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J. Biol. Chem., Vol. 275, Issue 42, 32871-32878, October 20, 2000

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

Ikuo Sakamoto, Shosei Kishida^§, Akimasa Fukui^¶, Michiko Kishida, Hideki Yamamoto, Shin-ichiro Hino, Tatsuo Michiue, Shinji Takada^**, Makoto Asashima^¶, and Akira Kikuchi^§§

From the  Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, ^§ PRESTO, Japan Science and Technology Corporation, Hiroshima 734-8551, the ^¶ Department of Life Science (Biology) and  CREST Project, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, the ^** Center for Molecular and Developmental Biology, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, and the  Kondoh Differentiation Signaling Project, ERATO, Japan Science and Technology Corporation, Kyoto 606-8305, Japan

Received for publication, May 15, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Catenin is efficiently phosphorylated by glycogen synthase kinase-3 in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, -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 -catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of -catenin, thereby inhibiting the binding of Tcf to -catenin. It did not affect the stability of -catenin but inhibited Wnt- or -catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and -catenin-dependent axis duplication, and prevented the -catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits -catenin signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Catenin has been originally identified as a protein that interacts with the cytoplasmic domain of cadherin and links cadherin to -catenin, which in turn mediates the anchorage of the cadherin complex to the cortical actin cytoskeleton (1). Many binding partners of -catenin have been found, suggesting that -catenin has other functions in addition to its role in cell-cell adhesion. Genetic and embryological studies have revealed that -catenin is a component of the Wnt signaling pathway and that it exhibits signaling functions (2-4).

Wnt proteins constitute a large family of cysteine-rich secreted ligands that control development in organisms ranging from nematode worms to mammals (5, 6). In vertebrates, the Wnt signaling pathway regulates organ development and cellular proliferation, morphology, motility, and fate (2-4). In the current model, the serine/threonine kinase, GSK-3¹ targets cytoplasmic -catenin for degradation in the absence of Wnt. As a result, cytoplasmic -catenin levels are low. When Wnt acts on its cell surface receptor Frizzled, Dvl, a cytoplasmic protein, is activated and it antagonizes the action of GSK-3. The phosphorylation of -catenin is reduced and -catenin is no longer degraded, resulting in its accumulation in the cytoplasm. Accumulated -catenin is translocated into the nucleus where it binds to Tcf/Lef, a transcription factor, and stimulates gene expression (7, 8). In the nucleus, several proteins that bind to Tcf/Lef regulate the complex formation of -catenin-Tcf-DNA. Therefore, it appears that -catenin signaling is regulated in both the cytoplasm and nucleus.

The mechanism by which the stability of -catenin is regulated has been increasingly clarified. Discovery and functional analyses of Axin have provided new clues as to how the stability of -catenin is regulated (9, 10). Axin was originally identified as a product of the mouse Fused locus (11). The mouse mutant Fused is recessive lethal; mutants have a duplication of the embryonic axis (12, 13). We have identified rat Axin (rAxin) and its homolog, Axil (for Axin-like), as GSK-3-interacting proteins (14, 15). Conductin has been identified as a -catenin-binding protein (16) and is identical with Axil. Both Axin and Axil bind not only to GSK-3 but also to -catenin and APC (14-20) and promote GSK-3-dependent phosphorylation of -catenin and APC (14, 15, 19, 21, 22). Phosphorylated -catenin forms a complex with TrCP/FWD1, a member of F-box protein family, resulting in the degradation of -catenin by ubiquitin and proteasome pathway (23, 24). Indeed, Axin inhibits Wnt-dependent -catenin accumulation and Tcf activation (25). Thus, Axin is a negative regulator of the Wnt signaling pathway. Further, Axin is phosphorylated by GSK-3 and the phosphorylation stabilizes Axin in contrast to -catenin (26). Dvl interacts with Axin (27-29) and inhibits GSK-3-dependent phosphorylation of -catenin, APC, and Axin in the Axin complex (26, 28). PP2A binds to Axin (22, 30), and it dephosphorylates APC and Axin (22). Further, the B56 subunit of PP2A binds to APC and its expression reduces the levels of cytoplasmic -catenin in HEK293 cells (31). In the Axin complex, the phosphorylation of -catenin, APC, and Axin is regulated by GSK-3, Dvl, and PP2A, and the stability of -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 -catenin accumulated in response to Wnt is translocated into the nucleus although the mechanism is unknown (32). In addition to Tcf/Lef, -catenin forms a complex with Pontin52 in the nucleus (33). Pontin52 can be coimmunoprecipitated within a large complex containing -catenin and Lef-1, but whether Pontin52 affects the -catenin activity to regulate the gene expression is not known. To understand the molecular mechanism of the -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 Dvl in 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 -catenin in intact cells. We designated this protein as Duplin (for axis duplication inhibitor) and examined its effects on -catenin signaling. We show here that Duplin inhibits the binding of -catenin to Tcf and -catenin-dependent activation of Tcf in mammalian cells and that it inhibits -catenin-dependent axis duplication in Xenopus embryos.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Chemicals-- pcDNAI/hTcf-4 and pTOPFLASH, and pUC/EF-1/-catenin^SA were supplied by Drs. H. Clevers (University Hospital, Utrecht, The Netherlands) and A. Nagafuchi (Kyoto University, Kyoto, Japan), respectively. -Catenin^SA is a -catenin mutant in which the serine and threonine residues of the GSK-3 phosphorylation sites (21) 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) (34), Q. Hu (Chiron Corp.), and M. Nakata (Sumitomo Electric Industries, Yokohama, Japan), respectively. MBP- and GST-fused proteins were purified from Escherichia coli according to the manufacturer's instructions except that GST-Dvl-1 was purified from Spodptera frugiperda 9 cells as described (28). 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 (25, 35). Wnt-3a-conditioned medium was generated as described (36). The anti-GSK-3 and anti--catenin antibodies were purchased from Transduction Laboratories (Lexington, KY). [-³²P]dCTP was obtained from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Other materials were from commercial sources.

Plasmid Construction-- 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/-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 -catenin plasmids, pCGN/rAxin, and pGEX-2T/hDvl-1-(1-140) were constructed as described (14, 28).

Subcellular Fractionation-- 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 mM MgCl₂, 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--catenin and anti-Myc antibodies.

Immunofluorescence Microscopy-- 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).

Interaction of Duplin with -Catenin-- To determine whether Duplin interacts with -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-3, anti--catenin, and anti-Dvl antibodies. To examine the interaction of Duplin with -catenin using the purified proteins in vitro, GST--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.

Inhibition by Duplin of the Binding of -Catenin to Tcf-- To show inhibition by Duplin of the binding of Tcf to -catenin in vitro, the indicated concentrations of GST-Duplin-(482-749) and GST-hTcf-4-(1-80) (0.5 µM) were incubated with MBP--catenin (30 pmol) immobilized on amylose resin in 100 µl of reaction mixture. MBP--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--catenin antibody. The immunoprecipitates were probed with the anti-HA and anti--catenin antibodies.

Luciferase Assay-- Wild-type L cells or L cells expressing HA-Duplin (35-mm diameter dish) were transfected with pTOPFLASH, pcDNAI/hTcf-4, and pME18S/lacZ (25, 37). 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 -catenin-dependent Tcf activation, wild-type L cells were further transfected with pUC/EF-1/-catenin^SA 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 SR promoter linked to the coding sequence of -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 (35).

Xenopus Injections and Analyses of Phenotypes-- 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 -catenin (X-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 (38). The phenotypes of the injected embryos were evaluated by DAI (39). 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-1, 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.

Other Procedures-- Yeast two-hybrid screening was carried out as described (14, 15). To obtain a full-length cDNA of Duplin, the clone isolated by the yeast two-hybrid method was labeled with random primers and [-³²P]dCTP and used to screen a ZAP rat brain cDNA library. Northern blot analysis was performed as described (40). Protein concentrations were determined with bovine serum albumin as a standard (41).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Duplin-- 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. 1A). 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 (42). Although no protein closely related to this protein was identified, the C-terminal half included several clusters of basic amino acids (Fig. 1, A and B). We designated this protein as Duplin (for axis duplication 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. 1C). The anti-Duplin antibody recognized a protein with a molecular mass of about 110 kDa (p110) (Fig. 1C). 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. 1D). Immunocytochemical analyses also showed that endogenous Duplin and HA-Duplin were located in the nucleus of L cells and SW480 cells (Fig. 1E). Furthermore, HA-Duplin-(1-482) was present in the cytoplasm, while HA-Duplin-(482-749) was present in the nucleus (Fig. 1E). 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. 1E). 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.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Structure and subcellular distribution of Duplin. A, amino acid sequence of Duplin. The clusters of basic amino acids are underlined. B, schematic representation of deletion mutants of Duplin. The white boxes indicate the region containing the clusters of basic amino acids. The nuclear localization, interaction with -catenin, and ventralizing activity are summarized. N.D., not determined. C, Northern blot analyses of Duplin in rat tissues (20 µg of total RNA each) (upper panel), ethidium bromide-stained gel showing that the same amounts of ribosomal RNA for each lane are loaded (middle panel), and Western blot analyses of Duplin in cultured cells (50 µg of protein each) (lower panel). D, subcellular localization of Duplin. Aliquots (20 µl each) of the total homogenate (T), cytoplasmic (C), membrane (M), and nuclear (N) fractions of COS cells expressing Myc-Duplin were probed with the anti-Myc antibody. E, nuclear localization of Duplin. L cells and SW480 cells were observed under the microscopy using Nomarski optics and stained with the anti-Duplin antibody. L cells expressing HA-Duplin or its mutants were stained with the anti-HA antibody.

Interaction of Duplin with -Catenin-- 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. 2A, 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 -catenin was precipitated with Myc-Duplin when Myc-Duplin was expressed in COS cells (Fig. 2A, lanes 1-4). Myc-Duplin did not form a complex with GSK-3, HA-rAxin, or HA-hTcf-4 (Fig. 2A, lanes 1-12). To examine whether the interaction of Duplin with -catenin is direct, MBP-Duplin-(482-749) or MBP was incubated with GST--catenin. GST--catenin was precipitated with MBP-Duplin-(482-749) but not with MBP (Fig. 2B, lanes 1-4). Regarding residues of 482-749 of Duplin, GST-Duplin-(667-749) but not GST-Duplin-(482-668) bound to MBP--catenin (Fig. 2B, lanes 5-10). To determine which region of -catenin binds to Duplin, various deletion mutants of GST--catenin were incubated with MBP-Duplin-(482-749). MBP-Duplin-(482-749) bound strongly to GST--catenin-(1-423) and GST--catenin-(132-423) and weakly to GST--catenin-(423-781), but not to GST--catenin-(1-131) or GST (Fig. 2C). Since -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 -catenin. Tcf-4 is a nuclear protein that binds to -catenin, and hTcf-4-(1-80) interacts directly with the Armadillo repeats of -catenin (7, 8). GST-Duplin-(482-749) competed with GST-hTcf-4-(1-80) for the binding to MBP--catenin in a dose-dependent manner (Fig. 2D). These results indicate that Duplin binds directly to -catenin, resulting in inhibition of the binding of -catenin to Tcf-4.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Interaction of -catenin with Duplin. A, interaction of -catenin with Duplin in intact cells. COS cells were transfected with expression vectors as indicated. Cell lysates (20 µg of protein) were probed with the anti-Myc, anti--catenin, anti-Dvl, anti-GSK-3, and anti-HA antibodies to show the protein expression levels (lanes 1, 2, 5, 6, 9, and 10). The lysates (150 µg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc, anti--catenin, anti-Dvl, anti-GSK-3, and anti-HA antibodies (lanes 3, 4, 7, 8, 11, and 12). B, direct interaction of Duplin with -catenin. GST--catenin (0.5 µM) was incubated with MBP-Duplin-(482-749) (lane 3) or MBP (lane 4) (30 pmol) immobilized on amylose resin. GST-Duplin-(482-668) (lane 8), GST-Duplin-(667-749) (lane 9), and GST (lane 10) (0.5 µM) were incubated with MBP--catenin (30 pmol) immobilized on amylose resin. MBP fusion proteins were precipitated by centrifugation and the precipitates were probed with the anti-GST antibody. C, the region of -catenin that binds to Duplin. GST--catenin and its deletion mutants (0.5 µM) were incubated with MBP-Duplin-(482-749) (30 pmol) immobilized on amylose resin. MBP-Duplin-(482-749) was precipitated by centrifugation, and the precipitates were probed with the anti-GST antibody. D, inhibition by Duplin of the binding of -catenin to Tcf-4. The indicated concentrations of GST-Duplin-(482-749) or GST and GST-hTcf-4-(1-80) (0.5 µM) were incubated with MBP--catenin (30 pmol) immobilized on amylose resin. MBP--catenin was precipitated by centrifugation, and the precipitates were probed with the anti-GST antibody. The results shown are representative of three independent experiments.

Inhibition of Wnt-3a-dependent Activation of Tcf by Duplin-- We showed previously that Wnt-3a-conditioned medium induces the accumulation of -catenin and activates Tcf-4 in L cells and that expression of Axin inhibits these Wnt-3a-dependent responses (25). To examine the effect of Duplin on the Wnt-3a-dependent responses, we established L cells stably expressing HA-Duplin (L/Du cells). Wnt-3a increased the levels of -catenin in L/Du cells as well as in wild-type L cells (Fig. 3A, lanes 1-4). Wnt-3a increased the levels of -catenin in the cytoplasm, membrane, and nuclear fractions, and expression of Duplin did not affect the subcellular distribution of -catenin (Fig. 3A, lanes 5-12). Further, expression of Duplin did not decrease the levels of -catenin in SW480 cells (data not shown). Since Duplin inhibited the binding of -catenin to Tcf-4 in vitro, we examined this inhibitory action of Duplin in intact cells. HA-Tcf-4 was expressed in wild-type L cells and L/Du cells, and these cells were treated with Wnt-3a (Fig. 3B, lanes 1 and 2). HA-Tcf-4 immunoprecipitated with -catenin in L/Du cells was less than that in wild-type L cells (Fig. 3B, lanes 3 and 4). Consistent with these observations, Wnt-3a-dependent Tcf-4 activation was inhibited in L/Du cells (Fig. 3C). Essentially the same results were obtained from three independent clones of L/Du cells. Expression of -catenin^SA, in which the serine and threonine residues of the GSK-3 phosphorylation sites (21) are changed to alanine, activated Tcf-4 in L cells (Fig. 3D, lanes 1 and 2). Co-expression with Duplin inhibited -catenin^SA-dependent Tcf-4 activation in a dose-dependent manner (Fig. 3D, lanes 3-6). However, Duplin did not inhibit Ras-dependent c-fos promoter activation (Fig. 3E). Taken together, these results demonstrate that Duplin does not affect -catenin stability, but does inhibit -catenin-dependent Tcf-4 activation, probably due to the inhibition of the binding of -catenin to Tcf-4.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of Duplin on the -catenin-Tcf signaling. A, effect of Duplin on -catenin accumulation. The total homogenates (lanes 1-4) or subcellular fractions (lanes 5-12) of wild-type L cells (WT) or L cells stably expressing HA-Duplin (L/Du) treated with Wnt-3a-conditioned medium (+) or control medium () were probed with the anti--catenin antibody. T, total homogenate; C, cytoplasm; M, membrane; N, nucleus. B, effect of Duplin on the binding of -catenin to Tcf in L cells. Wild-type L cells (lanes 1 and 3) or L/Du cells (lanes 2 and 4) which were transfected with pEF-BOS-HA/hTcf-4 and treated with Wnt-3a. The lysates were probed with the anti-HA and anti--catenin antibodies (lanes 1 and 2) or immunoprecipitated with the anti--catenin antibody (lanes 3 and 4). The immunoprecipitates were probed with the anti-HA and anti--catenin antibodies. C, effect of Duplin on Tcf-4 activation. After wild-type L cells or L/Du cells were transfected with pcDNAI/hTcf-4 and pTOPFLASH, the cells were treated with or without Wnt-3a. Data are expressed as -fold stimulation of the luciferase activity of wild-type L cells in the absence of Wnt-3a and represent the mean ± S.E. of four experiments (* denotes p < 0.05 versus wild-type cells treated with control medium; t test). D, effect of Duplin on -catenin-dependent Tcf-4 activation. Wild-type L cells were transfected with pcDNAI/hTcf-4, pTOPFLASH, pUC/EF-1/-cateninSA, and pBJ-Myc/Duplin as indicated. Data are expressed as -fold stimulation of the luciferase activity of SW480 cells without expression of -cateninSA and represent the mean ± S.E. of four independent experiments. E, effect of Duplin on Ras-dependent fos promoter activation. Wild-type L cells were transfected with pBJ/RasG12V, pfos-luc, and pBJ-Myc/Duplin. Data are expressed as -fold stimulation of the luciferase activity of wild-type L cells without expression of RasG12V and represent the mean ± S.E. of four independent experiments.

Regulation of Axis Formation by Duplin-- To confirm the mode of action of Duplin, we examined the effects of Duplin on the Wnt signaling pathway using Xenopus embryos. The Wnt signaling pathway regulates axis formation of Xenopus embryos (43). Dorsal injection of Duplin mRNA into four-cell stage embryos resulted in ventralizing phenotypes such as loss of head (Fig. 4A, a). Embryos injected ventrally with Duplin mRNA developed normally (Fig. 4A, b). When mRNA of Duplin-(482-749) was injected dorsally, the embryos showed ventralizing phenotypes (Fig. 4A, d), while injection of Duplin-(1-482) mRNA had no effect (Fig. 4A, c). In residues 482-749, Duplin-(667-749) showed ventralizing activity (Fig. 1B). Since Duplin-(667-749) binds to -catenin, one explanation for this result might be that the binding of Duplin to -catenin interferes with the translocation of -catenin from cytosol to nucleus. The DAIs of the embryos expressing Duplin-(667-749), Duplin-(482-749), and Duplin (full-length) were 3.44 (n = 59), 2.84 (n = 140), and 2.02 (n = 144), respectively, indicating that the ventralizing activity of Duplin (full-length) or Duplin-(482-749) is more potent than that of Duplin- (). These results suggest that, in addition to the binding to -catenin, nuclear localization is important for the activity of Duplin to regulate axis formation. siamois is a homeobox gene, which mediates the effects of the Wnt signaling pathway on axis formation and whose expression is induced by -catenin and Tcf (44, 45). Expression of siamois was suppressed by dorsal injection of Duplin in a dose-dependent manner but not by ventral injection (Fig. 4B, lanes 1-5). Dorsal injection of Duplin-(482-749) but not that of Duplin-(1-482) inhibited expression of siamois (Fig. 4B, lanes 6 and 7). Therefore, Duplin has ventralizing activity and the C-terminal region may be essential to regulate embryonic axis formation, consistent with the observations that the C-terminal region of Duplin has the nuclear localization signal and -catenin-binding site.

View larger version (60K): [in this window] [in a new window]   Fig. 4.   Effect of Duplin on axis formation of Xenopus embryos. A, ventralizing activity of Duplin. Embryos were injected dorsally with Duplin (1 ng) (a), ventrally with Duplin (1 ng) (b), dorsally with Duplin-(1-482) (1 ng) (c), or dorsally with Duplin-(482-749) (1 ng) (d). B, inhibition of siamois expression. Expression of siamois in injected embryos was detected with RT-PCR analysis. Dorsal injection with Xglobin (lane 1, 1 ng) and Duplin (lane 2, 100 pg; lane 3, 500 pg; lane 4, 1 ng). Ventral injection with Duplin (lane 5, 1 ng). Dorsal injection with Duplin-(1-482) and Duplin-(482-749) (lanes 6 and 7, 1 ng). The amounts of cDNA were standardized with EF-1. Sia, siamois; RT , experiments without RT-PCR. C, inhibition by Duplin of Wnt signal-dependent axis formation. Embryos were injected dorsally (a, c, e, g) or ventrally (b, d, f, h) with Xwnt-8 (100 pg) and Xglobin (1 ng) (a and b), Xwnt-8 (100 pg) and Duplin (1 ng) (c and d), X-catenin (500 pg) and Xglobin (1 ng) (e and f), or X-catenin (500 pg) and Duplin (1 ng) (g and h). D, left panel indicates the results of average DAI of A. DAI 0, completely ventralized; DAI 5, normal. The average DAI used is not an accurate concept but is used for illustrative purposes. Right panel shows the frequency of the secondary axis in C. Black and white columns indicate the complete and incomplete axes, respectively. E, inhibition by Duplin of -catenin-dependent rescue of the axis formation. Injections were done into UV-treated embryos with Xglobin (2 ng) (a), Xglobin (1 ng) and Duplin (1 ng) (b), X-catenin (500 pg) and Xglobin (1 ng) (c), or X-catenin (500 pg) and Duplin (1 ng) (d). F, inhibition by Duplin of -catenin-induced expression of siamois. Embryos without UV light irradiation (lane 1). UV light-irradiated embryos injected with Xglobin (2 ng) (lane 2), Xglobin (1 ng) and Duplin (1 ng) (lane 3), X-catenin (500 pg) and Xglobin (1 ng) (lane 4), or X-catenin (500 pg) and Duplin (1 ng) (lane 5). G, the results in E were expressed with DAI. H, expression of Duplin in Xenopus development. Aliquots of the extracts from the indicated stages of embryos were probed with the anti-Duplin antibody.

We also examined the effects of Duplin on the axis formation induced by the Wnt signal. It has been shown that ventral injection of Xenopus wnt-8 (Xwnt-8) mRNA induces a secondary dorsal axis but that its dorsal injection does not affect the axis formation (43, 46) (Fig. 4C, a and b). Co-injection of Xwnt-8 and Duplin in the dorsal side caused a partial defect of the head structure (Fig. 4C, c). Anomalous trunk-tail structure and repression of the second axis formation were observed in embryos with co-injection of Xwnt-8 and Duplin in the ventral side (Fig. 4C, d). Ventral but not dorsal injection of Xenopus -catenin (X-catenin) mRNA has been also shown to induce a secondary dorsal axis (43, 47) (Fig. 4C, e and f). Co-injection of X-catenin and Duplin in the dorsal side prevented Duplin-induced loss of head structure (Fig. 4C, g), and embryos co-injected in the ventral side demonstrated no secondary structure (Fig. 4C, h). The effects of Duplin on axis formation were summarized in Fig. 4D. It has been shown that UV light-irradiated embryos exhibit axial deficiencies (Fig. 4E, a) (38). Duplin did not affect the phenotypes (Fig. 4E, b). X-catenin rescued axial deficiencies (Fig. 4E, c), and co-injection with Duplin prevented X-catenin-dependent rescue of the axis formation (Fig. 4E, d). -Catenin recovered the expression level of siamois, which was inhibited by UV light irradiation, and Duplin inhibited the -catenin-induced expression of siamois (Fig. 4F). Average DAIs of the embryos in Fig. 4E were shown in Fig. 4G. The antibody against rat Duplin recognized a protein with a molecular mass of about 110 kDa in mid-gastrula embryos, suggesting that this antibody cross-reacts with Xenopus Duplin (Fig. 4H). Further, this antibody recognized another protein whose molecular weight is the same as that of the upper band observed in rat brain (p140). This protein was expressed through early to late developmental stages. Taken together, these results suggest that Duplin negatively regulates the Wnt signaling pathway in Xenopus development downstream of B-catenin, consistent with the results observed in mammalian cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of the study demonstrate that Duplin acts as a negative regulator of the Wnt signaling pathway by binding to -catenin. Duplin was originally identified as a binding protein of Dvl by yeast two-hybrid screening. The reason why Duplin does not form a complex with Dvl in intact cells may be due to their different subcellular distributions. It is necessary to investigate the possibility that Duplin and Dvl are translocated between the cytoplasm and nucleus in response to extracellular signal, and thereby they interact with each other. Alternatively, there may be proteins that inhibit the binding of Dvl to Duplin in intact cells. We find that Duplin is a nuclear protein. The C-terminal region of Duplin contains five basic amino acid cluster regions, which are known to be a nuclear localization signal (48). Indeed, the region of Duplin including basic amino acid cluster regions is sufficient for its nuclear localization. One of the sequences of KKRRKK⁵⁰⁵, KPKK⁵¹⁸, KKRKR⁵⁴⁶, KRR⁵⁷⁵, and KRKK⁵⁸⁴ may be critical for the nuclear localization of Duplin.

In response to Wnt, cytoplasmic -catenin is stabilized and accumulated -catenin is translocated into the nucleus and binds to Tcf (2-4). Although Duplin does not affect the stability and subcellular localization of -catenin, it inhibits the binding of -catenin to Tcf. It is thought that Tcf may be a transcriptional repressor rather than an activator, because Tcf binds to proteins that can mediate repression. One such repressor is Groucho in Drosophila (49). The binding sites for Armadillo and Groucho on Tcf do not overlap, but whether or not Armadillo and Groucho bind simultaneously to Tcf is not clear. It is possible that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho. Another Tcf-binding protein is a Xenopus member of the C-terminal binding protein family of transcriptional co-repressors (XCtBP), which is homologous to the transcriptional co-repressor CtBP (50). XCtBP binds to the C-terminal region of Xenopus Tcf-3 and represses its transcriptional activity (50). The other Tcf-binding protein is Drosophila cAMP response element-binding protein-binding protein (dCBP) (51). dCBP interacts with the high-mobility group domain of Tcf and acetylates a conserved lysine in the Armadillo-binding domain of Tcf. This acetylation lowers the affinity of Tcf for Armadillo. Interestingly, mammalian CBP and its related protein p300 (CBP/p300) synergize with -catenin to stimulate gene expression, and Xenopus CBP regulates the axis formation positively (52, 53). The reasons of the apparent discrepancy between the function of vertebrate CBP/p300 and dCBP are not known. It is unlikely that Duplin competes with CBP/p300 for the binding to -catenin because CBP/p300 interacts with the region containing amino acids 630-781 of -catenin, which is different from the Duplin-binding site (52, 53). Furthermore, it has been shown that NEMO-like kinase binds directly to and phosphorylates Tcf and that the phosphorylation of Tcf inhibits the binding of the -catenin/Tcf complex to DNA (54). These Tcf-binding proteins appear to suppress the complex formation of -catenin, Tcf, and DNA. Pontin52 is a nuclear protein that binds to -catenin, but the physiological significance is not known (33). XSox17 is a Xenopus high mobility group box-containing protein and binds to -catenin (55). XSox17 activates transcription of endodermal genes and represses -catenin-stimulated expression of dorsal genes. Thus, it is likely that -catenin signaling is inhibited by several mechanism at the level of Tcf and -catenin in the nucleus.

Our studies demonstrate that Duplin interacts directly with -catenin, thereby inhibiting the binding of -catenin to Tcf-4. Consistent with these characteristics, Duplin inhibits Wnt-3a- and -catenin-dependent Tcf-4 activation in L cells. These findings are confirmed by the studies using Xenopus embryos. Duplin induces ventralization and inhibits expression of siamois, which is known to be a Wnt-responsive gene (44, 45). Duplin prevents Xwnt-8 and X-catenin from inducing axis duplication. Furthermore, it inhibits X-catenin-dependent rescue of axial deficiencies induced by UV light irradiation. These results suggest that Duplin negatively regulates the Wnt signaling pathway downstream of -catenin in Xenopus embryos. Based on these overexpression assays of Duplin, we propose that Duplin forms a complex with -catenin in the nucleus and suppresses the -catenin-dependent Tcf activation in the absence of Wnt. It is possible that accumulated -catenin in response to Wnt is able to bind to Tcf, although the -catenin/Duplin complex does not bind to Tcf; thereby, the function of Tcf to repress expression of target genes is overcome, leading to their expression. Therefore, Duplin may allow Tcf to function as a transcriptional repressor unless the transcriptional co-activator -catenin is accumulated. It is likely that Duplin inhibits the -catenin signaling in a manner different from Groucho, XCtBP, dCBP, and NEMO-like kinase. Thus, there are multiple mechanisms for inhibiting the -catenin signaling. In the cytoplasm, the amount of -catenin is negatively regulated by degrading -catenin in the Axin complex (9, 10, 14, 17, 23, 25). In the nucleus, several proteins negatively regulate the -catenin-dependent gene expression by interfering with the complex formation of -catenin, Tcf, and DNA. Mutations in -catenin have been found in various human cancers, including colon cancer and melanoma, and the mutations result in the accumulation of -catenin (56, 57). Since -catenin functions as an oncogene, it is speculated that there might be several mechanisms for protecting against abnormal cellular proliferation by inhibiting -catenin signaling.

Duplin is expressed strongly in mid-gastrula stage, suggesting that it suppresses the expression of Wnt-responsive genes in appropriate places and times during development. p140 is recognized with the anti-Duplin antibody in Xenopus embryos and PC12 cells. The functional differences between Duplin and p140 remain to be clarified. Further studies are necessary for understanding the whole picture of the roles of Duplin in the Wnt signaling pathway.

	ACKNOWLEDGEMENTS

We are grateful to Drs. H. Clevers, A. Nagafuchi, B. Dallapiccola, Q. Hu, and M. Nakata for donating plasmids and antibodies and to Drs. K. Matsumoto and T. Akiyama for helpful discussion. We thank the Research Center for Molecular Medicine and Research Facilities for Laboratory Animal Sciences, Hiroshima University School of Medicine, for the use of their facilities.

	FOOTNOTES

^* This work was supported by grants-in-aid for scientific research (B) and for scientific research on priority areas (A) from the Ministry of Education, Science, and Culture, Japan (1998, 1999), and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (1998, 1999) and Uehara Memorial Foundation (1998).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF169825.

^§§ To whom all correspondence should be addressed. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi@mcai.med.hiroshima-u.ac.jp.

Published, JBC Papers in Press, July 31, 2000, DOI 10.1074/jbc.M004089200

	ABBREVIATIONS

The abbreviations used are: GSK-3, glycogen synthase kinase-3; Tcf/Lef, T cell factor/lymphocyte enhancer binding factor; APC, adenomatous polyposis coli protein; PP2A, protein phosphatase 2A; HA, hemagglutinin 1; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; DTT, dithiothreitol; DAI, dorso-anterior index; RT, reverse transcription; PCR, polymerase chain reaction; CBP, cAMP response element-binding protein-binding protein; dCBP, Drosophila cAMP response element-binding protein-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES