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Regulation of beta-catenin levels and localization by overexpression of plakoglobin and inhibition of the ubiquitin-proteasome system.

Salomon D, Sacco PA, Roy SG, Simcha I, Johnson KR, Wheelock MJ, Ben-Ze'ev A - J. Cell Biol. (1997)

Bottom Line: Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone.Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus.These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
beta-Catenin and plakoglobin (gamma-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and alpha-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, beta-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increased the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolished beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

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Schematic representation of plakoglobin functional domains involved in reducing β-catenin levels and in complexing with  α-catenin and N-cadherin. Full-length human plakoglobin (745 amino acids) is shown with the 13 armadillo repeats and the different deletion mutants from the NH2 (ΔN) and COOH terminus (ΔC) used in this study. The binding studies to α-catenin and N-cadherin of the  various plakoglobin mutants were described (Sacco et al., 1995; Wahl et al., 1996). The binding sites for the monoclonal antibodies 11E4  and PG 5.1 used in this study are also indicated.
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Figure 9: Schematic representation of plakoglobin functional domains involved in reducing β-catenin levels and in complexing with α-catenin and N-cadherin. Full-length human plakoglobin (745 amino acids) is shown with the 13 armadillo repeats and the different deletion mutants from the NH2 (ΔN) and COOH terminus (ΔC) used in this study. The binding studies to α-catenin and N-cadherin of the various plakoglobin mutants were described (Sacco et al., 1995; Wahl et al., 1996). The binding sites for the monoclonal antibodies 11E4 and PG 5.1 used in this study are also indicated.

Mentions: Since plakoglobin and β-catenin are localized in the submembrane plaque where they form mutually exclusive complexes with cadherins, we examined the possibility that the overexpressed plakoglobin competes with β-catenin for N-cadherin binding, and this competition leads to displacement and degradation of uncomplexed β-catenin. HT1080 cell lysates, prepared before and after plakoglobin induction, were immunoprecipitated with anti–N-cadherin antibody followed by Western blotting with anti–β-catenin and plakoglobin antibodies (Fig. 5 A). When plakoglobin expression was induced with dexamethasone, the complexes with N-cadherin contained more plakoglobin than β-catenin (Fig. 5 A, compare lanes 3 and 4 with lane 1). To examine the possibility that N-cadherin binding with plakoglobin is responsible for conferring the decrease in β-catenin levels, we employed HT1080 cells stably transfected with mutant plakoglobin constructs that included or lacked either the N-cadherin or the α-catenin binding domains (see Fig. 9; Sacco et al., 1995). Expression of full-length (FL) plakoglobin (Fig. 5 B, lanes 1 and 2), and deletions that left the armadillo repeats intact (Fig. 5 B, ΔC727, lanes 3 and 4) or removed part of the last armadillo repeat (Fig. 5 B, ΔC632, lanes 5 and 6), but were still capable of associating with N-cadherin, reduced the level of β-catenin when compared to uninduced cells (Fig. 5 B, lanes 1, 3, and 5, compare with 2, 4, and 6, respectively). In contrast, larger COOH-terminal deletions in plakoglobin that disrupt association with N-cadherin, but retain α-catenin binding (Fig. 9), leaving 458 amino acids or less (Fig. 5 C; ΔC458, ΔC414, ΔC375, ΔC161, and ΔC114), were unable to affect β-catenin levels when overexpressed (Fig. 5 C, lanes 2, 4, 6, 8, and 10, compare with 1, 3, 5, 7, and 9, respectively).


Regulation of beta-catenin levels and localization by overexpression of plakoglobin and inhibition of the ubiquitin-proteasome system.

Salomon D, Sacco PA, Roy SG, Simcha I, Johnson KR, Wheelock MJ, Ben-Ze'ev A - J. Cell Biol. (1997)

Schematic representation of plakoglobin functional domains involved in reducing β-catenin levels and in complexing with  α-catenin and N-cadherin. Full-length human plakoglobin (745 amino acids) is shown with the 13 armadillo repeats and the different deletion mutants from the NH2 (ΔN) and COOH terminus (ΔC) used in this study. The binding studies to α-catenin and N-cadherin of the  various plakoglobin mutants were described (Sacco et al., 1995; Wahl et al., 1996). The binding sites for the monoclonal antibodies 11E4  and PG 5.1 used in this study are also indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140206&req=5

Figure 9: Schematic representation of plakoglobin functional domains involved in reducing β-catenin levels and in complexing with α-catenin and N-cadherin. Full-length human plakoglobin (745 amino acids) is shown with the 13 armadillo repeats and the different deletion mutants from the NH2 (ΔN) and COOH terminus (ΔC) used in this study. The binding studies to α-catenin and N-cadherin of the various plakoglobin mutants were described (Sacco et al., 1995; Wahl et al., 1996). The binding sites for the monoclonal antibodies 11E4 and PG 5.1 used in this study are also indicated.
Mentions: Since plakoglobin and β-catenin are localized in the submembrane plaque where they form mutually exclusive complexes with cadherins, we examined the possibility that the overexpressed plakoglobin competes with β-catenin for N-cadherin binding, and this competition leads to displacement and degradation of uncomplexed β-catenin. HT1080 cell lysates, prepared before and after plakoglobin induction, were immunoprecipitated with anti–N-cadherin antibody followed by Western blotting with anti–β-catenin and plakoglobin antibodies (Fig. 5 A). When plakoglobin expression was induced with dexamethasone, the complexes with N-cadherin contained more plakoglobin than β-catenin (Fig. 5 A, compare lanes 3 and 4 with lane 1). To examine the possibility that N-cadherin binding with plakoglobin is responsible for conferring the decrease in β-catenin levels, we employed HT1080 cells stably transfected with mutant plakoglobin constructs that included or lacked either the N-cadherin or the α-catenin binding domains (see Fig. 9; Sacco et al., 1995). Expression of full-length (FL) plakoglobin (Fig. 5 B, lanes 1 and 2), and deletions that left the armadillo repeats intact (Fig. 5 B, ΔC727, lanes 3 and 4) or removed part of the last armadillo repeat (Fig. 5 B, ΔC632, lanes 5 and 6), but were still capable of associating with N-cadherin, reduced the level of β-catenin when compared to uninduced cells (Fig. 5 B, lanes 1, 3, and 5, compare with 2, 4, and 6, respectively). In contrast, larger COOH-terminal deletions in plakoglobin that disrupt association with N-cadherin, but retain α-catenin binding (Fig. 9), leaving 458 amino acids or less (Fig. 5 C; ΔC458, ΔC414, ΔC375, ΔC161, and ΔC114), were unable to affect β-catenin levels when overexpressed (Fig. 5 C, lanes 2, 4, 6, 8, and 10, compare with 1, 3, 5, 7, and 9, respectively).

Bottom Line: Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone.Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus.These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
beta-Catenin and plakoglobin (gamma-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and alpha-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, beta-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increased the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolished beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

Show MeSH
Related in: MedlinePlus