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• W2047897241 abstract "Duplin binds to β-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the β-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin α was isolated as a binding protein of Duplin. Importin α bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (DuplinΔ500–584) retained Duplin in the cytoplasm. DuplinΔ500–584 bound to β-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a β-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed β-catenin-dependent axis duplication and expression ofsiamois, a Wnt-regulated gene, but also inhibitedsiamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin α, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream β-catenin target genes. Duplin binds to β-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the β-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin α was isolated as a binding protein of Duplin. Importin α bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (DuplinΔ500–584) retained Duplin in the cytoplasm. DuplinΔ500–584 bound to β-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a β-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed β-catenin-dependent axis duplication and expression ofsiamois, a Wnt-regulated gene, but also inhibitedsiamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin α, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream β-catenin target genes. Wnt proteins constitute a large family of cysteine-rich secreted ligands that control development in organisms ranging from nematode worms to mammals (1Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1736) Google Scholar). The intracellular signaling pathway of Wnt is also conserved evolutionally and regulates cellular proliferation, morphology, motility, and fate, axis formation, and organ development (1Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1736) Google Scholar, 2Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (438) Google Scholar, 3Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (604) Google Scholar, 4Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1307) Google Scholar, 5Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 6Seidensticker M.J. Behrens J. Biochim. Biophys. Acta. 2000; 1495: 168-182Crossref PubMed Scopus (238) Google Scholar). In the current model, the serine/threonine kinase GSK-3β 1GSK-3βglycogen synthase kinase-3βAPCadenomatous polyposis coli proteinTcfT cell factorCBPcAMP response-binding protein-binding proteinCtBPC-terminal-binding proteinXwnt-8Xenopus wnt-8MBPmaltose-binding proteinGSTglutathione S-transferaseHAhemagglutininGFPgreen fluorescent proteinNLSnuclear localization signalSV40simian virus 40PBSphosphate-buffered salinePIASprotein inhibitor of activated STATSTATsignal transducers and activators of transcription targets cytoplasmic β-catenin for degradation in the absence of Wnt (7Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar, 8Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). As a result, cytoplasmic β-catenin levels are low. Axin has been shown to be important for the degradation of β-catenin (9Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (161) Google Scholar). It forms a complex with GSK-3β, β-catenin, APC, and protein phosphatase 2A (8Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar, 9Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (161) Google Scholar, 10Yamamoto 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, 11Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 12Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar, 13Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 14Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar, 15Hsu W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 16Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (164) Google Scholar, 17Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and regulates GSK-3β-dependent phosphorylation of β-catenin, Axin, and APC (8Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar, 9Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (161) Google Scholar, 10Yamamoto 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, 13Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 16Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (164) Google Scholar, 18Yamamoto 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, 19Hinoi T. Yamamoto H. Kishida M. Takada S. Kishida S. Kikuchi A. J. Biol. Chem. 2000; 275: 34399-34406Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Phosphorylated β-catenin forms a complex with Fbw1, a member of the F-box protein family, resulting in the degradation of β-catenin by the ubiquitin and proteasome pathways (20Kitagawa M. Hatakeyama S. Shirane M. Matsumoto M. Ishida N. Hattori K. Nakamichi I. Kikuchi A. Nakayama K.-I. Nakayama K. EMBO J. 1999; 18: 2401-2410Crossref PubMed Scopus (481) Google Scholar, 21Hart M. Concordet J.-P. Lassot I. Albert I. de los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Benarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). Indeed, expression of Axin decreases the protein level of β-catenin (22Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar). Thus, Axin is a negative regulator of the Wnt signaling pathway that keeps cytoplasmic β-catenin level low. glycogen synthase kinase-3β adenomatous polyposis coli protein T cell factor cAMP response-binding protein-binding protein C-terminal-binding protein Xenopus wnt-8 maltose-binding protein glutathione S-transferase hemagglutinin green fluorescent protein nuclear localization signal simian virus 40 phosphate-buffered saline protein inhibitor of activated STAT signal transducers and activators of transcription When Wnt acts on its cell-surface receptor Frizzled, the cytoplasmic protein Dvl antagonizes the action of GSK-3β. Although this mechanism has not yet been clarified, it has been suggested that Frat, which was identified as a GSK-3-binding protein (23Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), forms a complex with Dvl, and that this complex induces the dissociation of GSK-3β from Axin (24Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (358) Google Scholar, 25Itoh K. Antipova A. Ratcliffe M.J. Sokol S. Mol. Cell. Biol. 2000; 20: 2228-2238Crossref PubMed Scopus (84) Google Scholar). It has been also demonstrated that casein kinase I forms a complex with Dvl and that it enhances the action of Dvl (26Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (384) Google Scholar, 27Kishida M. Hino S. Michiue T. Yamamoto H. Kishida S. Fukui A. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 33147-33155Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Furthermore, it has been shown that Dvl inhibits GSK-3β-dependent phosphorylation of β-catenin, APC, and Axin in vitro and the phosphorylation of Axin in intact cells (28Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar, 29Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Once the phosphorylation of β-catenin is reduced, it dissociates from the Axin complex, and β-catenin is no longer degraded, resulting in its accumulation in the cytoplasm. Accumulated β-catenin is translocated to the nucleus where it binds to Tcf/lymphocyte enhancer binding factor, transcription factors, and stimulates the expression of genes including c-myc, fra-1,c-jun, cyclin D1, and peroxisome proliferator-activated receptor δ (4Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1307) Google Scholar, 5Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 6Seidensticker M.J. Behrens J. Biochim. Biophys. Acta. 2000; 1495: 168-182Crossref PubMed Scopus (238) Google Scholar). Thus, the Wnt signal stabilizes β-catenin, thereby regulating the expression of various genes. In addition to the regulation of its stability in the cytoplasm, β-catenin signaling through Tcf is also regulated in the nucleus. 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 (30Cavallo R.A. Cox R.T. Moline M.M. Roose J. Polevoy G.A. Clevers H. Peifer M. Bejsovec A. Nature. 1998; 395: 604-608Crossref PubMed Scopus (602) Google Scholar). The binding sites for Armadillo (Drosophila β-catenin) 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 Drosophila CBP (31Waltzer L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (326) Google Scholar).Drosophila CBP 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 β-catenin to stimulate gene expression, andXenopus CBP positively regulates the axis formation (32Hecht A. Vleminckx K. Stemmler M.P. van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar,33Takemaru K.-I. Moon R.T. J. Cell Biol. 2000; 149: 249-254Crossref PubMed Scopus (405) Google Scholar). The reasons for the apparent discrepancy between the function of vertebrate CBP/p300 and Drosophila CBP are not known. The other Tcf-binding protein is Xenopus CtBP family that is homologous to the transcriptional co-repressor human CtBP (34Brannon M. Brown J.D. Bates R. Kimelman D. Moon R.T. Development. 1999; 126: 3159-3170Crossref PubMed Google Scholar).Xenopus CtBP binds to the C-terminal region ofXenopus β-catenin-Tcf-3 and represses its transcriptional activity (34Brannon M. Brown J.D. Bates R. Kimelman D. Moon R.T. Development. 1999; 126: 3159-3170Crossref PubMed Google Scholar). Furthermore, NEMO-like kinase binds directly to and phosphorylates Tcf, which then inhibits the binding of the β-catenin-Tcf complex to DNA (35Ishitani T. Ninomiya-Tsuji J. Nagai S.-I. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (516) Google Scholar). These Tcf-binding proteins appear to regulate complex formation among β-catenin, Tcf, and DNA. It has been reported that there are several proteins that bind to β-catenin and inhibit its function. Pontin52 is a nuclear protein that binds to β-catenin and the TATA-box binding protein (36Bauer A. Huber O. Kemler R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14787-14792Crossref PubMed Scopus (170) Google Scholar). Recently it has been shown that Pontin52 and Reptin52, a newly identified Pontin52 homologue, antagonistically influence the transactivation potential of the β-catenin-Tcf complex, and that they are essential genes that act antagonistically in the control of Wingless signaling in Drosophila. These results indicate that the opposing actions of Pontin52 and Reptin52 on β-catenin-mediated transactivation constitute an additional mechanism for the control of the canonical Wingless/Wnt pathway (37Bauer A. Chauvet S. Huber O. Usseglio F. Rothbächer U. Aragnol D. Kemler R. Pradel J. EMBO J. 2000; 19: 6121-6130Crossref PubMed Google Scholar). XSox17 is aXenopus high-mobility group box containing protein that activates transcription of endodermal gene and represses β-catenin-stimulated expression of dorsal genes (38Zorn A.M. Barish G.D. Williams B.O. Lavender P. Klymkowsky M.W. Varmus H.E. Mol. Cell. 1999; 4: 487-498Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). ICAT also binds to β-catenin and represses β-catenin-mediated transactivation, thereby inhibiting Xenopus axis formation (39Tago K.-I. Nakamura T. Nishita M. Hyodo J. Nagai S.-I. Murata Y. Adachi S. Ohwada S. Morishita Y. Shibuya H. Akiyama T. Genes Dev. 2000; 14: 1741-1749PubMed Google Scholar). Thus, it is likely that β-catenin signaling through Tcf is inhibited by several mechanisms at the level of Tcf and β-catenin in the nucleus. We have recently identified a novel protein that binds to β-catenin in the nucleus and designated it Duplin (40Sakamoto I. Kishida S. Fukui A. Kishida M. Yamamoto H. Hino S.-I. Michiue T. Takada S. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 32871-32878Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Duplin inhibits the binding of β-catenin to Tcf-4, thereby inhibiting Wnt-3a- and β-catenin-dependent Tcf-4 activation in mammalian cells. Duplin inhibits expression of siamois, a Wnt-responsive gene (41Lemaire P. Garrett N. Gurdon J.B. Cell. 1995; 81: 85-94Abstract Full Text PDF PubMed Scopus (468) Google Scholar, 42Brannon M. Gomperts M. Sumoy L. Moon R.T. Kimelman D. Genes Dev. 1997; 11: 2359-2370Crossref PubMed Scopus (466) Google Scholar), and suppresses Xwnt-8 and Xβ-catenin-induced axis duplication in Xenopus embryos. Based on these functions of Duplin, we proposed that Duplin forms a complex with β-catenin in the nucleus and represses the β-catenin-dependent Tcf activation. Although it is likely that Duplin inhibits the β-catenin signaling in a manner different from Groucho, Xenopus CtBP,Drosophila CBP, NEMO-like kinase, and ICAT, the mechanisms by which the nuclear localization and action of Duplin are regulated are still unclear. To clarify these mechanisms of Duplin, we identified a Duplin-binding protein by the yeast two-hybrid screening. Here we show that Duplin binds to importin α directly through its basic amino acid clusters. We also demonstrate that nuclear localization of Duplin is necessary for its inhibition of Wnt-dependent activation of Tcf in mammalian cells and ventralization in Xenopusembryos. Moreover, we show that Duplin also functions to inhibit the Wnt signaling pathway downstream of β-catenin target genes. MBP and GST fusion proteins were purified from Escherichia coli according to the manufacturer's instructions. The anti-importin α-P, α-Q, and α-S antibodies were generated as described (43Kamei Y. Yuba S. Nakayama T. Yoneda Y. J. Histochem. Cytochem. 1999; 47: 363-372Crossref PubMed Scopus (86) Google Scholar). The anti-Myc antibody was prepared from 9E10 cells. The anti-MBP and GST antibodies were prepared in rabbit by immunization with recombinant MBP and GST, respectively. L cells (mouse fibroblasts) stably expressing HA-DuplinΔ500–584 and HA-Duplin-(1–668) were generated by selecting with G418 as described (22Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar, 44Okazaki 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 (45Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). The anti-β-catenin antibody was purchased from Transduction Laboratories (Lexington, KY). Other materials were from commercial sources. Standard recombinant DNA techniques were used to construct the following plasmids, pBTM116HA/Duplin-(482–749), pGEX-GFP/Duplin-(500–584), pGEX-GFP/Duplin-(500–565), pGEX-GFP/Duplin-(565–668), pGEX/Duplin-(500–521)- GFP, pGEX/Duplin-(542–546)-GFP, pMALc-2/importin α-P, pCGN/ DuplinΔ500−584, pCGN/Duplin-(1–668), pBJ-Myc/Duplin-(1–482), pBJ-Myc/Duplin-(482–749), pBJ-Myc/DuplinΔ500−584, pEF-BOS-Myc/ Duplin-(1–668), pSP-Myc/DuplinΔ500–584, pSP-Myc/Duplin-(1–668), pSP/GFP, and pSP/siamois. The structures of all plasmids were confirmed by restriction enzyme analysis and, in most cases, by DNA sequence analysis across crucial regions. pCGN/Duplin, pBJ-Myc/ Duplin, pEF-BOS-HA/hTcf-4E, pSP-Myc/Duplin, pGEX-GFP, and pGEX/SV40NLS-GFP were constructed as described (19Hinoi T. Yamamoto H. Kishida M. Takada S. Kishida S. Kikuchi A. J. Biol. Chem. 2000; 275: 34399-34406Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 40Sakamoto I. Kishida S. Fukui A. Kishida M. Yamamoto H. Hino S.-I. Michiue T. Takada S. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 32871-32878Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 46Hieda M. Tachibana T. Fukumoto M. Yoneda Y. J. Biol. Chem. 2001; 276: 16824-16832Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To determine whether Duplin interacts with importin α in intact cells, COS cells (6-cm diameter dish) transfected with pBJ-derived plasmids were lysed in 250 μl of lysis buffer (20 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1 mm dithiothreitol, 1% Nonidet P-40, 20 μg/ml leupeptin, 20 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride) and the lysates were centrifuged at 20,000 × g for 15 min at 4 °C. The supernatant (200 μg of protein) was immunoprecipitated with the anti-Myc antibody, then the precipitates were probed with the anti-Myc and anti-importin α antibodies. To examine the interaction of Duplin with importin α using the purified proteins in vitro, GST-GFP-Duplin deletion mutants (1 μm) were incubated with MBP-importin α-P (30 pmol) immobilized on amylose resin in 100 μl of reaction mixture (20 mm Tris/HCl, pH 7.5, and 1 mm dithiothreitol) for 1 h at 4 °C. MBP-importin α-P was precipitated by centrifugation, then the precipitates were probed with the anti-GST antibody. To determine whether various Duplin mutants interact with β-catenin in intact cells, the assays were carried out as described (40Sakamoto I. Kishida S. Fukui A. Kishida M. Yamamoto H. Hino S.-I. Michiue T. Takada S. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 32871-32878Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). L cells grown on coverslips were microinjected with GST-GFP-Duplin (0.5 mg/ml) using micromanipulator 5171® and transjector 5246® (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany). At 30 min post-microinjection, the cells were fixed for 20 min in PBS containing 4% paraformaldehyde. The coverslips were washed with PBS, mounted on glass slides, and viewed by a confocal laser-scanning microscopy (Zeiss LSM510, Jena, Germany). When the cells were transfected with pBJ/Myc-Duplin, they were fixed after 48 h, washed with PBS three times, and then permeabilized with PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum albumin for 1 h. The cells were washed and incubated for 1 h with the anti-Myc antibody. After washing with PBS, they were further incubated for 1 h with Alexa 546-labeled anti-mouse IgG and viewed by a confocal laser-scanning microscopy. L cells expressing HA-Duplin, HA-DuplinΔ500–584, or HA-Duplin-(1–668) (35-mm diameter dish) were transfected with pTOPFLASH, pEF-BOS-HA/hTcf-4E, and pME18S/lacZ (22Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar, 47Korinek 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 (2935) Google Scholar, 48Hino S.-I. Kishida S. Michiue T. Fukui A. Sakamoto I. Takada S. Asashima M. Kikuchi A. Mol. Cell. Biol. 2001; 21: 330-342Crossref PubMed Scopus (107) Google Scholar). At 46 h after transfection, the cells were treated with Wnt-3a conditioned medium for 8 h. Then they 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 the β-galactosidase gene was used as an internal control. Sense mRNA was obtained by in vitro transcription of linearized templates using SP6-mMESSAGE mMACHINE kit (Ambion). Fertilized eggs were dejellied using 4.5% l-cysteine hydrochloride monohydrate, 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). Yeast two-hybrid screening was carried out as described (8Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar, 10Yamamoto 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). Protein concentrations were determined with bovine serum albumin as a standard (49Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar). Various constructs used in this study are shown in Fig.1. To identify a protein that is involved in the functions of Duplin, we screened a mouse brain cDNA library with the yeast two-hybrid method using the C-terminal half of Duplin as bait. Among 2.6 × 105 clones, four clones were found to confer both His+ and LacZ+ phenotypes, and one of them was importin α-Q2. Importin α recognizes NLSs and mediates the selective transport of karyophilic proteins to the nuclei (50Yoneda Y. Genes Cells. 2000; 5: 777-787Crossref PubMed Scopus (169) Google Scholar). Mouse importin α can be classified into three subfamilies, α-P, α-Q, and α-S families, which have ∼50% amino acid identity to one another (51Tsuji L. Takumi T. Imamoto N. Yoneda Y. FEBS Lett. 1997; 416: 30-34Crossref PubMed Scopus (119) Google Scholar). The α-Q family is composed of the closely related members α-Q1 and α-Q2, and they share more than 80% amino acid sequence identity. The α-S family also has two members, α-S1 and α-S2. To examine whether Duplin forms a complex with importin α in intact cells, Myc-Duplin was expressed in COS cells, and three different antibodies that detect distinct importin α subfamilies were used to probe endogenous importin α (Fig.2 A). When the lysates expressing Myc-Duplin were immunoprecipitated with the anti-Myc antibody, endogenous importin α-P was detected in the Myc-Duplin immune complex (Fig. 2 A). Importin α-P was not immunoprecipitated from the same lysates with non-immune immunoglobulin (data not shown). Endogenous importin α-Q and importin α-S were also detected in the Myc-Duplin immunoprecipitates (Fig.2 A). These results indicate that Duplin forms a complex with three subfamilies of importin α.Figure 2Complex formation of Duplin with importin α. A, interaction of Duplin with importin α in intact cells. The lysates (20 μg of protein) of COS cells with (lanes 2, 6, and10) or without (lanes 1, 5, and9) expression of Myc-Duplin were probed with the anti-Myc (lanes 1, 2, 5, 6,9, and 10) and anti-importin α-P (lanes 1 and 2), anti-importin α-Q (lanes 5 and6), or anti-importin α-S (lanes 9 and10) antibodies. The lysates (200 μg of protein) described above were immunoprecipitated with the anti-Myc antibody (lanes 3, 4, 7, 8, 11, and12) and the precipitates were probed with the anti-Myc (lanes 3, 4, 7, 8,11, and 12) and anti-importin α-P (lanes 3 and 4), anti-importin α-Q (lanes 7 and8), or anti-importin α-S (lanes 11 and12) antibodies. IP, immunoprecipitation;Ab, antibody. B, interaction of Duplin mutants with importin α in intact cells. The lysates (20 μg of protein) of COS cells (lane 1), COS cells expressing Myc-Duplin (full-length) (lane 2), Myc-Duplin-(1–482) (lane 3), Myc-Duplin-(482–749) (lane 4), or Myc-DuplinΔ500–584 (lane 5) were probed with the anti-Myc and anti-importin α-P antibodies. The lysates (200 μg of protein) prepared in lanes 1–5 were immunoprecipitated with the anti-Myc antibody and the precipitates were probed with the anti-Myc and anti-importin α-P antibodies (lanes 6–10).Ig, immunoglobulin. C, direct interaction of Duplin with importin α. Purified proteins (0.5 μg of protein) of GST-GFP and GST-GFP-Duplin-(500–584) were subjected to SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining (lanes 1 and 2). GST-GFP and GST-GFP-Duplin-(500–584) (1 μm each) were incubated with MBP-importin α-P (30 pmol) immobilized on amylose resin. MBP-importin α-P was precipitated by centrifugation and the precipitates were probed with the anti-GST antibody (lanes 3 and 4) (upper panel). 10% of MBP-importin α-P used in this assay is shown by the anti-MBP antibody (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine which region of Duplin forms a complex with importin α, various deletion mutants of Duplin were expressed in COS cells (Fig. 2 B). When the lysates expressing Myc-Duplin mutants were immunoprecipitated with the anti-Myc antibody, importin α-P was co-precipitated with Myc-Duplin (full-length) and Myc-Duplin-(482–749) but not with Myc-Duplin-(1–482) (Fig.2 B). These results are consistent with the previous observations that Duplin-(482–749) was present in the nucleus and that Duplin-(1–482) was localized in the cytoplasm (40Sakamoto I. Kishida S. Fukui A. Kishida M. Yamamoto H. Hino S.-I. Michiue T. Takada S. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 32871-32878Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The region containing amino acids 500–584 had basic amino acid clusters, KKRRKK505, KPKK518, KKRKR546, KRR575, and KRKK584 (40Sakamoto I. Kishida S. Fukui A. Kishida M. Yamamoto H. Hino S.-I. Michiue T. Takada S. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 32871-32878Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). One or two clusters of basic amino acids are classical NLSs recognized by importin α (52Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1711) Google Scholar). By deleting these basic amino acid clusters (DuplinΔ500–584), Duplin did not form a complex with importin α-P (Fig. 2 B), indicating that the basic amino acid clusters of Duplin are necessary for its interaction with importin α. To examine whether the interaction of Duplin with importin α is direct, GST-fused Duplin and MBP-fused importin α were purified fromE. coli. GST-GFP-Duplin-(500–584), but not GST-GFP, was co-precipitated with MBP-importin α-P, indicating that Duplin binds directly to importin α (Fig. 2 C). Duplin-(500–565) and Duplin-(565–668) contain three and two basic amino acid clusters, respectively. Both GST-GFP-Duplin-(500–5" @default.
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• W2047897241 date "2002-02-01" @default.
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• W2047897241 title "Nuclear Localization of Duplin, a β-Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway" @default.
• W2047897241 cites W1537657975 @default.
• W2047897241 cites W1780615788 @default.
• W2047897241 cites W1817917437 @default.
• W2047897241 cites W1868158025 @default.
• W2047897241 cites W1902879109 @default.
• W2047897241 cites W1968203528 @default.
• W2047897241 cites W1974160989 @default.
• W2047897241 cites W1975516052 @default.
• W2047897241 cites W1976411071 @default.
• W2047897241 cites W1993520606 @default.
• W2047897241 cites W1994511343 @default.
• W2047897241 cites W1994700168 @default.
• W2047897241 cites W1994922837 @default.
• W2047897241 cites W2001054344 @default.
• W2047897241 cites W2012039807 @default.
• W2047897241 cites W2013460988 @default.
• W2047897241 cites W2023002592 @default.
• W2047897241 cites W2026112588 @default.
• W2047897241 cites W2029857102 @default.
• W2047897241 cites W2036864436 @default.
• W2047897241 cites W2047538824 @default.
• W2047897241 cites W2048264979 @default.
• W2047897241 cites W2050414776 @default.
• W2047897241 cites W2051032088 @default.
• W2047897241 cites W2056833504 @default.
• W2047897241 cites W2057208748 @default.
• W2047897241 cites W2060577651 @default.
• W2047897241 cites W2064901831 @default.
• W2047897241 cites W2070687006 @default.
• W2047897241 cites W2075319861 @default.
• W2047897241 cites W2075460910 @default.
• W2047897241 cites W2079354667 @default.
• W2047897241 cites W2079990775 @default.
• W2047897241 cites W2080372560 @default.
• W2047897241 cites W2082135441 @default.
• W2047897241 cites W2084258581 @default.
• W2047897241 cites W2086725260 @default.
• W2047897241 cites W2088681495 @default.
• W2047897241 cites W2091232397 @default.
• W2047897241 cites W2097098623 @default.
• W2047897241 cites W2103345757 @default.
• W2047897241 cites W2106236683 @default.
• W2047897241 cites W2112022185 @default.
• W2047897241 cites W2114511731 @default.
• W2047897241 cites W2118344768 @default.
• W2047897241 cites W2126122106 @default.
• W2047897241 cites W2127520892 @default.
• W2047897241 cites W2132475598 @default.
• W2047897241 cites W2139392120 @default.
• W2047897241 cites W2146167376 @default.
• W2047897241 cites W2151558240 @default.
• W2047897241 cites W2152123530 @default.
• W2047897241 cites W2153942451 @default.
• W2047897241 cites W2158569397 @default.
• W2047897241 cites W2164692746 @default.
• W2047897241 cites W2165061154 @default.
• W2047897241 cites W2239090104 @default.
• W2047897241 cites W4293247451 @default.
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