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• W1991527612 abstract "Wnt signaling, via the activation of the canonical β-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with β-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/β-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/β-catenin signaling in both the cytoplasm and the nucleus. Wnt signaling, via the activation of the canonical β-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with β-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/β-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/β-catenin signaling in both the cytoplasm and the nucleus. The secreted growth factors of the Wnt family play key roles in early embryogenesis and tissue homeostasis in adults by modulating cell proliferation, differentiation, morphology, and migration. Appropriate control of their signaling activity is essential for normal physiological activity as dysregulation of their signaling is associated with various types of human diseases such as cancer (1Moon R.T. Kohn A.D. De Ferrari G.V. Kaykas A. Nat. Rev. Genet. 2004; 5: 691-701Crossref PubMed Scopus (1571) Google Scholar, 2Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4430) Google Scholar, 3Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4179) Google Scholar, 4Reya T. Clevers H. Nature. 2005; 434: 843-850Crossref PubMed Scopus (2988) Google Scholar, 5Nusse R. Cell Res. 2005; 15: 28-32Crossref PubMed Scopus (795) Google Scholar). In the canonical Wnt signaling pathway, the soluble form of the core factor β-catenin is mainly targeted for ubiquitination and proteasomal degradation, which is promoted by the destruction complex of Axin, adenomatous polyposis coli (APC) 2The abbreviations used are:APCadenomatous polyposis coliDvlDishevelledsiRNAsmall interfering RNAPBSphosphate-buffered salineChIPchromatin immunoprecipitationDprDapperGFPgreen fluorescence proteinHDAC1histone deacetylase 1LEFlymphoid enhancer factorTCFT-cell factorLMBleptomycin BNESnuclear export signalNLSnuclear localization signalRTreverse transcriptionJNKc-Jun N-terminal kinaseWTwild type 2The abbreviations used are:APCadenomatous polyposis coliDvlDishevelledsiRNAsmall interfering RNAPBSphosphate-buffered salineChIPchromatin immunoprecipitationDprDapperGFPgreen fluorescence proteinHDAC1histone deacetylase 1LEFlymphoid enhancer factorTCFT-cell factorLMBleptomycin BNESnuclear export signalNLSnuclear localization signalRTreverse transcriptionJNKc-Jun N-terminal kinaseWTwild type, and glycogen synthase kinase 3β (6Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2135) Google Scholar, 7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1093) Google Scholar, 8Kishida 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 (440) Google Scholar, 9Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1016) Google Scholar). The binding of Wnt to its cell surface receptors, Frizzled (Fz) and low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) leads to the recruitment of Dishevelled (Dvl) to Fz and Axin to LRP5/6, resulting in the disruption of the destruction complex and consequently the accumulation of β-catenin. The accumulated β-catenin moves into the nucleus and activates the transcription of target genes through interacting with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors (10Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar, 11Hsu S.C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (337) Google Scholar, 12Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1601) Google Scholar, 13Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar).Dapper (Dpr), which was first identified as an interacting protein of Dvl, a central mediator in Wnt signaling, controls Xenopus embryogenesis by functioning as a general antagonist of Dvl to modulate the β-catenin-dependent and JNK-dependent Wnt signaling (14Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 15Gloy J. Hikasa H. Sokol S.Y. Nat. Cell Biol. 2002; 4: 351-357Crossref PubMed Scopus (94) Google Scholar). Several Dpr orthologs, including Dpr1, Dpr2, and Dpr3, have been identified in zebrafish, mouse, and human (16Fisher D.A. Kivimae S. Hoshino J. Suriben R. Martin P.M. Baxter N. Cheyette B.N. Dev. Dyn. 2006; 235: 2620-2630Crossref PubMed Scopus (59) Google Scholar, 17Katoh M. Int. J. Oncol. 2003; 22: 907-913PubMed Google Scholar, 18Waxman J.S. Hocking A.M. Stoick C.L. Moon R.T. Development. 2004; 131: 5909-5921Crossref PubMed Scopus (73) Google Scholar, 19Zhang L. Zhou H. Su Y. Sun Z. Zhang H. Zhang Y. Ning Y. Chen Y.G. Meng A. Science. 2004; 306: 114-117Crossref PubMed Scopus (108) Google Scholar, 20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The function of Dpr has been shown to be evolutionally conserved from fish to human (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 21Su Y. Zhang L. Gao X. Meng F. Wen J. Zhou H. Meng A. Chen Y.G. FASEB J. 2007; 21: 682-690Crossref PubMed Scopus (53) Google Scholar). However, recent studies suggested that the Dpr family members may also positively regulate Wnt signaling through distinct mechanisms. Zebrafish Dpr1 and Dpr2 were reported to positively modulate the canonical β-catenin or noncanonical calcium-planar cell polarity pathways of Wnt signaling during fish embryo development, respectively (18Waxman J.S. Hocking A.M. Stoick C.L. Moon R.T. Development. 2004; 131: 5909-5921Crossref PubMed Scopus (73) Google Scholar). Frodo, a Dpr homolog that shares 90! identity at the amino acid level to Xenopus Dpr, has been suggested to positively synergize with Dvl to induce secondary axis and be required for normal eye and neural tissue development (15Gloy J. Hikasa H. Sokol S.Y. Nat. Cell Biol. 2002; 4: 351-357Crossref PubMed Scopus (94) Google Scholar). In addition, we have demonstrated that zebrafish and mouse Dpr2 specifically inhibited transforming growth factor β/Nodal signaling during mesoderm induction by promoting lysosomal degradation of its type I receptors (19Zhang L. Zhou H. Su Y. Sun Z. Zhang H. Zhang Y. Ning Y. Chen Y.G. Meng A. Science. 2004; 306: 114-117Crossref PubMed Scopus (108) Google Scholar, 21Su Y. Zhang L. Gao X. Meng F. Wen J. Zhou H. Meng A. Chen Y.G. FASEB J. 2007; 21: 682-690Crossref PubMed Scopus (53) Google Scholar). Dpr has been also implicated to be associated with tumorigenesis. Human Dpr1 was down-regulated in hepatocellular carcinoma, and this down-regulation was correlated with the cytoplasm accumulation of β-catenin (22Yau T.O. Chan C.Y. Chan K.L. Lee M.F. Wong C.M. Fan S.T. Ng I.O. Oncogene. 2005; 24: 1607-1614Crossref PubMed Scopus (79) Google Scholar).Our recent work demonstrated that human Dpr1 promotes Dvl2 degradation by a lysosome inhibitor-sensitive mechanism in the cytoplasm (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), whereas Frodo, in addition to interact with Dvl, has been shown to associate with other multiple proteins in both the cytoplasm and the nucleus; that is, TCF3 (23Hikasa H. Sokol S.Y. Development. 2004; 131: 4725-4734Crossref PubMed Scopus (61) Google Scholar), cell cycle and DNA replication-related protein Dbf4 (24Brott B.K. Sokol S.Y. Dev. Cell. 2005; 8: 703-715Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and the catenin protein family member p120-catenin (25Park J.I. Ji H. Jun S. Gu D. Hikasa H. Li L. Sokol S.Y. McCrea P.D. Dev. Cell. 2006; 11: 683-695Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The subcellular localization of Dpr proteins seems complex. Endogenous Dpr1 was showed to be found throughout the cytoplasm as punctate spots and diffusely in the nucleus in both cultured cells and Xenopus animal cap (14Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Although ectopic Dpr1 and Dpr2 were predominantly present in the cytoplasm, they were also visible in the nucleus of some cells (14Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 18Waxman J.S. Hocking A.M. Stoick C.L. Moon R.T. Development. 2004; 131: 5909-5921Crossref PubMed Scopus (73) Google Scholar). The observation that some of the C-terminal fragments of Dpr1 are localized almost exclusively in the nucleus further suggested that Dpr1 might have a role in the nucleus (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). However, the physiological significance of Dpr1 in the nucleus is still unclear.In this study we provide evidence that human Dpr1 shuttles between the nucleus and the cytoplasm. We have further identified a classical nuclear localization signal (NLS) and a nuclear export signal (NES) within Dpr1. In addition, we found that mutation of the NES rendered the exclusive nuclear localization of Dpr1, and this Dpr1 mutant still retained its ability of inhibiting Wnt signaling. Finally we showed that Dpr1 could disrupt the LEF1-β-catenin complex and recruit co-repressor HDAC1 to LEF1.EXPERIMENTAL PROCEDURESConstruction of Plasmids—Human Dpr1 cDNA and siRNA against Dpr1 were described previously (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Myc-tagged Dpr1 mutants and deletions were generated by restriction digestions and PCR and subcloned into pCMV-Myc. All of the sequences were verified by DNA sequencing. HDAC constructs were kindly provided by Dr. Xin-Hua Feng (Baylor College of Medicine), Dvl construct was provided by Dr. Xi He (Children's Hospital, Harvard Medical School) (26Capelluto D.G. Kutateladze T.G. Habas R. Finkielstein C.V. He X. Overduin M. Nature. 2002; 419: 726-729Crossref PubMed Scopus (165) Google Scholar), c-Myc promoter luciferase reporter was provided by Drs. Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University School of Medicine) (27He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4048) Google Scholar), and LEF1, β-catenin, LEF-luciferase and Topflash-luciferase were provided by Dr. Zhijie Chang (Tsinghua University).Cell Culture and Establishment of Stable Cell Lines—HEK293T, HeLa, and SW480 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10! fetal bovine serum (Hyclone), nonessential amino acids, l-glutamine, and penicillin/streptomycin in a 5! CO2-containing atmosphere at 37 °C. To generate stable cells expressing Dpr1-specific siRNA construct, HeLa cells were transfected with siRNA constructs in pSUPER with specific anti-Dpr1 sequence or anti-nonspecific sequence (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) using Lipofectamine (Invitrogen), and stable transfectants were selected with 0.5 μg/ml puromycin (Invitrogen) for 14 days. Individual clones were then obtained after confirmation of Dpr1 expression by immunoblotting and reverse transcription (RT)-polymerase chain reaction.Transfection, Immunoprecipitation, and Immunoblotting—HEK293T or HeLa cells were transiently transfected using the calcium phosphate method or Lipofectamine. At 36 h post-transfection the cells were lysed with 1 ml of lysis buffer (20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 25 mm NaF, 1! Triton X-100) plus protease inhibitors (Roche Applied Science) for 30 min at 4 °C. After 12,000 × g centrifugation for 15 min, the lysates were immunoprecipitated with specific antibody and protein A-Sepharose (Zymed Laboratories Inc.) for 3 h at 4 °C. Thereafter, the precipitants were washed 3 times with washing buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1! Nonidet P-40, 0.5! sodium deoxycholate, and 0.1! SDS), and the immune complexes were eluted with sample buffer containing 1! SDS for 5 min at 95 °C and analyzed by SDS-PAGE. Immunoblotting was performed with primary antibodies and then secondary antibodies conjugated to horseradish peroxidase (Amersham Biosciences). Proteins were visualized by chemiluminescence. Goat anti-LEF1, mouse anti-β-catenin, and mouse anti-HDAC1 antibodies were purchased from Santa Cruz Biotechnology. Anti-Dpr1 antibody was described previously (20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar).Subcellular Fractionation—HEK293T cells were harvested by scraping and resuspended in hypotonic lysis buffer (10 mm HEPES, pH 7.9, 1 mm EGTA, 1 mm EDTA, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol) containing protease inhibitors (Roche Applied Science) on ice for 15 min. Then Nonidet P-40 was added to 0.001! (for HEK293T) or 0.005! (for HeLa), and cells were vortexed for 30 s and centrifuged at 1000 × g at 4 °C for 5 min. The low speed centrifugation was washed with the ice-cold phosphate-buffered saline (PBS) three times and then resuspended in an equivalent volume of hypotonic lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1! Nonidet P-40, 0.5! sodium deoxycholate, and 0.1! SDS) and lysed by sonification. After centrifugation at 15,000 g for 15 min at 4 °C, the supernatant (nuclear extracts) was collected for analysis.Immunofluorescence—HeLa cells grown on glass coverslips were washed twice with PBS, fixed with 4! paraformaldehyde in PBS for 15 min, permeabilized with 0.1! Triton X-100 for 10 min, and blocked with 3! bovine serum albumin in PBS for 60 min. The cells were then incubated with primary antibodies diluted in TBST (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0.1! Tween 20) for 3 h, washed twice with PBS, and then incubated with rhodamine-conjugated anti-mouse or anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) for an additional 40 min. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma). Images were obtained with confocal Olympus fluoview 500 microscope.Luciferase Reporter Assays—HEK293T cells were transfected with various plasmids as indicated in the figures. 36 h after transfection, the cells were harvested, and luciferase activities were measured by aluminometer (Berthold Technologies). Reporter activity was normalized to the co-transfected Renilla. Experiments were repeated in triplicate, and the data represent the mean ± S.D. of three independent experiments.In Vitro Synthesis of mRNA and Microinjection of Zebrafish Embryos—Capped mRNAs were in vitro synthesized with the Cap-Scribe kit (Roche Applied Science). The synthesized mRNA was purified using the RNAeasy Mini kit (Qiagen) and dissolved in nuclease-free water. 20 pmol of synthetic mRNA was injected into 1-cell embryos using a gas-driven microinjector (Sutter Instruments). Injection dose was an estimated amount received by a single embryo. For the mRNA injection experiment, the control embryos were injected with green fluorescence protein (GFP) mRNA.Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was carried out essentially as described previously (28Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1436) Google Scholar). The cell lysates were subjected to immunoprecipitation. Precipitated genomic DNA pellets were subjected to PCR. The primers used to amplify the human c-Myc promoter harboring the third LEF/TCF binding elements, as described in Sierra et al. (29Sierra J. Yoshida T. Joazeiro C.A. Jones K.A. Genes Dev. 2006; 20: 586-600Crossref PubMed Scopus (326) Google Scholar), are 5′-GTGAATACACGTTTGCGGGTTAC-3′ and 5′-AGAGACCCTTGTGAAAAAAACCG-3′.RT-PCR—RNA preparation and RT-PCR have been performed as previously described (30Zhang S. Fei T. Zhang L. Zhang R. Chen F. Ning Y. Han Y. Feng X.H. Meng A. Chen Y.G. Mol. Cell. Biol. 2007; 27: 4488-4499Crossref PubMed Scopus (205) Google Scholar). Total RNA was prepared using Trizol reagent (Roche Applied Science) and treated with DNase (Takara). Two microgram of RNA was reverse-transcribed at 42 °C for 45 min in a 20-μl reaction mixture using the reverse transcription system (Promega). PCR was performed with the following primer sets: glyceraldehyde-3-phosphate dehydrogenase (5′-CATCACTGCCACCCAGAAGA-3′ and 5′-GCTGTAGCCAAATTCGTTGT-3′); c-Myc (5′-CCTACCCTCTAACGACAGC-3′ and 5′-CTCTGACCTTTTGCCAGGAG-3′); cyclin D1 (5′-ATCGAAGCCCTGCTGGAGT-3′ and 5′-GGGAAAGAGCAAAGGAAAA-3′).Flow Cytometric Analysis—HeLa cells co-transfected with GFP and other constructs as indicated were treated with Wnt3a-containing or control L cell-cultured-conditioned medium. Then cells were harvested and fixed with 75! alcohol in -20 °C overnight. Before flow cytometry analysis, cells were treated with 100 μg/ml RNase and 5 μg/ml propidium iodide in 4 °C for 30 min. The GFP-positive cells were selected by FAC-Scan flow cytometer (BD Biosciences). The Dprl-siRNA-stable cells were subjected to flow cytometric analysis as above.RESULTSDpr1 Shuttles between the Cytoplasm and the Nucleus—Previous studies have shown that endogenous Xenopus and human Dpr proteins are distributed in both the nucleus and cytoplasm, whereas ectopic Dpr1 stays mainly in the cytoplasm as punctate dots (14Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 18Waxman J.S. Hocking A.M. Stoick C.L. Moon R.T. Development. 2004; 131: 5909-5921Crossref PubMed Scopus (73) Google Scholar, 20Zhang L. Gao X. Wen J. Ning Y. Chen Y.G. J. Biol. Chem. 2006; 281: 8607-8612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). To further investigate the cellular distribution of Dpr1, Myc-tagged full-length human Dpr1 was expressed in HeLa cells and analyzed via immunofluorescent confocal studies. Consistent with previous reports, full-length Dpr1 displayed a punctate pattern in the cytoplasm (Fig. 1A). We then examined the distribution of endogenous Dpr1 in HeLa cells using a specific polyclonal antibody against Dpr1 and found that endogenous Dpr1 was distributed in the punctate pattern throughout the cytoplasm and also apparently stained in the nucleus (Fig. 1B). This observation coincides with the localization pattern of endogenous Xenopus Dpr (14Cheyette B.N. Waxman J.S. Miller J.R. Takemaru K. Sheldahl L.C. Khlebtsova N. Fox E.P. Earnest T. Moon R.T. Dev. Cell. 2002; 2: 449-461Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Interestingly, upon expression of Wnt-1 in the cells, Dpr1 tended to shift mostly into the cytoplasm instead (Fig. 1C). These data raise the possibility that Dpr1 may be capable of shuttling between the cytoplasm and the nucleus.To validate the above possibility, we asked whether leptomycin B (LMB), an inhibitor of the nuclear export mediated by export receptor chromosome maintenance region 1 (31Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (844) Google Scholar), has any effect on the localization of Dpr1. When HeLa cells transfected with full-length Dpr1 were treated with LMB for 30 min, almost all the Dpr1 was accumulated in the nucleus (Fig. 1D). In addition, endogenous Dpr1 accumulated around and in the nucleus upon LMB treatment regardless of Wnt-1 stimulation (Fig. 1, E and F).The Functional NES and NLS Mediate the Nucleocytoplasmic Shuttling of Dpr1—The translocation of proteins larger than 40–60 kDa in and out of the nucleus is usually dependent on the active transport mediated by nuclear transport receptors, which recognize certain specific NLSs and NESs (32Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1660) Google Scholar). Dpr1 is about 92 kDa and seems too large to diffuse freely through nuclear pores. We, thus, reasoned that Dpr1 may have functional NES and NLS signals to mediate its nucleocytoplasmic shuttling.To map the region of Dpr1 responsible for its shuttling between the cytoplasm and the nucleus, we generated several truncation mutants of Dpr1 (Fig. 2A) and then examined their subcellular localization in HeLa cells. All deletion mutants were correctly expressed, as indicated by immunoblotting analysis (data not shown). As shown in Fig. 2B, the N-terminal deletions Dpr1(N1) and Dpr1(N2) were predominantly localized in the cytoplasm, and LMB treatment induced a partial nuclear location of Dpr1(N1) and the complete nuclear accumulation of Dpr1(N2), indicating that the putative NES element was localized in the N-terminal region of amino acids 1–311. The C-terminal deletions Dpr1(C2) and Dpr1(C3) exclusively localized in the nucleus (Fig. 2B), whereas Dpr1 Dpr1(C1), which only missed the N-terminal 96 amino acids, showed similar punctate distribution in the cytoplasm similar to that of wild-type Dpr1. LMB treatment resulted in the nuclear accumulation of Dpr1(C1). As to the central region truncations, Dpr1(M1) displayed a dispersed distribution throughout the cells with most in the nucleus, whereas Dpr1(M2), which has only 10 more amino acids than Dpr1(M1), displayed a strict nuclear localization as Dpr1(M3), implying that those 10 amino acids (620–630 amino acids) are important for the nuclear distribution of Dpr1. LMB treatment had virtually no influence on the distribution manner of Dpr1(M1), suggesting that there are no localization signals in this region. These results together indicate that the N-terminal and C-terminal regions of Dpr1 account for the nuclear export and import of Dpr1, respectively.FIGURE 2The subcellular localization of Dpr1 is determined by its nuclear export signal and its nuclear localization signal. A, schematic representation of Dpr1 truncation mutants. The number indicates the amino acid residues. B, distribution of Dpr1 truncation mutants. HeLa cells were transfected with Myc-Dpr1 mutants (0.5 μg each) as indicated. 36 h later the cells were treated with or without 100 pm LMB for 30 min and then processed for immunofluorescence with anti-Myc antibody and rhodamine-conjugated secondary antibody. C and D, mutations of the potential NES resulted in the nuclear accumulation of Dpr1. Amino acid alignment of Dpr1 showed that the potential NES was highly conserved from Xenopus to human (C). Myc-Dpr1 NES mutants NESm or NESm2 (0.5 μg each) were transfected into HeLa cells, and the subcellular localization was determined by indirect immunofluorescence (D). E and F, mutations in the potential NLS retain Dpr1 in the cytoplasm regardless of LMB treatment. The potential NLS was highly conserved in Dpr1 proteins from different species (E). Myc-Dpr1 NLS mutants NLSm or NLSm2 were transfected into HeLa cells (0.5 μg each). After treated with or without 100 pm LMB for 30 min, their subcellular localization was detected by indirect immunofluorescence (F). The nuclei were counterstained with diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)By inspecting the amino acid sequence of human Dpr1, we found one potential leucine-rich NES at positions amino acids 132–141 (Fig. 2C) and one potential bipartite NLS at amino acids 610–623 (Fig. 2E), and both the potential NES and NLS sequences were highly conserved from zebrafish to human. To examine the importance of these potential NES and NLS, point mutations within the putative motifs were generated with two important residues replaced by Ala (Fig. 2, C and E), yielding two NES mutants, Dpr1(NESm) and Dpr1(NESm2), and two NLS mutants, Dpr1(NESm) and Dpr1(NESm2). Strikingly, we found that replacement of the anterior two residues (L132A, I136A) in the NES (Dpr1(NESm)) led to complete nuclear accumulation of the mutant Dpr1 protein as indicated by immunostaining in HeLa cells (Fig. 2D), indicating that these two residues are essential for the nuclear export of Dpr1. In contrast, the localization of Dpr1(NESm2) was relatively unaffected when Leu-139 and Leu-141 were substituted with Ala.We did similar manipulation on the potential NLS by point mutations, yielding two mutants, Dpr1(NLSm), with the Ala substitution at Lys-622 and Lys-623, and Dpr1(NLSm2), with the Ala substitution at Lys-610 and Lys-611 (Fig. 2E). Both mutants exhibited similar cytoplasmic punctate distribution as wild-type protein in HeLa cells (Fig. 2F). However, LMB treatment did not alter the cytoplasmic distribution of Dpr1(NLSm), although it caused partial nuclear accumulation of Dpr1(NLSm2) (Fig. 2F). These data suggest that the bipartite NLS is important for the nuclear transport of Dpr1 and imply that the residues Lys-622 and Lys-623 play a key role in the nuclear localization of Dpr1. Localization of these mutants was also examined in HEK293T cells, and similar results were obtained. Furthermore, Wnt-1 stimulation showed no detectable influence on that of these mutants (data not shown). Together these results validate that Dpr1 protein shuttles between the cytoplasm and the nucleus, and this shuttling is mediated by the functional NES and NLS sequences.Dpr1 Antagonizes Wnt Signaling in the Nucleus—To explore the functional significance of Dpr1 localization in the nucleus, the effect of Dpr1(NESm) and Dpr1(NLSm) on Wnt signaling was examined. HEK293T cells were transiently transfected with the Wnt-responsive reporter LEF-luciferase or Topflash-luciferase, Wnt1 together with wild-type or mutant Dpr1. As shown in Fig. 3, A and B, wild-type Dpr1 and its two mutants interfered with the Wnt1-induced expression of the reporters LEF-luciferase and Topflash-luciferase in a dose-dependent manner, although the nuclear-localized Dpr1(NESm) is less effective, whereas none of them had effect on the control reporter Fopflash-luciferase (data not shown). Similar results were obtained in HeLa cells (data not shown).FIGURE 3Dpr1 down-regulates Wnt signaling in the nucleus. A–D, the nuclear Dpr1(NESm) retains its ability to inhibit Wnt1 or β-cateni" @default.
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• W1991527612 date "2008-12-01" @default.
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• W1991527612 title "Dapper1 Is a Nucleocytoplasmic Shuttling Protein That Negatively Modulates Wnt Signaling in the Nucleus" @default.
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