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• W2123292213 abstract "β-Catenin is a key molecule involved in both cell adhesion and Wnt signaling pathway. However, the exact relationship between these two roles has not been clearly elucidated. Tyrosine phosphorylation of β-catenin was shown to decrease its binding to E-cadherin, leading to decreased cell adhesion and increased β-catenin signaling. We have previously shown that receptor-like protein-tyrosine phosphatase PCP-2 localizes to the adherens junctions and directly binds and dephosphorylates β-catenin, suggesting that PCP-2 might regulate the balance between signaling and adhesive β-catenin. Here we demonstrate that PCP-2 can inhibit both the wild-type and constitutively active forms of β-catenin in activating target genes such as c-myc. The phosphatase activity of PCP-2 is required for this effect since loss of catalytic activity attenuates its inhibitory effect on β-catenin activation. Expression of PCP-2 in SW480 colon cancer cells can lead to stabilization of cytosolic pools of β-catenin perhaps, by virtue of their physical interaction. PCP-2 expression also leads to increased membrane-bound E-cadherin and greater stabilization of adherens junctions by dephosphorylation of β-catenin, which could further sequester cytosolic β-catenin and thus inhibit β-catenin mediated nuclear signaling. Furthermore, SW480 cells stably expressing PCP-2 have a reduced ability to proliferate and migrate. Thus, PCP-2 may play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may be an alternative mechanism for activating β-catenin signaling. β-Catenin is a key molecule involved in both cell adhesion and Wnt signaling pathway. However, the exact relationship between these two roles has not been clearly elucidated. Tyrosine phosphorylation of β-catenin was shown to decrease its binding to E-cadherin, leading to decreased cell adhesion and increased β-catenin signaling. We have previously shown that receptor-like protein-tyrosine phosphatase PCP-2 localizes to the adherens junctions and directly binds and dephosphorylates β-catenin, suggesting that PCP-2 might regulate the balance between signaling and adhesive β-catenin. Here we demonstrate that PCP-2 can inhibit both the wild-type and constitutively active forms of β-catenin in activating target genes such as c-myc. The phosphatase activity of PCP-2 is required for this effect since loss of catalytic activity attenuates its inhibitory effect on β-catenin activation. Expression of PCP-2 in SW480 colon cancer cells can lead to stabilization of cytosolic pools of β-catenin perhaps, by virtue of their physical interaction. PCP-2 expression also leads to increased membrane-bound E-cadherin and greater stabilization of adherens junctions by dephosphorylation of β-catenin, which could further sequester cytosolic β-catenin and thus inhibit β-catenin mediated nuclear signaling. Furthermore, SW480 cells stably expressing PCP-2 have a reduced ability to proliferate and migrate. Thus, PCP-2 may play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may be an alternative mechanism for activating β-catenin signaling. Reversible and dynamic tyrosine phosphorylation is controlled by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs) 2The abbreviations used are: PTP, protein-tyrosine phosphatases; RPTP, receptor-like PTP; MAM-PTP, MAM-subfamily PTP; APC, adenomatous polyposis coli; EGF, epidermal growth factor; EGFR, EGF receptor; PBS, phosphate-buffered saline; siRNA, small interfering RNA; Tcf, TCF, T cell factor; ChIP, chromatin immunoprecipitation; WT, wild type; mu, mutant. (1Majeti R. Weiss A. Chem. Rev. 2001; 101: 2441-2448Crossref PubMed Scopus (38) Google Scholar). PTPs are a large family that is broadly classified into receptor-like protein-tyrosine phosphatases (RPTPs) and cytosolic PTPs (2Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (603) Google Scholar). A subfamily of RPTPs containing an MAM (Meprin/A5/PTP mu) domain in the ectodomain followed by an Ig-like domain and fibronectin type III repeats is defined as the MAM-subfamily PTPs (MAM-PTPs), which include PCP-2, PTPμ, PTPκ, and PTPρ (3Wang H. Lian Z. Lerch M.M. Chen Z. Xie W. Ullrich A. Oncogene. 1996; 12: 2555-2562PubMed Google Scholar, 4Besco J. Popesco M.C. Davuluri R.V. Frostholm A. Rotter A. BMC Genomics. 2004; 5: 14Crossref PubMed Scopus (21) Google Scholar). These RPTPs also contain a single membrane-spanning region with two cytoplasmic PTP domains. The intracellular juxtamembrane domain of these RPTPs contains a region that is homologous to the conserved intracellular domain of the cadherins (5Zondag G.C. Moolenaar W.H. Biochimie (Paris). 1997; 79: 477-483Crossref PubMed Scopus (27) Google Scholar). Cadherins are a family of calcium-dependent adhesion molecules that play an essential role in the formation of the cell-cell contacts termed adherens junctions. Cadherin-mediated cell-cell adhesion is important for development and maintenance of epithelial tissue integrity, and its disturbance contributes to the invasive and metastatic phenotype of epithelial tumors. Through their intracellular domains, cadherins associate with molecules of the Armadillo superfamily including β-catenin, which links them to the actin cytoskeleton via the α-catenin bridge (6Hirohashi S. Am. J. Pathol. 1998; 153: 333-339Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, 7Huber O. Bierkamp C. Kemler R. Curr. Opin. Cell Biol. 1996; 8: 685-691Crossref PubMed Scopus (307) Google Scholar). In addition to its adhesive functions, β-catenin has also been found to serve as a key component in Wnt signaling (8Peifer M. Science. 1997; 275: 1752-1753Crossref PubMed Scopus (302) Google Scholar, 9Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (887) Google Scholar). When released from E-cadherin, uncomplexed β-catenin is rapidly degraded by cytosolic proteasomes. Failure to properly degrade β-catenin, primarily attributable to an impairment in its ubiquitination, results in its posttranslational stabilization and passage into the nucleus, where it interacts with transcription factors of T cell factor/lymphoid enhancer factor (Tcf/Lef) family to activate target genes involved in cell growth control and apoptosis such as c-myc and cyclin D1 (10He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4092) Google Scholar, 11Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3265) Google Scholar). Aberrant activation of β-catenin signaling has been implicated in cancer formation in numerous basic and clinical studies (12Giles R.H. van Es J.H. Clevers H. Biochim. Biophys. Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1348) Google Scholar). There is increasing evidence to suggest that phosphorylation of tyrosyl residues in some components of the cadherin/catenin complex leads to loss of adhesive function and breakdown of adherens junction. Roura et al. (13Roura S. Miravet S. Piedra J. Garcia d.H. Dunach M. J. Biol. Chem. 1999; 274: 36734-36740Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar) reported that phosphorylation of tyrosine residue 654 on β-catenin diminishes its association with E-cadherin. Furthermore, they observed that phosphorylation of Tyr-654 also stimulated the association of β-catenin to the basal transcription factor TATA-binding protein. Thus, it is reasonable to conclude that phosphorylation-dependent release of β-catenin from the cadherin complex not only regulates the integrity and function of the adhesion complex but may also be an alternative mechanism for activating β-catenin signaling (14Nelson W.J. Nusse R. Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2253) Google Scholar). We and others have previously shown that several RPTPs were functionally associated with E-cadherin/β-catenin complex and play a regulatory role in the control of the integrity of cell junctions, presumably by keeping them in a dephosphorylated state (15Brady-Kalnay S.M. Mourton T. Nixon J.P. Pietz G.E. Kinch M. Chen H. Brackenbury R. Rimm D.L. Del Vecchio R.L. Tonks N.K. J. Cell Biol. 1998; 141: 287-296Crossref PubMed Scopus (154) Google Scholar, 16Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar, 17Fuchs M. Muller T. Lerch M.M. Ullrich A. J. Biol. Chem. 1996; 271: 16712-16719Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Yan H.X. He Y.Q. Dong H. Zhang P. Zeng J.Z. Cao H.F. Wu M.C. Wang H.Y. Biochemistry. 2002; 41: 15854-15860Crossref PubMed Scopus (35) Google Scholar). In this study, we investigated the regulatory effect of PCP-2 on nuclear signaling activity of β-catenin. We demonstrate that PCP-2 repressed not only wild-type but also active mutant β-catenin-induced transcriptional activity. PCP-2 expression in human colon carcinoma cell line SW480, in which the β-catenin signaling pathway is up-regulated, led to stabilization of cytosolic pools of β-catenin, perhaps by their physical interaction and by enhancing adherens junction stability, thus decreasing the transcriptional activity of β-catenin by sequestering the protein at the plasma membrane. This effect was attributed to the inhibition of cell proliferation and migration when PCP-2 was transfected into SW480 cells. These results delineate a novel role for PCP-2 in regulation of the canonical β-catenin signaling pathway. Cell Culture and Transfections—HEK293, SW480 colon carcinoma cells, A431 human epidermoid carcinoma cells, and SW850 pancreatic carcinoma cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Stable SW480 clones were generated by transfection using JetPEI (Polyplus) of pcDNA3.0 or PCP-2 constructs and screened by Western blotting. Positive clones expressing similar levels of PCP-2 were cultured in the presence of 300 μg/ml G418 for maintenance of transgene expression. Plasmids—Different recombinants for wild-type or mutant forms of PCP-2 were constructed using pcDNA3/Myc(–) (Invitrogen). All β-catenin constructs tagged with Myc were produced by PCR amplification with the use of human wild-type β-catenin in a pRK5RS vector as a template. The fidelity of the constructs was verified by DNA sequencing. The resulting PCR products were subcloned into the pcDNA3/Myc(–) vector. N-terminal-deleted β-catenin (β-ΔN) lacks the first 140 amino acids, C-terminal-deleted β-catenin (β-ΔC) lacks the last 147 amino acids, and armadillo-domain-deleted β-catenin (β-ΔArm) contains both the N-terminal 140 amino acids and the C-terminal 147 amino acids but lacks the 141–633 amino acids. Point mutant Y654E was obtained using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). Antibodies—Monoclonal antibodies specific for E-cadherin and β-catenin were purchased from BD Transduction Laboratories and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Polyclonal antibodies reactive with PCP-2 were used as described (18Yan H.X. He Y.Q. Dong H. Zhang P. Zeng J.Z. Cao H.F. Wu M.C. Wang H.Y. Biochemistry. 2002; 41: 15854-15860Crossref PubMed Scopus (35) Google Scholar). The monoclonal antibodies against Myc tag (9B11) and phosphotyrosine (P-Tyr-102) were obtained from Cell Signaling Technology. The monoclonal antibodies specific for c-Myc was purchased from Neomarker (Fremint, CA). β-Catenin/Tcf Luciferase Reporter Assay—Two different Tcf-luciferase reporter constructs were used in this study: an intact wild-type Tcf-luciferase construct (pGL-OT) and a mutant Tcf-luciferase reporter construct (pGL-OF) (both gifts of B. Vogelstein) (10He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4092) Google Scholar). The cyclin D1 reporter plasmid was kindly provided by Dr. O. Tetsu (11Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3265) Google Scholar). A Dual Luciferase reporter assay was carried out according to the manufacturer's suggestions (Promega). pRL-TK (Promega) was cotransfected with each reporter construct to normalize for transfection. All experiments were performed in triplicate. Immunofluorescence—Cells were cultured on glass coverslips, fixed with 3% paraformaldehyde in phosphate-buffered saline, and permeabilized with 0.2% Triton X-100. The coverslips were incubated with E-cadherin or β-catenin monoclonal antibodies at 4 °C overnight. The secondary antibody was fluorescein isothiocyanate- or Cy3-conjugated goat anti-mouse immunoglobulin G (Sigma). After being washed in Tris-buffered saline, coverslips were mounted with 90% glycerol in Tris-buffered saline and analyzed with a conventional fluorescence microscope (Olympus IX70). Immunoprecipitation, Immunoblotting, and Chromatin Immunoprecipitation (ChIP)—Cell extracts were prepared using Nonidet P-40 lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 1mm EGTA, 1 mm Na3VO4, 1 mm NaF, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin). The supernatant was collected and incubated with antibodies at 4 °C for 3 h and then with protein A for an additional 3 h. The beads were washed three times with lysis buffer and resuspended in SDS sample buffer. For immunoblot analysis, samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schüll). The membrane was first probed with a specific antibody and then detected using the ECL system with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). For ChIP assay, chromatin was isolated from formaldehyde-treated SW480 cells, fragmented to a mean size <600 bp, and subjected to ChIP using chromatin immunoprecipitation assay kits (Upstate Biotechnology, Hamburg, Germany) together with 10 μgof β-catenin-specific antibody following Upstate Biotechnology's protocol. A specific primer pair for the c-myc promoter region was used for investigating the binding of β-catenin to DNA. For analyzing chromatin input, one-fiftieth of the precipitated chromatin was taken as a template, and for all other reactions, one-tenth of the precipitated chromatin was taken as a template. Cell Fractionation—To obtain Triton X-100-soluble and -insoluble fractions, cells were incubated with Triton buffer (1% Triton X-100, 0.3 m sucrose, 25 mm HEPES, pH 7.4, 100 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgCl2, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) for 15 min on a rocking platform. After centrifugation, the supernatant (Triton X-100-soluble fraction) was collected. The cell pellet was resuspended in SDS lysis buffer (20 mm Tris, pH 7.5, 2.5 mm EDTA, 1% SDS, and 1 mm dithiothreitol) and subjected to standard SDS-PAGE and immunoblot analysis. Aggregation Assay and Wound Healing—Cell-cell adhesion was evaluated in an aggregation assay, as described previously (19Nam J.S. Ino Y. Sakamoto M. Hirohashi S. Clin. Cancer Res. 2002; 8: 2430-2436PubMed Google Scholar). In brief, cultures were rinsed with 10 mm HCMF buffer (10 mm HEPES, pH 7.4, 137 mm NaCl, 5.4 mm KCl, 0.3 mm Na2HPO4·7H2O, 5.5 mm glucose) containing 2 mm CaCl2 and trypsinized into single cells by incubation with 0.04% trypsin in HCMF buffer supplemented with 2 mm CaCl2. They were then incubated under gyratory shaking at 80 rpm for 30 min in HCMF containing 1% bovine serum albumin and 1.25 mm Ca2+ and photographed. For in vitro wound assays, monolayers of cells were wounded by scraping with a plastic pipette tip, rinsed several times with medium to remove dislodged cells, and cultured in serum-free Dulbecco's modified Eagle's medium for 24 h in a humidified incubator containing 5% CO2. Cells that had migrated into the wound area were photographed with a light microscope equipped with phase-contrast optics (Olympus IX70). Cell Surface Biotinylation—Cells were rinsed once in serum-free Dulbecco's modified Eagle's medium and twice in ice-cold PBS, pH 7.5, and then incubated with 1.0 mg/ml sulfo-NHS-SS-biotin (biotin disulfide N-hydroxysuccinimide ester, Pierce) and dissolved in PBS for 30 min on a rocking platform on ice. Biotinylation was stopped by washing twice in PBS containing 100 mm glycine and twice in PBS for altogether 35 min. Cells were solubilized in lysis buffer (10 mm Tris/HCl, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, and protease inhibitors: 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin), pH 7.4. The unlysed cells were removed by centrifugation at 15,700 × g at 4 °C. A 50-μl volume of streptavidin-agarose beads was then added to the supernatant to isolate cell-membrane protein. Membrane-associated E-cadherin or β-catenin was detected in the pool of surface proteins by SDS-PAGE and immunoblotting. Generation of Recombinant Adenovirus Expressing PCP-2—The PCP-2 coding sequence insert was cloned into the adenoviral shuttle vector (Stratagene). Then it was linearized with PmeI and cotransformed with E1-deleted adenoviral backbone AdEasy-1 into the competent bacterial strain BJ5183, which allows efficient recombination to occur. After screening, recombinants for adenoviruses Ad-PCP-2 and Ad-Blank, which contain no insert sequence as a control, were generated. Growth Curves and Colony Formation Assay—Growth Curves, stable SW480 cell lines (5 × 104) were plated per well of 6-well plates. At each time point, the cells were trypsinized and counted. Each data point was performed in triplicate. The measurement of viable cell mass was also performed with a Cell Counting Kit-8 (Dojin Laboratories, Kumamoto, Japan) to count living cells by WST-8. For colony formation after transfection with drug selection, an equivalent number of SW480 cells (106) were transfected with 2 μg of vector or PCP-2 constructs conferring neomycin drug resistance. After transfection, cells were replated and selected in G418-containing medium for 3 weeks, and the resultant colonies were fixed and stained with crystal violet. Transfections were done in triplicate for each combination of plasmids. RNA Interference—Three double-stranded siRNA oligonucleotides against PCP-2 (sense strand, 5′-CCACAAAGAAGAAAGACAAGGUCAA-3′, 5′-GGGACAUCAAGAUUAUGCUGGUGAA-3′, 5′-GAUCCGCAUUGAUCCUCAGAGUAAU-3′) were provided by Invitrogen. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent according to the manufacturer's directions with 150 nmol of siRNA per well in a six-well dish. A scrambled siRNA was used as the control. siRNA transfected cells were incubated for 36–48 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. In Vivo Tumorigenicity Assay—SW480 transfectants were released from tissue culture dishes and washed in serum-free medium. Tumor cells were diluted with PBS and injected into the mid-dorsum of BALB/c nude mice (4–6 weeks old) in a total of volume 0.1 ml (5 × 106). Animals were inspected weekly for tumor development. Growing tumors were measured using vernier calipers, and tumor volume was calculated by the formula length × width2 × 0.52, which approximates the volume of an elliptical solid. Statistical analysis was performed by Student's t test (two-tailed). The criterion for statistical significance was taken as p < 0.05. All procedures regarding animals were conducted according to institutionally approved protocols. PCP-2 Inhibited β-Catenin-mediated Transcriptional Activity—We have previously demonstrated functional interaction between β-catenin and PCP-2. To study its effect on β-catenin-mediated transactivation, PCP-2 was cotransfected with wild-type β-catenin into HEK293 cells together with Tcf reporter plasmid pGL-OT. As shown in Fig. 1A, PCP-2 very strongly suppressed β-catenin-mediated luciferase activity driven from the pGL-OT reporter in a dose-dependent manner. We also used LiCl, an inhibitor of GSK3β activity and widely used to mimic Wnt signaling, to increase endogenous β-catenin levels and detect its transactivation potential after transfection with PCP-2. Treatment of the cells with LiCl resulted in an evident increase in β-catenin levels. Although β-catenin activity was elevated in empty vector-transfected cells, this effect was significantly reduced in cells transfected with PCP-2 (Fig. 1B). As functional β-catenin/Tcf-binding sites have been identified in the promoter of cyclin D1, we then tested the effect of PCP-2 on the cyclin D1 reporter transcription. Fig. 1C shows that cyclin D1 reporter was strongly transcribed in response to wild-type β-catenin. However, exogenous expression of PCP-2 imposed a substantial inhibition of the cyclin D1 promoter activity. To address the inhibitory effect of PCP-2 on β-catenin transcriptional activity under normal signaling conditions, we knocked down endogenous PCP-2 level using RNA interference strategy and then examined changes of β-catenin reporter gene activity after EGF stimulation in A431 human epidermoid carcinoma cells, which overexpress EGFR. As shown in Fig. 1, D and E, PCP-2 down-regulation by siRNA led to an increased transcriptional activity of β-catenin upon EGF treatment as compared with cells transfected with control siRNA. The specificity of PCP-2-mediated effects on Tcf reporters was confirmed by using pGL-OF, which harbors mutated Tcf-binding sites and was not influenced by PCP-2. Taken together, these results suggest that PCP-2 can exert specifically inhibitory effect on β-catenin-dependent transcriptional activity. PCP-2 Down-regulated the Active Mutant Form of β-Catenin-induced Signaling—Mutations in the ubiquitin-targeting sequence of β-catenin occur in a number of different cancers (20Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (609) Google Scholar). Among these mutations, S37A and N-terminal truncation are common stabilizing ones that render β-catenin resistant to ubiquitination-dependent degradation. To test whether PCP-2 could modulate the transcriptional activities of the degradation-resistant mutants of β-catenin, we cotransfected Myc-tagged wild-type and S37A, ΔN, ΔC, or ΔArm mutant β-catenin constructs (Fig. 2A) together with empty vector or PCP-2 and the Tcf reporter plasmids pGL-OT into HEK293 cells and monitored β-catenin signaling by assaying the reporter activity. As shown in Fig. 2B, transfection of S37A and ΔN mutant forms of β-catenin significantly increased the Tcf reporter activity in HEK293 cells, whereas ΔC and ΔArm did not, probably due to the loss of Tcf-binding sites and transactivation domain of β-catenin. Cotransfection of PCP-2 significantly reduced both S37A and ΔN mutant forms of β-catenin-induced Tcf reporter activation, indicating that the inhibitory effect of PCP-2 on β-catenin is independent of its degradation sensitivity. The Phosphatase Activity Was Involved in PCP-2-mediated Repression of β-Catenin Signaling—We previously showed that PCP-2 bound β-catenin and conferred its dephosphorylation (18Yan H.X. He Y.Q. Dong H. Zhang P. Zeng J.Z. Cao H.F. Wu M.C. Wang H.Y. Biochemistry. 2002; 41: 15854-15860Crossref PubMed Scopus (35) Google Scholar). To examine whether the tyrosine phosphorylation of β-catenin was attributed to its transcriptional activity, we transiently transfected wild-type β-catenin or its point mutants Y654E and Y654F, which mimic the phosphorylated or dephosphorylated states of Tyr-654 respectively, together with pGL-OT reporter plasmids and determined β-catenin-sensitive reporter activity 24 h after transfection. As shown in Fig. 3A, expression of Y654E β-catenin mutant raised the activity of this reporter gene to a higher extent than the WT and Y654F forms of β-catenin did. Interestingly, as compared with the wild-type β-catenin, PCP-2-mediated fold repression of β-catenin-dependent transcriptional activity was evidently decreased in the presence of the Y654E mutant, which was resistant to dephosphorylation by PCP-2 (Fig. 3B). These data suggest that tyrosine phosphorylation of β-catenin lead to a greater stimulation of β-catenin-Tcf-mediated transcription, and PCP-2-mediated dephosphorylation was necessary for its role in negatively regulating β-catenin signaling. To further confirm these results, a catalytically inactive mutant of PCP-2 (PCP-2/CS) was generated in which critical cysteine residues in both PTP domains were mutated to serine. As shown in Fig. 3C, introduction of the Cys to Ser mutation in both PTP domains evidently interfered with its inhibitory effect on β-catenin activity. To examine whether such mutations affect PCP-2 association with β-catenin, Myc-tagged wild-type or mutant PCP-2 were transiently cotransfected into HEK293 cells with wild-type β-catenin or its point mutant Y654E. Twenty-four hours later, cells were lysed and immunoprecipitated with antibody specific for β-catenin. After SDS-PAGE of immunoprecipitates, immunoblots were probed for PCP-2 and β-catenin. As shown in Fig. 3D, either wild-type or mutant PCP-2 was efficiently coimmunoprecipitated with β-catenin or its mutant, indicating that their association was independent of PCP-2 catalytic activity or β-catenin tyrosine phosphorylation status. PCP-2 Repressed β-Catenin Activity in Colon Cancer Cell Line—To examine the effects of PCP-2 on β-catenin signaling in detail, we isolated stable clones of SW480 cells expressing Myc-tagged WT-PCP-2 or PCP-2/CS (mu-PCP-2) and characterized them by immunoblotting (Fig. 4A) and by immunofluorescence (Fig. 4B). SW480 cells were chosen for study because they harbor a truncated APC that renders β-catenin resistant to degradation and contains relatively low E-cadherin levels; thus, most of the β-catenin is not retained in the membrane by this molecule (21Gottardi C.J. Wong E. Gumbiner B.M. J. Cell Biol. 2001; 153: 1049-1060Crossref PubMed Scopus (462) Google Scholar). Two independent clones for each construct with similar protein expression levels were used for further analysis. Drug-resistant clones of empty vector-transfected cells were pooled to rule out clone-specific effects. As shown in Fig. 4C, exogenous expression of WT-PCP-2 led to a substantial drop in Tcf reporter activities. The inhibitory effect of PCP-2 was partially compromised by inactivation of its catalytic activity, as expected from the above observation that the phosphatase activity was involved in PCP-2-mediated repression of β-catenin signaling (Fig. 3). To directly address whether there was physiological relevance to our finding of decreased β-catenin transactivation, we then evaluated the amounts of c-myc, which was a crucial target for β-catenin/Tcf4-mediated Wnt signaling activities. In Fig. 4, D and E, we showed that exogenous expression of PCP-2 or PCP-2/CS caused a significant reduction in the amounts of c-myc mRNA and protein levels, suggesting that the decreased transcriptional activity is accompanied by specific decreases in gene transcription and that the decrease in c-Myc protein levels occurs at the mRNA level. A ChIP assay was further performed to determine whether PCP-2 signaling caused a direct effect on reducing binding of the β-catenin-Tcf transcription factor to the endogenous c-myc promoter region. Fig. 4F revealed that β-catenin was indeed recruited less to the promoter regions of c-myc gene in the PCP-2-expressing cell pool than in the empty vector control cell pool. These data demonstrate that PCP-2 induced dissociation of β-catenin-Tcf with c-myc promoter and suggest a new function for this RPTP in β-catenin-dependent gene regulation. PCP-2 Directly Associated with β-Catenin and Enhanced Its Stability—To explore the molecular mechanism by which PCP-2 down-regulate β-catenin signaling activity, we first tested whether PCP-2 could associate with E-cadherin/β-catenin complex in SW480 cells. PCP-2 or β-catenin was immunoprecipitated from SW480 cells stably transfected with Myc-tagged WT- or mu-PCP-2. The immunoprecipitates were then analyzed by immunoblotting with anti-β-catenin, anti-E-cadherin, or anti-Myc tag antibodies. As shown in Fig. 5, A and B, both PCP-2 and E-cadherin were found in β-catenin immunoprecipitates, but only β-catenin could be found in PCP-2 immunoprecipitates, indicating that PCP-2 directly associated with β-catenin but not E-cadherin. Note that mu-PCP-2 was able to coimmunoprecipitate with β-catenin to the same extent as WT-PCP-2. We then examined whether the expression of PCP-2 could influence the stability of β-catenin. The cells were treated with cycloheximide to block new protein synthesis; protein extracts were prepared at 0, 3, 6, and 9 h after the block. Western blotting (Fig. 5C) showed that β-catenin was rapidly degraded in empty vector-transfected cells, whereas its levels remained stable in WT- and mu-PCP-2 cells. These data suggest that PCP-2 expression could lead to stabilization of cytosolic pools of β-catenin perhaps by virtue of their physical interaction. Expression of PCP-2 Led to Decreased Free Cytoplasmic β-Catenin and Increased Membrane-associated E-cadherin—To determine whether the reduction in β-catenin/Tcf signaling was due to β-catenin sequestration by PCP-2 from the free competent signaling pool, β-catenin from SW480-derived cells was affinity-precipitated with a GST-E-cadherin cytoplasmic fusion protein, and the levels of free, uncomplexed β-catenin were analyzed by immunoblotting (Fig. 6A). As demonstrated previously, this strategy allows, in contrast to immunoprecipitation, specifically and selectively, the precipitation of the free, non-E-cadherin-bound pool of β-catenin (22Muller T. Choidas A. Reichmann E. Ullrich A. J" @default.
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• W2123292213 date "2006-06-01" @default.
• W2123292213 modified "2023-10-16" @default.
• W2123292213 title "Protein-tyrosine Phosphatase PCP-2 Inhibits β-Catenin Signaling and Increases E-cadherin-dependent Cell Adhesion" @default.
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