β-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from β-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

C. elegans and Drosophila generate distinct signaling and adhesive forms of β-catenin at the level of gene expression. Whether vertebrates, which rely on a single β-catenin gene, generate unique adhesive and signaling forms at the level of protein modification remains unresolved. We show that β-catenin unphosphorylated at serine 37 (S37) and threonine 41 (T41), commonly referred to as transcriptionally Active β-Catenin (ABC), is a minor nuclear-enriched monomeric form of β-catenin in SW480 cells, which express low levels of E-cadherin. Despite earlier indications, the superior signaling activity of ABC is not due to reduced cadherin binding, as ABC is readily incorporated into cadherin contacts in E-cadherin-restored cells. β-catenin phosphorylated at serine 45 (S45) or threonine 41 (T41) (T41/S45) or along the GSK3 regulatory cassette S33, S37 or T41 (S33/37/T41), however, is largely unable to associate with cadherins. β-catenin phosphorylated at T41/S45 and unphosphorylated at S37 and T41 is predominantly nuclear, while β-catenin phosphorylated at S33/37/T41 is mostly cytoplasmic, suggesting that β-catenin hypophosphorylated at S37 and T41 may be more active in transcription due to its enhanced nuclear accumulation. Evidence that phosphorylation at T41/S45 can be spatially separated from phosphorylations at S33/37/T41 suggests that these phosphorylations may not always be coupled, raising the possibility that phosphorylation at S45 serves a distinct nuclear function

Force-dependent allostery of the α-catenin actinbinding domain controls adherens junction dynamics and functions

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis

Activity of the β-catenin phosphodestruction complex at cell–cell contacts is enhanced by cadherin-based adhesion

It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering β-catenin (β-cat) from T-cell factor family transcription factors. Whether changes in intercellular adhesion can affect β-cat signaling and the mechanism through which this occurs has remained unresolved. We show that axin, APC2, GSK-3β and N-terminally phosphorylated forms of β-cat can localize to cell–cell contacts in a complex that is molecularly distinct from the cadherin–catenin adhesive complex. Nonetheless, cadherins can promote the N-terminal phosphorylation of β-cat, and cell–cell adhesion increases the turnover of cytosolic β-cat. Together, these data suggest that cadherin-based cell–cell adhesion limits Wnt signals by promoting the activity of a junction-localized β-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development

Alpha-T-catenin is expressed in peripheral nerves as a constituent of Schwann cell adherens junctions

The adherens junction component, alpha-T-catenin (αTcat) is an established contributor to cardiomyocyte junction structure and function, but recent genomic studies link CTNNA3 polymorphisms to diseases with no clear cardiac underpinning, including asthma, autism and multiple sclerosis, suggesting causal contributions from a different cell-type. We show Ctnna3 mRNA is highly expressed in peripheral nerves (e.g. vagus and sciatic), where αTcat protein enriches at paranodes and myelin incisure adherens junctions of Schwann cells. We validate αTcat immunodetection specificity using a new Ctnna3-knock-out fluorescence reporter mouse line yet find no obvious Schwann cell loss-of-function morphology at the light microscopic level. CTNNA3/Ctnna3 mRNA is also abundantly detected in oligodendrocytes of the central nervous system via public databases, supporting a general role for αTcat in these unique cell–cell junctions. These data suggest that the wide range of diseases linked to CTNNA3 may be through its role in maintaining neuroglial functions of central and peripheral nervous systems. This article has a corresponding First Person interview with the co-first authors of the paper

Issues associated with assessing nuclear localization of N-terminally unphosphorylated β-catenin with monoclonal antibody 8E7

Abstract Background β-catenin is a dual function adhesion/transcriptional co-activator protein, and both functions are critical for normal tissue homeostasis. Since the transcriptional functions of β-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of β-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of β-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of β-catenin. Results We show that monoclonal antibody, 8E7, which recognizes the signaling form of β-catenin specifically unphosphorylated at S37 and T41 (Active B-Catenin, ABC), also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not β-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen. Conclusion These findings reveal that interpretation of nuclear, signaling active β-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods. Reviewers This article was reviewed by Frank J. T. Staal (nominated by Rachel Gerstein), Jyoti M. Sen (nominated by Avinash Bhandoola) and Manabu Sugai.</p