Abstract
β-catenin (CTNNB1) is a dual-function cell–cell adhesion/transcriptional co-activator protein and an essential transducer of canonical Wnt signals. Although a number of established techniques and reagents are available to quantify the nuclear signaling activity of β-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of β-catenin, and generation of N-terminally hypophosphorylated β-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of β-catenin outside of the cadherin complex appears universally required for β-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of β-catenin in cells, using a GST-tagged form of ICAT (Inhibitor of β-Catenin and Tcf) as an affinity matrix. This method is more sensitive and quantitative than immunofluorescence and may be useful in studies that implicate TCF-independent signaling events.
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References
Goentoro L, Kirschner MW (2009) Evidence that fold-change, and not absolute level, of beta-catenin dictates Wnt signaling. Mol Cell 36:872–884
Cox RT, Kirkpatrick C, Peifer M (1996) Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J Cell Biol 134:133–148
Cox RT, Pai LM, Kirkpatrick C, Stein J, Peifer M (1999) Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. Genetics 153:319–332
Song LN, Herrell R, Byers S, Shah S, Wilson EM, Gelmann EP (2003) Beta-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription. Mol Cell Biol 23:1674–1687
Truica CI, Byers S, Gelmann EP (2000) Beta-catenin affects androgen receptor transcriptional activity and ligand specificity. Cancer Res 60(17):4709–4713
Essers MA, de Vries-Smits LM, Barker N, Polderman PE, Burgering BM, Korswagen HC (2005) Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 308:1181–1184
Hoogeboom D, Essers MA, Polderman PE, Voets E, Smits LM, Burgering BM (2008) Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell factor activity. J Biol Chem 283:9224–9230
Staal FJ, Noort Mv M, Strous GJ, Clevers HC (2002) Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin. EMBO Rep 3:63–68
van Noort M, Meeldijk J, van der Zee R, Destree O, Clevers H (2002) Wnt signaling controls the phosphorylation status of beta-catenin. J Biol Chem 277:17901–17905
Maher MT, Flozak AS, Stocker A, Chenn A, Gottardi CJ (2009) Activity of the β-catenin phospho-destruction at cell-cell contacts is enhanced by cadherin-based adhesion. J Cell Biol 186:219–228
Maher MT, Mo R, Flozak AS, Peled ON, Gottardi CJ (2010) Beta-catenin phosphorylated at serine 45 is spatially uncoupled from beta-catenin phosphorylated in the GSK3 domain: implications for signaling. PLoS One 5:e10184
Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS (2003) Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J Biol Chem 278(35):33067–33077
Tago K, Nakamura T, Nishita M, Hyodo J, Nagai S, Murata Y, Adachi S, Ohwada S, Morishita Y, Shibuya H, Akiyama T (2000) Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein. Genes Dev 14:1741–1749
Tutter AV, Fryer CJ, Jones KA (2001) Chromatin-specific regulation of LEF-1-beta-catenin transcription activation and inhibition in vitro. Genes Dev 15:3342–3354
Graham TA, Clements WK, Kimelman D, Xu W (2002) The crystal structure of the beta-catenin/ICAT complex reveals the inhibitory mechanism of ICAT. Mol Cell 10:563–571
Daniels DL, Weis WI (2002) ICAT inhibits beta-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules. Mol Cell 10:573–584
Gottardi CJ, Gumbiner BM (2004) Distinct molecular forms of beta-catenin are targeted to adhesive or transcriptional complexes. J Cell Biol 167:339–349
Choi HJ, Huber AH, Weis WI (2006) Thermodynamics of beta-catenin-ligand interactions: the roles of the N- and C-terminal tails in modulating binding affinity. J Biol Chem 281:1027–1038
McCrea PD, Gumbiner BM (1991) Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure. J Biol Chem 266:4514–4520
Pronobis MI, Rusan NM, Peifer M (2015) A novel GSK3-regulated APC:Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient betacatenin destruction. Elife 4:e08022
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Flozak, A.S., Lam, A.P., Gottardi, C.J. (2016). A Simple Method to Assess Abundance of the β-Catenin Signaling Pool in Cells. In: Barrett, Q., Lum, L. (eds) Wnt Signaling. Methods in Molecular Biology, vol 1481. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6393-5_6
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DOI: https://doi.org/10.1007/978-1-4939-6393-5_6
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