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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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Expression of β-catenin and plakoglobin in cells  treated with inhibitors of the ubiquitin-proteasome system. (A)  HT1080 cells were treated for 2 h with proteasome inhibitors  (MG 132 or ALLN), or with the inactive analogue ALLM, as described in Materials and Methods, and then induced to express  plakoglobin with dexamethasone in the presence of the inhibitors. At different times after dexamethasone stimulation, equal  amounts of total cell lysate were analyzed for β-catenin and plakoglobin expression by immunoblotting as described in Fig. 8.  (B) The immunoblot for β-catenin was overexposed to reveal the  higher molecular mass forms of β-catenin (arrowhead, probably  ubiquitinated) formed in the presence of the proteasome inhibitors.
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Figure 10: Expression of β-catenin and plakoglobin in cells treated with inhibitors of the ubiquitin-proteasome system. (A) HT1080 cells were treated for 2 h with proteasome inhibitors (MG 132 or ALLN), or with the inactive analogue ALLM, as described in Materials and Methods, and then induced to express plakoglobin with dexamethasone in the presence of the inhibitors. At different times after dexamethasone stimulation, equal amounts of total cell lysate were analyzed for β-catenin and plakoglobin expression by immunoblotting as described in Fig. 8. (B) The immunoblot for β-catenin was overexposed to reveal the higher molecular mass forms of β-catenin (arrowhead, probably ubiquitinated) formed in the presence of the proteasome inhibitors.

Mentions: A recent study has demonstrated that the turnover of β-catenin is mediated by the ubiquitin-proteasome degradation system (Aberle et al., 1997). When this proteolytic pathway is inhibited by specific inhibitors of the proteasome-mediated proteolysis such as Lactacystin, MG-132 and the peptide aldehyde ALLN, it leads to the stabilization and accumulation of ubiquitinated forms of β-catenin (Aberle et al., 1997). To examine if stabilization of β-catenin against proteasome-mediated degradation will block the decrease in β-catenin levels of HT1080 cells induced to express plakoglobin, we treated cells with MG-132 or ALLN and determined the levels of β-catenin and plakoglobin. The results summarized in Fig. 10 show that while β-catenin levels were reduced in cells treated with an inactive peptide analogue (ALLM) (Fig. 10 A, lanes 1 and 2), they were even higher than the controls in the presence of the active proteasome inhibitors ALLN and MG-132 (Fig. 10 A, lanes 3–8, compare to lanes 1, 2, and 9). Furthermore, a higher molecular mass form of β-catenin, probably representing ubiquitinated β-catenin, could be detected in the presence of these inhibitors (Fig. 10 B, lanes 3–8), in agreement with Aberle et al. (1997). These results imply that plakoglobin overexpression cannot confer a decrease in β-catenin levels when the proteasome degradation pathway is inhibited, which results in β-catenin stabilization against degradation.


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)

Expression of β-catenin and plakoglobin in cells  treated with inhibitors of the ubiquitin-proteasome system. (A)  HT1080 cells were treated for 2 h with proteasome inhibitors  (MG 132 or ALLN), or with the inactive analogue ALLM, as described in Materials and Methods, and then induced to express  plakoglobin with dexamethasone in the presence of the inhibitors. At different times after dexamethasone stimulation, equal  amounts of total cell lysate were analyzed for β-catenin and plakoglobin expression by immunoblotting as described in Fig. 8.  (B) The immunoblot for β-catenin was overexposed to reveal the  higher molecular mass forms of β-catenin (arrowhead, probably  ubiquitinated) formed in the presence of the proteasome inhibitors.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Expression of β-catenin and plakoglobin in cells treated with inhibitors of the ubiquitin-proteasome system. (A) HT1080 cells were treated for 2 h with proteasome inhibitors (MG 132 or ALLN), or with the inactive analogue ALLM, as described in Materials and Methods, and then induced to express plakoglobin with dexamethasone in the presence of the inhibitors. At different times after dexamethasone stimulation, equal amounts of total cell lysate were analyzed for β-catenin and plakoglobin expression by immunoblotting as described in Fig. 8. (B) The immunoblot for β-catenin was overexposed to reveal the higher molecular mass forms of β-catenin (arrowhead, probably ubiquitinated) formed in the presence of the proteasome inhibitors.
Mentions: A recent study has demonstrated that the turnover of β-catenin is mediated by the ubiquitin-proteasome degradation system (Aberle et al., 1997). When this proteolytic pathway is inhibited by specific inhibitors of the proteasome-mediated proteolysis such as Lactacystin, MG-132 and the peptide aldehyde ALLN, it leads to the stabilization and accumulation of ubiquitinated forms of β-catenin (Aberle et al., 1997). To examine if stabilization of β-catenin against proteasome-mediated degradation will block the decrease in β-catenin levels of HT1080 cells induced to express plakoglobin, we treated cells with MG-132 or ALLN and determined the levels of β-catenin and plakoglobin. The results summarized in Fig. 10 show that while β-catenin levels were reduced in cells treated with an inactive peptide analogue (ALLM) (Fig. 10 A, lanes 1 and 2), they were even higher than the controls in the presence of the active proteasome inhibitors ALLN and MG-132 (Fig. 10 A, lanes 3–8, compare to lanes 1, 2, and 9). Furthermore, a higher molecular mass form of β-catenin, probably representing ubiquitinated β-catenin, could be detected in the presence of these inhibitors (Fig. 10 B, lanes 3–8), in agreement with Aberle et al. (1997). These results imply that plakoglobin overexpression cannot confer a decrease in β-catenin levels when the proteasome degradation pathway is inhibited, which results in β-catenin stabilization against degradation.

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