Beta-catenin N- and C-terminal tails modulate the coordinated binding of adherens junction proteins to beta-catenin

beta-Catenin plays a central role in the establishment and regulation of adherens junctions because it interacts with E-cadherin and, through alpha-catenin, with the actin cytoskeleton. beta-Catenin is composed of three domains: a central armadillo repeat domain and two N- and C-terminal tails. The C-tail interacts with the armadillo domain and limits its ability to bind E-cadherin and other cofactors. The two beta-catenin tails are mutually inter-regulated because the C-tail is also necessary for binding of the N-tail to the armadillo domain. Moreover, the N-tail restricts the interaction of the C-tail with the central domain. Depletion of either of the two tails has consequences for the binding of factors at the other end: deletion of the C-tail increases alpha-catenin binding, whereas deletion of the N-tail blocks E-cadherin interaction to the armadillo repeats. As an effect of the interconnection of the tails, the association of alpha-catenin and E-cadherin to beta-catenin is interdependent. Thus, binding of alpha-catenin to the N-tail, through conformational changes that affect the C-tail, facilitates the association of E-cadherin. These results indicate that different cofactors of beta-catenin bind coordinately to this protein and indicate how the two terminal ends of beta-catenin exquisitely modulate intermolecular binding within junctional complexes.     

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E- cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.     

Interactions of Plakoglobin and ��-Catenin with Desmosomal Cadherins: BASIS OF SELECTIVE EXCLUSION OF ��- AND ��-CATENIN FROM DESMOSOMES*

Plakoglobin and β-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with α-catenin. Plakoglobin, but normally not β-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of β-catenin and α-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between β-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of β-catenin. Binding of plakoglobin and β-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and β-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, β-catenin binds to desmoglein-1 more weakly than does plakoglobin. β-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal β-catenin “tails” that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and α-catenin compete directly for binding to plakoglobin, consistent with the absence of α-catenin in desmosomes.     

Crystal structure of a beta-catenin/BCL9/Tcf4 complex

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.6 A resolution. Our studies reveal that the beta-catenin binding site of BCL9 is distinct from that of most other beta-catenin partners and forms a good target for developing drugs that block canonical Wnt/beta-catenin signaling. The BCL9 beta-catenin binding domain (CBD) forms an alpha helix that binds to the first armadillo repeat of beta-catenin, which can be mutated to prevent beta-catenin binding to BCL9 without affecting cadherin or alpha-catenin binding. We also demonstrate that beta-catenin Y142 phosphorylation, which has been proposed to regulate BCL9-2 binding, does not directly affect the interaction of beta-catenin with either BCL9 or BCL9-2.     

Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in the embryonic heart

Plakoglobin (gamma-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaque of desmosomes as well as of other adhering cell junctions, and is involved in anchorage of cytoskeletal filaments to specific cadherins. We have generated a null mutation of the plakoglobin gene in mice. Homozygous -/- mutant animals die between days 12-16 of embryogenesis due to defects in heart function. Often, heart ventricles burst and blood floods the pericard. This tissue instability correlates with the absence of desmosomes in heart, but not in epithelia organs. Instead, extended adherens junctions are formed in the heart, which contain desmosomal proteins, i.e., desmoplakin. Thus, plakoglobin is an essential component of myocardiac desmosomes and seems to play a crucial role in the sorting out of desmosomal and adherens junction components, and consequently in the architecture of intercalated discs and the stabilization of heart tissue.     

Cadherin-catenin complexes during zebrafish oogenesis: heterotypic junctions between oocytes and follicle cells.

During vertebrate oogenesis, the germ cells and associated somatic cells remain connected by a variety of adhering junctional complexes. However, the molecular composition of these cellular structures is largely unknown. To identify the proteins forming the heterotypic adherens junctions between oocytes and follicle cells in the zebrafish (Danio rerio), the cDNAs encoding alphaE-catenin and plakoglobin were isolated. Using these cDNAs, in combination with the previously isolated beta-catenin cDNA, and antibodies specific for alpha- and beta-catenin, plakoglobin, and N- and E-cadherin, we found differences in catenin and plakoglobin gene expression during oogenesis. The immunolocalization of these plaque proteins, as well as of cadherins, in the ovarian follicle indicated an enrichment of alpha- and beta-catenin and of E-cadherin-like protein(s) in the oocyte cortex, notably at sites of oocyte-follicle cell contacts, suggesting the presence of hitherto unknown heterotypic adherens junctions between these cells. By contrast, plakoglobin and N-cadherin localization was restricted to cell-cell contacts in the follicle cell layer. During oocyte maturation, mRNAs for alphaE- and beta-catenin and plakoglobin accumulated, and all three plaque-forming proteins were stored in unfertilized eggs, either in complexed forms with cadherins or as free cytoplasmic pools. These findings suggest possible roles of these junctional proteins during early embryogenesis.     

The Arm-Repeat Protein NPRAP (Neurojungin) Is a Constituent of the Plaques of the Outer Limiting Zone in the Retina, Defining a Novel Type of Adhering Junction

In the retina, special plaque-bearing adhering junctions are aligned to form a planar system (the "outer limiting zone," OLZ) of heterotypic connections between the photoreceptor cells and the surrounding glial cells ("Müller cells"), together with homotypic junctions. In the plaques of these junctions, which contain N-cadherin-and possibly also related cadherins-we have identified, by immunolocalization techniques, a recently discovered neural tissue-specific protein, neurojungin, a member of the plakoglobin/armadillo protein family. In these plaques we have also detected other adherens plaque proteins, such as alpha- and beta-catenin, protein p120, and vinculin, as well as proteins known as constituents of tight junction plaques, such as symplekin and protein ZO-1, and the desmosomal plaque protein plakophilin 2. This unusual combination of proteins and the demonstrated absence of plakoglobin define the OLZ junctions as a new and distinct category of adhering junction, which probably has special architectural functions.     

Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.