		FIGURE 4.
Saturation binding analysis of CAR cytoplasmic domain/tubulin heterodimer interactions. Increasing concentrations of purified tubulin (>99% pure) were added to GST-CAR-Sepharose beads as described under "Experimental Procedures." The specifically bound tubulin was detected by immunoblotting and quantitated by comparison to a standard curve to derive the saturation binding curve. The dissociation constant was obtained by curve fitting with non-linear regression.

 
CAR is a type I membrane protein. As previously shown, in U87CAR cells, CAR immunoreactivity is detected at the cell surface. When U87CAR cells were immunolabeled with anti-CAR and anti-tubulin antibodies, in all cells examined, CAR immunoreactivity partially overlapped with that of tubulin (Fig. 3A). This is consistent with the observation that in tumor cells, the highest tubulin concentration appears in the cell periphery (39). The co-localization was also confirmed by visualization of the immunoreactivity by confocal microscopy (Fig. 3B). These results are consistent with an interaction of the cytoplasmic domain of CAR with tubulin, either directly or indirectly.


Figure 5
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  		FIGURE 5.
CAR cytoplasmic domain binds assembled microtubules. A, characterization of the microtubule spin-down assay. Pre-assembled microtubules stabilized in the presence of Taxol (+MT) or buffer lacking microtubules (-MT) were incubated with MAP2, BSA, or buffer alone, followed by ultracentrifugation as described under "Experimental Procedures." Proteins in the pellet and supernatant were resolved by SDS-PAGE and stained with Coomassie Blue. MAP2 was predominantly found in the pellet in the presence of microtubules only (lane 1 as compared with lane 9). BSA was predominantly found in the supernatant (lanes 7 and 10) in the presence (lane 7 as compared with lane 2) or absence of MT (lane 10). Most of the tubulin was found as assembled microtubules in the pellet (lanes 1-3 as compared with lanes 6-8). B, GST protein was incubated in the presence of pre-assembled microtubules (lanes 1 and 3) or buffer lacking microtubules (lanes 2 and 4), followed by ultracentrifugation and SDS-PAGE. The majority of the GST protein was detected in the supernatant fraction (lanes 3 and 4). C, GST-CAR fusion protein was processed in the microtubule spin-down assay as described in A, but following SDS-PAGE, the protein was detected by immunoblotting with an anti-CAR antibody. In the presence of pre-assembled microtubules (+MT), GST-CAR was predominantly in the pellet, whereas it remained in the supernatant when ultracentrifuged in the presence of buffer alone (-MT). D, a His-tagged CAR fusion protein deleted in the last 26 amino acids was processed in the microtubule spin-down assay as described in A. The fusion protein was detected predominantly in the supernatant whether in the presence (+MT) or absence (-MT) of microtubules. P, pellet; S, supernatant. E, a synthetic peptide containing the last 26 amino acids of CAR or an irrelevant control peptide were preincubated with an excess of microtubules prior to carrying out the spin-down assay as described under "Experimental Procedures." There was a dose-dependent decrease in the amount of GST-CAR fusion protein recovered in the pellet fraction only when the preincubation was performed in the presence of the CAR peptide. The experiment was repeated twice.

 
Direct Binding of CAR to Tubulin Heterodimers and Microtubules—To test whether CAR can associate directly with tubulin, binding assays were carried out between the GST-CAR fusion protein and purified, depolymerized bovine brain tubulin (>99% pure) as described under "Experimental Procedures." Purified tubulin did not bind GST-Sepharose (data not shown) but it did bind and could be eluted from GST-CAR-Sepharose, indicating that CAR can interact directly with tubulin heterodimers. The binding was saturable (Fig. 4). In this assay, the dissociation constant (Kd), as determined by curve fitting with non-linear regression analysis, was ~1 µM, a value that is consistent with that obtained for other tubulin-binding proteins such as CRMP-2 (0.8 µM) (40) and stathmin (0.5 µM) (41).

We then assessed whether CAR binds microtubules in vitro by performing a microtubule spin-down assay as described under "Experimental Procedures." In the presence of preformed microtubules, both GST and BSA remained in the supernatant, and did not associate with the microtubules that were found in the pellet after ultracentrifugation (Fig. 5, A and B). In contrast, GST-CAR fractionated predominantly to the pellet, but only in the presence of microtubules (Fig. 5C). The distal portion of the cytoplasmic domain was required for this interaction as a fusion protein lacking the last 26 amino acids of the C terminus was predominantly found in the supernatant (Fig. 5D). As well, a synthetic peptide encoding the distal 26 amino acids competed specifically with GST-CAR binding to microtubules (Fig. 5E).

Measurement of the binding affinity of CAR to a constant amount of microtubules was determined using the microtubule spin-down assay shown in Fig. 5. CAR bound microtubules more avidly than it did the tubulin heterodimer, with a dissociation constant of 0.032 µM (Fig. 6A). Furthermore, the interaction of CAR with microtubules resulted in tightly packed arrays as visualized by negative stain electron microscopy (Fig. 6B). The higher affinity of CAR for microtubules suggests a possible role for CAR in regulating microtubule dynamics. Incubation of cells with nocodazole, which induces depolymerization of microtubules, results in a time-dependent disruption of the microtubule network. Nocodazole treatment, as described under "Experimental Procedures," affected CAR expressing cells differentially in that the microtubule network was less susceptible to a 5-min treatment with 4 µM nocodazole in U87CAR cells than in control U87LNCX cells (Fig. 6C).


Figure 6
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  		FIGURE 6.
Analysis of CAR cytoplasmic domain/microtubule interactions. A, saturation binding. Increasing concentrations of GST-CAR fusion protein were added to a constant concentration of pre-assembled microtubules as described under "Experimental Procedures." The samples were processed in the microtubule spindown assay as described in the legend to Fig. 5. The amount of bound CAR was quantitated by immunoblotting with an anti-CAR antibody to generate the saturation binding curve. B, electron microscopic analysis of CAR binding to microtubules. Binding of GST protein (left panel) or GST-CAR fusion protein (right panel) was observed by electron microscopy. In the presence of GST, microtubules were seen in single arrays, whereas in the presence of GST-CAR, microtubules appeared more tightly packed. C, sensitivity to nocodazole. Cells were treated for 5 min with 0 or 4 µM nocodazole and processed as described under "Experimental Procedures." The microtubule network was visualized by immunoreaction with an anti-tubulin antibody and collections of cells were photographed at the same magnification and exposure time. At these experimental conditions, a more extensive microtubule network remained in the U87CAR cells (+noc) than in U87LNCX cells (+noc). The treatment was repeated twice and over 100 cells were visualized each time.

 
Response of U87CAR Cells to Paclitaxel—Drugs that interfere with the equilibrium of microtubule assembly are commonly used clinical anti-tumor agents (42). One such drug, the taxane paclitaxel, which shifts the cytoskeleton equilibrium toward assembly of very stable microtubules, has potent anti-glioma activity in vitro (43). To determine whether expression of CAR and its interaction with microtubules has an impact on glioma cell response to paclitaxel, we compared cytotoxicity of paclitaxel in U87CAR cells and control vector U87LNCX cells. U87CAR cells had an IC50 of 4.44 ± 0.28 nM versus an IC50 of 5.65 ± 0.19 nM for vector control cells (p < 0.0008 by two-tailed t test; n = 3). Taken together with the previous findings on the effect of nocodazole, these results suggest that the association of CAR with microtubules alters microtubule dynamics.

Effect of CAR on Glioma Cell Migration—The cytoskeleton is important for maintaining cell shape and facilitating cell movement (44). A chemotaxis assay in Boyden chambers was used to assess cell motility of U87CAR cells as compared with the vector control U87LNCX cells. Significantly fewer U87CAR cells migrated through the transwell as shown in Fig. 7C (LNCX versus CAR). The difference in migration was directly related to the expression of CAR as knock-down of CAR levels to less than 20% by transduction of U87CAR cells with a recombinant adenovirus bearing an antisense construct of full-length CAR (Fig. 7A) restored migration to the extent observed in U87LNCX cells (Fig. 7C, CAR-AS). Furthermore, the cytoplasmic domain was required for this differential effect as U87 cells expressing on the cell surface a CAR construct that is deleted for the C-terminal domain (33) had a similar migration rate as U87LNCX (Fig. 7C, CAR781).

As paclitaxel is known to affect cell migration (45), the Boyden assay was also carried out in the presence of 0.5 nM paclitaxel, a concentration that is not cytotoxic to these cells. Although, as expected, paclitaxel inhibited the migration of U87LNCX cells and U87CAR781 cells (data not shown), it had no effect, at this concentration, on the migration of full-length CAR-expressing U87CAR cells (Fig. 7D). In the presence of paclitaxel, the difference in the migration rate of U87CAR and U87LNCX cells was abolished, indicating that CAR may affect U87 glioma cell migration through its interaction with microtubules.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Compelling evidence is accumulating for CAR being a tumor suppressor (30, 32). With increasing malignancy, tumors progressively lose CAR expression, as compared with adjacent normal cells (31, 46-49). In tumor cell lines of various origins, CAR overexpression decreases cell proliferation (30, 32). Previously, we used retrovirus-mediated gene transfer to express CAR in the U87 glioma cell line at levels equivalent to those found in the developing brain (33). In these cells, CAR expression had a significant impact on growth and invasion in three-dimensional spheroids, and more importantly, on tumor growth when the cells were implanted into the brain. In both conditions (spheroid and tumor growth), the cytoplasmic domain of CAR was required (33).


Figure 7
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  		FIGURE 7.
Effect of CAR on glioma cell migration. A and B, expression of CAR in various cell lines used in the migration assays. Western blot of cell lysates obtained from U87LNCX, U87CAR, and U87CAR (A) infected with an AdVBFP or antisense full-length CAR (AdVAS), as well as U87CAR781 (B) cells that express a truncated CAR (amino acids 1-260). The blots were immunoreacted with antibody 2240 that recognizes the N-terminal domain of CAR (60). By densitometric analysis, AdVBFP-infected U87CAR cells retained 80% CAR, whereas cells infected with AdVAS had only 20% of the initial CAR levels after normalization. C and D, chemotaxis in Boyden chambers. Cell migration assays were performed as described under "Experimental Procedures." In C, U87CAR cells were infected with the recombinant adenoviruses for 48 h prior to the start of assay. The differences were statistically significant by analysis of variance (p < 0.01) and post-hoc Bonferroni analysis on selected comparisons are indicated by the asterisk. Infection of U87CAR cells with AdVBFP did not affect migration but decrease of CAR expression through AdVAS infection led to increased migration. D, U87CAR and control U87LNCX cells were compared in Boyden chambers in the absence or presence of paclitaxel (PTX) as described under "Experimental Procedures." Each experiment was carried out in triplicate and replicated 3 times for the control condition and twice in the presence of PTX. The differences in migration were statistically significant by analysis of variance (p < 0.004). Post hoc Bonferroni analysis revealed that under control conditions, migration of U87CAR cells was significantly less than that of the control U87LNCX cells (p < 0.01), and that PTX significantly decreased the migration of the control U87LNCX cells (p < 0.001) without affecting the migration of the U87CAR cells (NS; p > 0.05).

 
In this report we show that one possible mechanism for the observed effects of CAR may be through its interaction with tubulin and microtubules. We have used several different approaches to provide evidence for this binding (pull downs with recombinant protein (Fig. 2, A and B), co-precipitation from cell lysates that express exogenous as well as endogenous CAR (Fig. 2, C and D), co-localization in situ (Fig. 3)), and have shown that the affinities of the association are comparable with those reported for other tubulin (40, 41) or microtubule-binding proteins (e.g. MAP2 (50) and myosin-Va (51)) (Figs. 4, 5 and 6).

We also show that the interaction of CAR with tubulin and microtubules is functionally relevant, in terms of drug response to taxanes that interfere with microtubules as well as cell migration. CAR-expressing U87 glioma cells were more sensitive to treatment with paclitaxel, a microtubule stabilizing agent, than control U87LNCX cells. Altered microtubule dynamics can result in microtubule drug resistance or sensitivity. In particular, decreased microtubule dynamics can manifest itself in increased paclitaxel sensitivity (reviewed in Ref. 52). This suggests that CAR expression may result in enhanced microtubule stabilization. Although the mechanism for the stabilization is not known, our in vitro observation of increased bundling of microtubules in the presence of GST-CAR suggests a possible way CAR may regulate microtubules. Presumably, the bundling occurs due to the presence of the CAR cytoplasmic domain in the fusion protein. Although GST is known to dimerize and might have contributed to increased cross-linking of the microtubules, control experiments did not reveal any effects of the GST moiety itself (Fig. 6B, left panel). However, in this in vitro assay, the dimerization of GST may facilitate the ability of CAR to cross-link microtubules by allowing the CAR domains to dimerize. This may actually mimic the endogenous configuration of CAR because the extracellular domains have been shown to exist as dimers (53) and CAR binds the adenovirus knob as a trimer (54). Similarly, adhesion molecules such as L1 are known to cluster at adhesion sites on the cell surface and to homomultimerize through their ectodomains (55). Therefore, it is conceivable that CAR, as an adhesion molecule, exists as multimers in vivo and is able to cross-link microtubules at adhesion sites. The localized interactions of CAR and microtubules may thus serve to shift the cytoskeletal equilibrium.

To further support this theory, we compared migration of U87LNCX and U87CAR cells in transwell chambers. Microtubules have been shown to control directed cell migration (56). Accordingly, U87CAR cells migrated through a transwell less than control U87LNCX cells or U87CAR781 cells with a deletion of the cytoplasmic domain of CAR (Fig. 7C). However, upon microtubule stabilization with a non-cytotoxic dose of paclitaxel (IC90), the migration difference was nullified (Fig. 7D). It has been shown that microtubule-binding proteins, such as p27Kip1 (57) and JAM-A (58), can inhibit cellular motility though microtubule stabilization. We propose that CAR may act similarly within a multiprotein complex to reduce microtubule dynamics to minimal basal activity level. Thus when treated with a low dose of microtubule-stabilizing agent, U87CAR cells are not affected, whereas control U87LNCX cells have microtubule dynamics reduced to the same basal level.

Microtubules are known to play important roles in cell migration. For instance, during cell migration, microtubules are targeted to adhesion complexes (59) where they promote cell migration through their contribution to the dissociation of adhesion sites from the substrate. Our data support the theory that CAR may be one such adhesion molecule that can interact with the cytoskeleton of the cell to regulate its function. Through its PDZ-binding domain, it is conceivable that CAR participates in protein complexes that could in turn affect microtubule regulatory molecules.

In conclusion, the demonstration that CAR binds tubulin and microtubules may provide a mechanistic basis for the selective growth advantage imparted by the loss of CAR expression in various tumor types. Additionally, the results potentially implicate this adhesion molecule in protein complexes important for cell proliferation, migration, and signal transduction.


   FOOTNOTES
 
* This work was supported in part by Canadian Institutes of Health Research Grants MOP 53071 and MOP-74565. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to the results of this work. Back

2 Supported by a Canadian Institutes of Health Research MD/PhD studentship. Back

3 Received a studentship from Fonds de la recherche en santé du Québec. Back

4 Research Scholar of the Fonds de la recherche en santé du Québec and a Killam Scholar. To whom correspondence should be addressed: 3801 University St., Montreal, QC H3A 2B4, Canada. Tel.: 514-398-5920; E-mail: Josephine.nalbantoglu{at}mcgill.ca.

5 The abbreviations used are: CAR, Coxsackie and adenovirus receptor; AdVBFP, adenovirus expressing blue fluorescent protein; AdVAS, antisense full-length CAR; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BSA, bovine serum albumin; MT, microtubule; MAP2, microtubule-associated protein 2; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Claude Guérin for technical help, Jim Dixson for electron microscopy, and the McGill Proteomics Center for sample processing and analysis. We thank Dr. Kerstin Sollerbrant for providing antibodies and Dr. Jer-Tsong Hsieh for providing cell lines.



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