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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 is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques.Individual clones expressing various levels of plakoglobin were established by stable transfection.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.

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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The localization of β-catenin and plakoglobin in cells treated  with inhibitors of the ubiquitin-proteasome system. HT1080 cells were pretreated with the inactive analogue  ALLM (A), with the proteasome inhibitors ALLN (B and D), or with  MG-132 in the presence of dexamethasone to induce plakoglobin expression  (C). After 9 h, the cells were immunostained for β-catenin (A–C) or plakoglobin (D). Bar, 10 μm.
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Figure 11: The localization of β-catenin and plakoglobin in cells treated with inhibitors of the ubiquitin-proteasome system. HT1080 cells were pretreated with the inactive analogue ALLM (A), with the proteasome inhibitors ALLN (B and D), or with MG-132 in the presence of dexamethasone to induce plakoglobin expression (C). After 9 h, the cells were immunostained for β-catenin (A–C) or plakoglobin (D). Bar, 10 μm.

Mentions: To determine the localization of plakoglobin and β-catenin when the ubiquitin-proteasome proteolytic pathway is inhibited, HT1080 cells were treated with ALLN or MG-132 and plakoglobin expression was induced for 9 h. The cells were immunostained with anti-plakoglobin and anti– β-catenin antibodies (Fig. 11). The results shown in Fig. 11 demonstrate that when proteasome inhibitors were applied, a significant amount of β-catenin translocated into the nuclei of the cells (Fig. 11, B and C), in contrast to cells treated with the inactive ALLM peptide that showed only cell–cell junctional staining (Fig. 11 A). Interestingly, under these conditions plakoglobin was not organized in cell–cell junctions, but was diffusely distributed in the cytoplasm with a significant accumulation in the nuclei of the cells (Fig. 11 D). Similar results were obtained using Lactacystin, but not with other protease inhibitors including pepstatin A, aprotinin and leupeptin which have no effect on this degradation pathway (results not shown).


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)

The localization of β-catenin and plakoglobin in cells treated  with inhibitors of the ubiquitin-proteasome system. HT1080 cells were pretreated with the inactive analogue  ALLM (A), with the proteasome inhibitors ALLN (B and D), or with  MG-132 in the presence of dexamethasone to induce plakoglobin expression  (C). After 9 h, the cells were immunostained for β-catenin (A–C) or plakoglobin (D). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: The localization of β-catenin and plakoglobin in cells treated with inhibitors of the ubiquitin-proteasome system. HT1080 cells were pretreated with the inactive analogue ALLM (A), with the proteasome inhibitors ALLN (B and D), or with MG-132 in the presence of dexamethasone to induce plakoglobin expression (C). After 9 h, the cells were immunostained for β-catenin (A–C) or plakoglobin (D). Bar, 10 μm.
Mentions: To determine the localization of plakoglobin and β-catenin when the ubiquitin-proteasome proteolytic pathway is inhibited, HT1080 cells were treated with ALLN or MG-132 and plakoglobin expression was induced for 9 h. The cells were immunostained with anti-plakoglobin and anti– β-catenin antibodies (Fig. 11). The results shown in Fig. 11 demonstrate that when proteasome inhibitors were applied, a significant amount of β-catenin translocated into the nuclei of the cells (Fig. 11, B and C), in contrast to cells treated with the inactive ALLM peptide that showed only cell–cell junctional staining (Fig. 11 A). Interestingly, under these conditions plakoglobin was not organized in cell–cell junctions, but was diffusely distributed in the cytoplasm with a significant accumulation in the nuclei of the cells (Fig. 11 D). Similar results were obtained using Lactacystin, but not with other protease inhibitors including pepstatin A, aprotinin and leupeptin which have no effect on this degradation pathway (results not shown).

Bottom Line: Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques.Individual clones expressing various levels of plakoglobin were established by stable transfection.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.

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