Interactions of plakoglobin and beta-catenin with desmosomal cadherins: basis of selective exclusion of alpha- and beta-catenin from desmosomes

Abstract

Plakoglobin and beta-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with alpha-catenin. Plakoglobin, but normally not beta-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 beta-catenin and alpha-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between beta-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of beta-catenin. Binding of plakoglobin and beta-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and beta-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, beta-catenin binds to desmoglein-1 more weakly than does plakoglobin. beta-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 beta-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 alpha-catenin compete directly for binding to plakoglobin, consistent with the absence of alpha-catenin in desmosomes.

FIGURE 1.

Constructs used in ITC experiments and pulldown assays. The N- and C-terminal tails and the central armadillo domains of plakoglobin and β-catenin are represented as Nt, Ct, and arm, respectively. The numbers shown on top of each construct represent the beginning and ending residue numbers.

FIGURE 2.

Structure of the plakoglobin arm domain and C-terminal helix bound to phosphorylated E-cadherin. A, overall structure of the plakoglobin-pE-CBD complex. Plakoglobin arm repeat helices are shown in yellow (H1 and H2) and blue (H3), and helix C is shown in red. The pE-CBD is shown in magenta. B, superposition of the plakoglobin arm domain (yellow H1 and H2 and blue H3) with the β-catenin arm domain (gray) as seen in the β-cat-arm-pE_cyto complex (33). The orientation is that same as in A. H1 and H2 helices are shown in gray and H3 in blue. The pE_cyto structure is shown in cyan. C, superposition of the pE-cadherin CBD bound to plakoglobin (magenta) and β-catenin (cyan). The arm repeat domains of β-catenin and plakoglobin were superimposed, and the transformation was used to compare the bound cadherins.

FIGURE 3.

Helix C in plakoglobin and β-catenin. A, close up of the hydrophobic interaction between plakoglobin (helix C in red and repeat 12 in blue and yellow) and E-cadherin (magenta). B, comparison of the hydrophobic interactions between helix C (plakoglobin, red; β-catenin, pink) and arm repeat 12 (plakoglobin, blue and yellow; β-catenin, gray). The β-catenin structure is from Ref. , Protein Data Bank code 2Z6H. C, same as B but with the E-cadherin CBD (magenta) bound to plakoglobin.

FIGURE 4.

Ligand binding to full-length plakoglobin determined by ITC. Representative experiments are shown. All isothermal calorimetric experiments were performed at 30 °C in H buffer. The raw heat signals obtained by a series of injections of each ligand into a solution of full-length plakoglobin are shown on top, and the binding curve calculated using the best fit parameters obtained by a nonlinear least squares fit is shown on the bottom. The base-line heats that are subtracted from the raw data, obtained by injecting the ligand into H buffer, are shown vertically offset from the binding data.

FIGURE 5.

Cadherin binding to full-length β-catenin determined by ITC. Representative experiments are shown. All isothermal calorimetric experiments were performed at 30 °C in H buffer. The raw heat signals obtained by a series of injections of each ligand into a solution of full-length β-catenin are shown on top, and the binding curve calculated using the best fit parameters obtained by a nonlinear least squares fit is shown on the bottom. The base-line heats that are subtracted from the raw data, obtained by injecting the ligand into H buffer, are shown vertically offset from the binding data.

FIGURE 6.

Proteolytic sensitivity of β-catenin and plakoglobin. a, pg-arm or βcat-arm was incubated with the indicated amount of trypsin for 15 and 45 min at room temperature. After incubation, each reaction was added by SDS sample buffer and was boiled. Untreated pg-arm and βcat-arm were shown as controls. b, 66-kDa plakoglobin fragment was incubated with trypsin for 0, 5, 10, 15, and 30 min and 1, 2, and 3 h. The asterisks indicate degradation products used for N-terminal sequence analysis. c, schematic drawings of primary structures of β-catenin and plakoglobin showing the location of trypsin cleavage sites. Numbered boxes represent the 12 armadillo repeats, and NH₂ and COOH represent the N- and C-terminal tails. Numbered arrows show the location of the cleavage sites. The sequence around the cleaved Arg or Lys residues (shown in boldface) is indicated.

FIGURE 7.

Mapping of the desmosomal cadherin-binding sites on plakoglobin. a, GST-pg-arm at a concentration of 10 μm was incubated with 10 μm catenin-binding domain of Dsg1 or full cytoplasmic domains of E-cadherin or Dsc1 for 1 h. After isolation of protein complexes with glutathione-agarose, 10 μm ICAT was added into each reaction, and each reaction mixture was incubated for another 1 h at 4 °C. Protein complexes were purified with glutathione-agarose, and each purified protein complex in the absence or presence of ICAT was analyzed by SDS-PAGE and Coomassie staining. Molecular markers are shown on the right. b, GST-Dsg1-CBD at a concentration of 10 μm was incubated with 10 μm pg-full. After protein complexes were co-precipitated on glutathione-agarose, ICAT, ICAT-h, Dsc1_cyto, or E_cyto each at a concentration of 10 μm was added into each reaction, and the beads were centrifuged. Supernatants (S) and pelleted beads (P) were analyzed by SDS-PAGE and visualized with Coomassie Blue stain. ICAT and ICAT-h stain very poorly relative to plakoglobin, and their bands in the pellet with plakoglobin have been marked with white boxes. c, similar binding assays were performed using GST-Dsc1_cyto, instead of GST-Dsg1-CBD. After isolation of protein complex of GST-Dsc1_cyto and pg-full, ICAT, ICAT-h, or Dsg1-CBD was added into each reaction. Although ICAT and ICAT-h stain poorly, it is clear that neither protein co-sediments with plakoglobin-bound Dsc1.

FIGURE 8.

Overlapping binding sites of α-catenin and desmosomal cadherins on plakoglobin. Upper panel, GST-E_cyto, GST-Dsg1_cyto, or GST-Dsc1_cyto each at a concentration of 7 μm was incubated with 10 μm plakoglobin and increasing amounts of α-catenin as indicated. Protein complexes were isolated with glutathione-agarose and analyzed by SDS-PAGE and Coomassie staining. Lower panel, same binding assays were carried out in the absence of plakoglobin as a control for nonspecific background binding of α-catenin to glutathione-agarose.