Nucleo-cytoplasmic distribution of β-catenin is regulated by retention

β-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with LEF/TCF transcription factors in the nucleus. The regulation of β-catenin activity is thought to occur mainly on the level of protein degradation, but it has been suggested that β-catenin nuclear localization and hence its transcriptional activity may additionally be regulated via nuclear import by TCF4 and BCL9 and via nuclear export by APC and axin. Using live-cell microscopy and fluorescence recovery after photobleaching (FRAP), we have directly analysed the impact of these factors on the subcellular localization of β-catenin, its nucleo-cytoplasmic shuttling and its mobility within the nucleus and the cytoplasm. We show that TCF4 and BCL9/Pygopus recruit β-catenin to the nucleus, and APC, axin and axin2 enrich β-catenin in the cytoplasm. Importantly, however, none of these factors accelerates the nucleo-cytoplasmic shuttling of β-catenin, i.e. increases the rate of β-catenin nuclear import or export. Moreover, the cytoplasmic enrichment of β-catenin by APC and axin is not abolished by inhibition of CRM-1-dependent nuclear export. TCF4, APC, axin and axin2 move more slowly than β-catenin in their respective compartment, and concomitantly decrease β-catenin mobility. Together, these data indicate that β-catenin interaction partners mainly regulate β-catenin subcellular localization by retaining it in the compartment in which they are localized, rather than by active transport into or out of the nucleus.

[1]  R. Fodde,et al.  The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade. , 2002, Human molecular genetics.

[2]  Michael Kühl,et al.  Functional interaction of β-catenin with the transcription factor LEF-1 , 1996, Nature.

[3]  W. Liu,et al.  Cloning of the human homolog of conductin (AXIN2), a gene mapping to chromosome 17q23-q24. , 1999, Genomics.

[4]  Hans Clevers,et al.  Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors , 2002, Molecular and Cellular Biology.

[5]  S. Orsulic,et al.  E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. , 1999, Journal of cell science.

[6]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[7]  R Grosschedl,et al.  Functional interaction of beta-catenin with the transcription factor LEF-1. , 1996, Nature.

[8]  P. Polakis,et al.  Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. , 1996, Science.

[9]  N. Imamoto,et al.  beta-catenin can be transported into the nucleus in a Ran-unassisted manner. , 1999, Molecular biology of the cell.

[10]  F. Fagotto,et al.  CRM1- and Ran-independent nuclear export of β-catenin , 2001, Current Biology.

[11]  Jeremy Nathans,et al.  A new member of the frizzled family from Drosophila functions as a Wingless receptor , 1996, Nature.

[12]  C. L. Adams,et al.  The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration , 1996, The Journal of cell biology.

[13]  B. Henderson,et al.  A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. , 2000, Experimental cell research.

[14]  B. Henderson.  Nuclear-cytoplasmic shuttling of APC regulates β-catenin subcellular localization and turnover , 2000, Nature Cell Biology.

[15]  M. Waterman,et al.  Differential Importin-α Recognition and Nuclear Transport by Nuclear Localization Signals within the High-Mobility-Group DNA Binding Domains of Lymphoid Enhancer Factor 1 and T-Cell Factor 1 , 1998, Molecular and Cellular Biology.

[16]  D. Kimelman,et al.  Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function. , 2004, Molecular cell.

[17]  Xi He,et al.  Control of β-Catenin Phosphorylation/Degradation by a Dual-Kinase Mechanism , 2002, Cell.

[18]  Choun-Ki Joo,et al.  Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway , 2002, Molecular and Cellular Biology.

[19]  Xi He,et al.  Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. , 2002, Cell.

[20]  A. Brown,et al.  Transformation by Wnt family proteins correlates with regulation of beta-catenin. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[21]  W. Birchmeier,et al.  Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. , 1998, Science.

[22]  T. Dale,et al.  A divergent canonical WNT-signaling pathway regulates microtubule dynamics , 2004, The Journal of cell biology.

[23]  J. Trent,et al.  Identification of an amplified, highly expressed gene in a human glioma. , 1987, Science.

[24]  A. Schohl,et al.  Nucleo-cytoplasmic Shuttling of Axin, a Negative Regulator of the Wnt-β-Catenin Pathway* , 2004, Journal of Biological Chemistry.

[25]  Walter Birchmeier,et al.  Hot spots in β-catenin for interactions with LEF-1, conductin and APC , 2000, Nature Structural Biology.

[26]  H Clevers,et al.  Mutant E-cadherin breast cancer cells do not display constitutive Wnt signaling. , 2001, Cancer research.

[27]  Hans Clevers,et al.  XTcf-3 Transcription Factor Mediates β-Catenin-Induced Axis Formation in Xenopus Embryos , 1996, Cell.

[28]  Hans Clevers,et al.  Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC , 1997, Science.

[29]  B. Gumbiner,et al.  Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of β-catenin , 1998, Current Biology.

[30]  K. Kinzler,et al.  Association of the APC tumor suppressor protein with catenins. , 1993, Science.

[31]  B. Henderson,et al.  Lymphoid Enhancer Factor-1 Blocks Adenomatous Polyposis Coli-mediated Nuclear Export and Degradation of β-Catenin , 2002, The Journal of Biological Chemistry.

[32]  B. Cullen,et al.  Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Mariann Bienz,et al.  The APC tumour suppressor has a nuclear export function , 2000, Nature.

[34]  F. Fagotto,et al.  CRM1- and Ran-independent nuclear export of beta-catenin. , 2001, Current biology : CB.

[35]  Paul Polakis,et al.  Downregulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK3β , 1998, Current Biology.

[36]  K. Mostov,et al.  NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion , 1997, The Journal of cell biology.

[37]  F. Brembeck,et al.  Essential role of BCL9-2 in the switch between beta-catenin's adhesive and transcriptional functions. , 2004, Genes & development.

[38]  M. Montminy,et al.  Glutamine Rich and Basic Region/Leucine Zipper (bZIP) Domains Stabilize cAMP-response Element-binding Protein (CREB) Binding to Chromatin* , 2005, Journal of Biological Chemistry.

[39]  P. Polakis,et al.  Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. , 1998, Current biology : CB.

[40]  M. Bienz,et al.  The APC tumor suppressor binds to C-terminal binding protein to divert nuclear beta-catenin from TCF. , 2004, Developmental cell.

[41]  Nam-Chul Ha,et al.  Mechanism of Phosphorylation-Dependent Binding of APC to β-Catenin and Its Role in β-Catenin Degradation , 2004 .

[42]  J. Behrens,et al.  The Wnt connection to tumorigenesis. , 2004, The International journal of developmental biology.

[43]  W. Birchmeier,et al.  E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton , 1994, The Journal of cell biology.

[44]  Nam-Chul Ha,et al.  Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. , 2004, Molecular cell.

[45]  B. Gumbiner,et al.  Distinct molecular forms of β-catenin are targeted to adhesive or transcriptional complexes , 2004, The Journal of cell biology.

[46]  Mariann Bienz,et al.  Nuclear export of the APC tumour suppressor controls β‐catenin function in transcription , 2003, The EMBO journal.

[47]  Hideki Yamamoto,et al.  Axin, a Negative Regulator of the Wnt Signaling Pathway, Directly Interacts with Adenomatous Polyposis Coli and Regulates the Stabilization of β-Catenin* , 1998, The Journal of Biological Chemistry.

[48]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[49]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Bernhard Schmierer,et al.  Analysis of Smad nucleocytoplasmic shuttling in living cells , 2004, Journal of Cell Science.

[51]  B. Henderson,et al.  Redefining the subcellular location and transport of APC: new insights using a panel of antibodies , 2005, EMBO reports.

[52]  Raymond L. White,et al.  APC‐mediated downregulation of β‐catenin activity involves nuclear sequestration and nuclear export , 2000, EMBO reports.

[53]  Akinao Nose,et al.  Expressed recombinant cadherins mediate cell sorting in model systems , 1988, Cell.

[54]  Marc W. Kirschner,et al.  Physiological regulation of β-catenin stability by Tcf3 and CK1ε , 2001, The Journal of cell biology.

[55]  E. Wieschaus,et al.  Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan. , 2001, Development.

[56]  Hans C Clevers,et al.  Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin. , 2002, EMBO reports.

[57]  Minoru Yoshida,et al.  Nuclear Export of Human β-Catenin Can Occur Independent of CRM1 and the Adenomatous Polyposis Coli Tumor Suppressor* , 2001, The Journal of Biological Chemistry.

[58]  R. Nusse,et al.  Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex. , 1999, Genes & development.

[59]  B. Gumbiner,et al.  Translocation of beta-catenin into the nucleus independent of interactions with FG-rich nucleoporins. , 2003, Experimental cell research.

[60]  M. Bienz,et al.  Pygopus and Legless target Armadillo/β-catenin to the nucleus to enable its transcriptional co-activator function , 2004, Nature Cell Biology.

[61]  B. Herrmann,et al.  Nuclear localization of β-catenin by interaction with transcription factor LEF-1 , 1996, Mechanisms of Development.

[62]  M. Montminy,et al.  Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Sandipan Chatterjee,et al.  Wnt/wingless Signaling Requires Bcl9/legless-mediated Recruitment of Pygopus to the Nuclear Beta-catenin-tcf Complex , 2022 .

[64]  Yoichi Kato,et al.  LDL-receptor-related proteins in Wnt signal transduction , 2000, Nature.

[65]  F. Masiarz,et al.  Association of the APC gene product with beta-catenin. , 1993, Science.

[66]  H. Clevers,et al.  Wnt signals are transmitted through N‐terminally dephosphorylated β‐catenin , 2002 .

[67]  H. Varmus,et al.  Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of beta-catenin. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[68]  N. Imamoto,et al.  β-Catenin Shows an Overlapping Sequence Requirement but Distinct Molecular Interactions for Its Bidirectional Passage through Nuclear Pores* , 2004, Journal of Biological Chemistry.

[69]  William C. Skarnes,et al.  An LDL-receptor-related protein mediates Wnt signalling in mice , 2000, Nature.

[70]  G Winbeck,et al.  Hot spots in beta-catenin for interactions with LEF-1, conductin and APC. , 2000, Nature structural biology.

[71]  Paul Polakis,et al.  Binding of GSK3β to the APC-β-Catenin Complex and Regulation of Complex Assembly , 1996, Science.

[72]  A. Prescott,et al.  The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells , 2002, The Journal of cell biology.

[73]  R Kemler,et al.  beta-catenin is a target for the ubiquitin-proteasome pathway. , 1997, The EMBO journal.

[74]  M. Ptashne,et al.  A vector for expressing GAL4(1-147) fusions in mammalian cells. , 1989, Nucleic acids research.

[75]  K. Basler,et al.  Identification and in vivo role of the Armadillo-Legless interaction , 2004, Development.