A Novel β-Catenin-binding Protein Inhibits β-Catenin-dependent Tcf Activation and Axis Formation

β-Catenin is efficiently phosphorylated by glycogen synthase kinase-3β in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, β-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of β-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of β-catenin, thereby inhibiting the binding of Tcf to β-catenin. It did not affect the stability of β-catenin but inhibited Wnt- or β-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and β-catenin-dependent axis duplication, and prevented the β-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits β-catenin signaling. β-Catenin is efficiently phosphorylated by glycogen synthase kinase-3β in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, β-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of β-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of β-catenin, thereby inhibiting the binding of Tcf to β-catenin. It did not affect the stability of β-catenin but inhibited Wnt- or β-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and β-catenin-dependent axis duplication, and prevented the β-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits β-catenin signaling. glycogen synthase kinase-3β T cell factor/lymphocyte enhancer binding factor adenomatous polyposis coli protein protein phosphatase 2A hemagglutinin 1 glutathioneS-transferase maltose-binding protein phosphate-buffered saline dithiothreitol dorso-anterior index reverse transcription polymerase chain reaction cAMP response element-binding protein-binding protein Drosophila cAMP response element-binding protein-binding protein β-Catenin has been originally identified as a protein that interacts with the cytoplasmic domain of cadherin and links cadherin to α-catenin, which in turn mediates the anchorage of the cadherin complex to the cortical actin cytoskeleton (1Takeichi M. Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2984) Google Scholar). Many binding partners of β-catenin have been found, suggesting that β-catenin has other functions in addition to its role in cell-cell adhesion. 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Indeed, Axin inhibits Wnt-dependent β-catenin accumulation and Tcf activation (25Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (115) Google Scholar). Thus, Axin is a negative regulator of the Wnt signaling pathway. Further, Axin is phosphorylated by GSK-3β and the phosphorylation stabilizes Axin in contrast to β-catenin (26Yamamoto H. Kishida S. Kishida M. Ikeda S. Takada S. Kikuchi A. J. Biol. Chem. 1999; 274: 10681-10684Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Dvl interacts with Axin (27Fagotto F. Jho E. Zeng L. Kurth T. Joos T. Kaufmann C. Costantini F. J. Cell Biol. 1999; 145: 741-756Crossref PubMed Scopus (234) Google Scholar, 28Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar, 29Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (206) Google Scholar) and inhibits GSK-3β-dependent phosphorylation of β-catenin, APC, and Axin in the Axin complex (26Yamamoto H. Kishida S. Kishida M. Ikeda S. Takada S. Kikuchi A. J. Biol. Chem. 1999; 274: 10681-10684Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 28Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar). PP2A binds to Axin (22Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (164) Google Scholar, 30Hsu W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and it dephosphorylates APC and Axin (22Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (164) Google Scholar). Further, the B56 subunit of PP2A binds to APC and its expression reduces the levels of cytoplasmic β-catenin in HEK293 cells (31Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (366) Google Scholar). In the Axin complex, the phosphorylation of β-catenin, APC, and Axin is regulated by GSK-3β, Dvl, and PP2A, and the stability of β-catenin and Axin is controlled by their phosphorylation. Therefore, Axin may be a scaffold protein, in that it binds to several signaling molecules to create a multiprotein complex. Cytoplasmic β-catenin accumulated in response to Wnt is translocated into the nucleus although the mechanism is unknown (32Fagotto F. Gluck U. Gumbiner B.M. Curr. Biol. 1998; 8: 181-190Abstract Full Text Full Text PDF PubMed Google Scholar). In addition to Tcf/Lef, β-catenin forms a complex with Pontin52 in the nucleus (33Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (170) Google Scholar). Pontin52 can be coimmunoprecipitated within a large complex containing β-catenin and Lef-1, but whether Pontin52 affects the β-catenin activity to regulate the gene expression is not known. To understand the molecular mechanism of the β-catenin signaling in the Wnt pathway, we have screened the new binding partners of the components of the Wnt signaling pathway. We isolated a novel protein that binds to Dvl by yeast two-hybrid screening. Although this protein bound to Dvlin vitro, it did not form a complex with Dvl in intact cells. However, during these experiments, we found that this novel protein is located in the nucleus and that it forms a complex with β-catenin in intact cells. We designated this protein as Duplin (for axis duplication inhibitor) and examined its effects on β-catenin signaling. We show here that Duplin inhibits the binding of β-catenin to Tcf and β-catenin-dependent activation of Tcf in mammalian cells and that it inhibits β-catenin-dependent axis duplication inXenopus embryos. pcDNAI/hTcf-4 and pTOPFLASH, and pUC/EF-1α/β-cateninSA were supplied by Drs. H. Clevers (University Hospital, Utrecht, The Netherlands) and A. Nagafuchi (Kyoto University, Kyoto, Japan), respectively. β-CateninSA is a β-catenin mutant in which the serine and threonine residues of the GSK-3β phosphorylation sites (21Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar) are changed to alanine. The cDNA of hDvl-1, the anti-HA antibody, and the anti-GST and anti-MBP antibodies were provided by Drs. B. Dallapiccola (Vergata University, Rome, Italy) (34Pizzuti A. Amati F. Calabrese G. Mari A. Colosimo A. Silani V. Giardino L. Ratti A. Penso D. Calza L. Palka G. Scarlato G. Novelli G. Dallapiccola B. Hum. Mol. Genet. 1996; 5: 953-958Crossref PubMed Scopus (61) Google Scholar), Q. Hu (Chiron Corp.), and M. Nakata (Sumitomo Electric Industries, Yokohama, Japan), respectively. MBP- and GST-fused proteins were purified fromEscherichia coli according to the manufacturer's instructions except that GST-Dvl-1 was purified from Spodptera frugiperda 9 cells as described (28Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar). The anti-Duplin and anti-Dvl antibodies were prepared in rabbits by immunization with recombinant fragments of Duplin-(482–668) and Dvl-1-(1–140), respectively. The anti-Myc antibody was prepared from 9E10 cells. L cells (mouse fibroblasts) stably expressing HA-Duplin were generated by selecting with G418 as described (25Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (115) Google Scholar, 35Okazaki M. Kishida S. Murai H. Hinoi T. Kikuchi A. Cancer Res. 1996; 56: 2387-2392PubMed Google Scholar). Wnt-3a-conditioned medium was generated as described (36Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). The anti-GSK-3β and anti-β-catenin antibodies were purchased from Transduction Laboratories (Lexington, KY). [α-32P]dCTP was obtained from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Other materials were from commercial sources. Standard recombinant DNA techniques were used to construct the following plasmids, pBTM116HA/hDvl-1-(251–336), pBSKS/Duplin-(1–482), pBSKS/Duplin-(482–749), pBSKS/Duplin (full-length), pCGN/hTcf-4 (full length), pGEX-2T/hTcf-4-(1–80), pMAL-c2/β-catenin, pMAL-c2/Duplin, pMAL-c2/Duplin-(1–276), pMAL-c2/Duplin-(482–749), pMAL-c2/Duplin-(482–668), pMAL-c2/Duplin-(667–749), pGEX-4T-1/Duplin-(482–749), pGEX-4T-1/Duplin-(482–668), pGEX-4T-1/Duplin-(667–749), pBJ-Myc/Duplin, pCGN/Duplin, pCGN/Duplin-(1–482), pCGN/Duplin-(482–749), pCGN/Duplin-(482–668), pCGN/Duplin-(482–564), pCGN/Duplin-(667–749), pCGN/Duplin-(565–668), and pEF-BOS-HA/hTcf-4. The structures of all plasmids were confirmed by restriction analysis and in many cases by DNA sequence analysis across crucial regions. pGEX-derived β-catenin plasmids, pCGN/rAxin, and pGEX-2T/hDvl-1-(1–140) were constructed as described (14Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar, 28Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar). COS or L cells (two 10-cm diameter dishes) were washed with cold PBS and suspended in 1 ml of homogenizing buffer (20 mm Tris/HCl, pH 7.5, 1 mm DTT, 250 mm sucrose, 3 mmMgCl2, 3 mm CaCl2, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). This suspension was homogenized with a Potter-Elvehjem homogenizer at 4 °C and used as the total homogenate. The homogenate was centrifuged at 700 × g for 10 min at 4 °C. The precipitate was washed and resuspended in homogenizing buffer. This suspension was used as the nuclear fraction. The supernatant was centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was used as the cytoplasmic fraction. The precipitate was washed and resuspended in homogenizing buffer. This suspension was used as the membrane fraction. The volume of all the fractions were normalized to 1 ml. Aliquots (20 μl) of the total homogenate and cytoplasm, membrane, and nuclear fractions were subjected to SDS-polyacrylamide gel electrophoresis and probed with the anti-β-catenin and anti-Myc antibodies. SW480 and L cells on coverslips were fixed for 20 min in PBS containing 4% paraformaldehyde. The cells were washed with PBS three times, and then permeabilized with PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum albumin for 12 h. The cells were washed and incubated for 1 h with the anti-HA or the anti-Duplin antibody. After washing with PBS, they were further incubated for 1 h with Alexa 594 labeled-anti-mouse or -anti-rabbit IgG. The coverslips were washed with PBS, mounted on glass slides, and viewed with a confocal laser-scanning microscope (TCS-NT®, Leica-laser-technik GmbH, Heidelberg, Germany). To determine whether Duplin interacts with β-catenin in intact cells, COS cells (10-cm diameter dish) transfected with pCGN- and pBJ-derived plasmids were disrupted by sonication in 500 μl of the lysis buffer (20 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1 mm DTT, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) and the homogenate was centrifuged at 100, 000 × g for 30 min at 4 °C. The supernatant (150 μg of protein) was immunoprecipitated with the anti-Myc antibody, and then the precipitates were probed with the anti-Myc, anti-HA, anti-GSK-3β, anti-β-catenin, and anti-Dvl antibodies. To examine the interaction of Duplin with β-catenin using the purified proteins in vitro, GST-β-catenin and its deletion mutants (0.5 μm) were incubated with MBP-Duplin-(482–749) (30 pmol) immobilized on amylose resin in 100 μl of reaction mixture (20 mm Tris/HCl, pH 7.5, 1 mm DTT) for 1 h at 4 °C. MBP-Duplin was precipitated by centrifugation, and then the precipitates were probed with the anti-GST antibody. To show inhibition by Duplin of the binding of Tcf to β-catenin in vitro, the indicated concentrations of GST-Duplin-(482–749) and GST-hTcf-4-(1–80) (0.5 μm) were incubated with MBP-β-catenin (30 pmol) immobilized on amylose resin in 100 μl of reaction mixture. MBP-β-catenin was precipitated by centrifugation, then the precipitates were probed with the anti-GST antibody. To demonstrate the inhibitory action of Duplin in intact cells, wild-type L cells or L cells expressing HA-Duplin (10-cm diameter dish) were transfected with pcDNAI/hTcf-4. At 46 h after transfection, the cells were deprived of serum for 6 h, then treated with Wnt-3a-conditioned medium for 8 h. The cells were disrupted as described above, and the lysates were immunoprecipitated with the anti-β-catenin antibody. The immunoprecipitates were probed with the anti-HA and anti-β-catenin antibodies. Wild-type L cells or L cells expressing HA-Duplin (35-mm diameter dish) were transfected with pTOPFLASH, pcDNAI/hTcf-4, and pME18S/lacZ (25Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (115) Google Scholar, 37Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2937) Google Scholar). At 46 h after transfection, the cells were deprived of serum for 6 h, then treated with Wnt-3a conditioned medium for 8 h. When the effect of Duplin on β-catenin-dependent Tcf activation, wild-type L cells were further transfected with pUC/EF-1α/β-cateninSA and pBJ-Myc/Duplin. The cells were lysed, and luciferase activity was measured using a PicaGene (Toyo B-NET Co., Ltd., Tokyo, Japan) and lumiphotometer TD4000 (Futaba Medical, Tokyo, Japan). To standardize the transfection efficiency, pME18S/lacZ carrying SRα promoter linked to the coding sequence of β-galactosidase gene was used as an internal control. The transcriptional activity of the c-fos promoter activated by Ras was measured using luciferase as a reporter gene (35Okazaki M. Kishida S. Murai H. Hinoi T. Kikuchi A. Cancer Res. 1996; 56: 2387-2392PubMed Google Scholar). Duplin, Duplin-(1–482), Duplin-(482–749), Duplin-(482–668), Duplin-(482–564), Duplin-(565–668), Duplin-(667–749), hDvl-1,Xenopus wnt-8 (Xwnt-8), Xenopus β-catenin (Xβ-catenin), and Xenopus globin (Xglobin) cDNAs were individually subcloned into the BglII site of pSP64T. Sense mRNA was obtained by in vitro transcription of linearized templates using SP6-mMESSAGE mMACHINE kit (Ambion). Fertilized eggs were dejellied using 4.5% cysteine acid, and mRNAs were injected into dorsal or ventral blastomeres at the four-cell stage. After injection, embryos were cultured for 3 days (at stage 40–41). UV light irradiation was performed as described (38Scharf S.R. Gerhart J.C. Dev. Biol. 1980; 79: 181-198Crossref PubMed Scopus (154) Google Scholar). The phenotypes of the injected embryos were evaluated by DAI (39Kao K.R. Elinson R.P. Dev. Biol. 1988; 127: 64-77Crossref PubMed Scopus (423) Google Scholar). For RT-PCR, injected embryos were incubated at stage 10.5, and then total RNAs were isolated. Oligo(dT)-primed cDNAs were synthesized using 5 μg of total RNA from 10 embryos. PCR analyses (35 cycles) were performed with ExTaq DNA polymerase (Takara). Primers for PCR are: EF-1α, 5′-CAG ATT GGT GCT GGA TAT GC-3′ and 5′-ACT GCC TTG ATG ACT CCT AG-3′; siamois, 5′-AAG ATA ACT GGC ATT CCT GAG C-3′ and 5′-GGT AGG GCT GTG TAT TTG AAG G-3′. To examine whether Duplin is expressed in Xenopus embryos, 20 embryos were extracted in 100 μl of buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, protease inhibitor mixture (Roche Molecular Biochemicals)), and the extracts were centrifuged at 15,000 × g for 20 min at 4 °C. Clear supernatant (50 μg of protein) was probed with the anti-Duplin antibody. Yeast two-hybrid screening was carried out as described (14Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar, 15Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (173) Google Scholar). To obtain a full-length cDNA of Duplin, the clone isolated by the yeast two-hybrid method was labeled with random primers and [α-32P]dCTP and used to screen a λZAP rat brain cDNA library. Northern blot analysis was performed as described (40Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Protein concentrations were determined with bovine serum albumin as a standard (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). To identify a novel protein that is involved in the Wnt signaling pathway, we screened a rat brain cDNA library with yeast two-hybrid method using the PDZ domain of Dvl-1 as a bait. Several clones were found to confer both His+ and LacZ+ phenotypes, and a full-length cDNA of one clone was isolated. This clone spanned a distance of 2,503 base pairs and contained an uninterrupted open reading frame of 2,247 base pairs, encoding a predicted protein of 749 amino acids (Fig.1 A). The first ATG was preceded by stop codons in all three reading frames. The neighboring sequence of the first ATG was consistent with the translation initiation start proposed by Kozak (42Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4168) Google Scholar). Although no protein closely related to this protein was identified, the C-terminal half included several clusters of basic amino acids (Fig. 1, A andB). We designated this protein as Duplin (for axisduplication inhibitor). mRNA of Duplin was expressed ubiquitously in various rat tissues, and two bands were observed, suggesting that two mRNAs are derived from two highly conserved genes or result from alternative splicing of a single gene (Fig. 1 C). The anti-Duplin antibody recognized a protein with a molecular mass of about 110 kDa (p110) (Fig. 1 C). The molecular mass of Myc-Duplin expressed in COS cells was similar to that of p110, indicating that Duplin cDNA encodes this protein. Another protein with a molecular mass of 140 kDa (p140) was recognized in PC12 cells by the antibody, but we do not know the relationship between Duplin and p140. When the cells were divided into cytoplasmic, membrane, and nuclear fractions by subcellular fractionation, Myc-Duplin was present mainly in the nuclear fraction of COS cells (Fig. 1 D). Immunocytochemical analyses also showed that endogenous Duplin and HA-Duplin were located in the nucleus of L cells and SW480 cells (Fig. 1 E). Furthermore, HA-Duplin-(1–482) was present in the cytoplasm, while HA-Duplin-(482–749) was present in the nucleus (Fig. 1 E). In the residues 482–749, the region containing amino acids 482–564 was detected in the nucleus, whereas the region containing amino acids 565–668 was observed in both the cytoplasm and nucleus (Fig. 1 E). HA-Duplin-(667–749) was localized in the cytoplasm (data not shown). Therefore, Duplin is located in the nucleus and the C-terminal region has a nuclear localization signal. Since Duplin was isolated as a binding protein to Dvl by a yeast two-hybrid screening, we examined whether Duplin binds directly to Dvl. To this end, GST-Dvl-1 and various deletion mutants of MBP-Duplin were purified. However, MBP-Duplin-(1–482) could not be purified. GST-Dvl-1 bound to MBP-Duplin-(482–668) but not to MBP-Duplin-(1–276) or MBP-Duplin-(667–749) in vitro (data not shown), while Duplin did not form a complex with Dvl when Myc-Duplin was expressed in COS cells (Fig. 2 A,lanes 1–4). Further, ectopically expressed HA-Duplin or HA-Duplin-(1–487) did not associate with Myc-Dvl-1, either (data not shown). Since Dvl is located in the cytoplasm mainly, the difference between in vitro and intact cell experiments might be due to the difference of their subcellular localizations. Therefore, we did not study the interaction of Dvl with Duplin further. Instead, we found that endogenous β-catenin was precipitated with Myc-Duplin when Myc-Duplin was expressed in COS cells (Fig. 2 A, lanes 1–4). Myc-Duplin did not form a complex with GSK-3β, HA-rAxin, or HA-hTcf-4 (Fig. 2 A, lanes 1–12). To examine whether the interaction of Duplin with β-catenin is direct, MBP-Duplin-(482–749) or MBP was incubated with GST-β-catenin. GST-β-catenin was precipitated with MBP-Duplin-(482–749) but not with MBP (Fig. 2 B, lanes 1–4). Regarding residues of 482–749 of Duplin, GST-Duplin-(667–749) but not GST-Duplin-(482–668) bound to MBP-β-catenin (Fig. 2 B, lanes 5–10). To determine which region of β-catenin binds to Duplin, various deletion mutants of GST-β-catenin were incubated with MBP-Duplin-(482–749). MBP-Duplin-(482–749) bound strongly to GST-β-catenin-(1–423) and GST-β-catenin-(132–423) and weakly to GST-β-catenin-(423–781), but not to GST-β-catenin-(1–131) or GST (Fig. 2 C). Since β-catenin-(132–423) contains Armadillo repeats 1–7, these results indicate that the C-terminal region of Duplin binds directly to the region including the Armadillo repeats of β-catenin. Tcf-4 is a nuclear protein that binds to β-catenin, and hTcf-4-(1–80) interacts directly with the Armadillo repeats of β-catenin (7Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar, 8Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1619) Google Scholar). GST-Duplin-(482–749) competed with GST-hTcf-4-(1–80) for the binding to MBP-β-catenin in a dose-dependent manner (Fig.2 D). These results indicate that Duplin binds directly to β-catenin, resulting in inhibition of the binding of β-catenin to Tcf-4. We showed previously that Wnt-3a-conditioned medium induces the accumulation of β-catenin and activates Tcf-4 in L cells and that expression of Axin inhibits these Wnt-3a-dependent responses (25Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (115) Go