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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 RNA and stability of β-catenin  protein in cells overexpressing inducible plakoglobin. (A) RNA  was extracted from MDCK cells (lane 1), untransfected HT1080  cells (lane 4), and plakoglobin-transfected HT1080 cells before  (lane 3), and 12 h after stimulation with dexamethasone (lane 2).  Equal amounts of total cell RNA were analyzed by Northern blot  hybridization with plakoglobin (PG) and β-catenin (β-CAT)  cDNAs. The levels of 18 and 28S rRNA are shown for comparison. (B) Uninduced (−Dex) and cells induced for 12 h (+Dex)  were pulse labeled with [35S]methionine for 30 min, followed by  chase with fresh medium. Equal amounts of radioactive cellular  proteins were immunoprecipitated with anti–β-catenin antibody  and analyzed by SDS-PAGE. (C) The radioactivity in the β-catenin band in B and in an identical independent experiment was determined by a phosphorimager, and the values ±SD are presented  as percent of the values obtained after 30 min pulse labeling.
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Figure 4: Expression of β-catenin RNA and stability of β-catenin protein in cells overexpressing inducible plakoglobin. (A) RNA was extracted from MDCK cells (lane 1), untransfected HT1080 cells (lane 4), and plakoglobin-transfected HT1080 cells before (lane 3), and 12 h after stimulation with dexamethasone (lane 2). Equal amounts of total cell RNA were analyzed by Northern blot hybridization with plakoglobin (PG) and β-catenin (β-CAT) cDNAs. The levels of 18 and 28S rRNA are shown for comparison. (B) Uninduced (−Dex) and cells induced for 12 h (+Dex) were pulse labeled with [35S]methionine for 30 min, followed by chase with fresh medium. Equal amounts of radioactive cellular proteins were immunoprecipitated with anti–β-catenin antibody and analyzed by SDS-PAGE. (C) The radioactivity in the β-catenin band in B and in an identical independent experiment was determined by a phosphorimager, and the values ±SD are presented as percent of the values obtained after 30 min pulse labeling.

Mentions: To determine if the decrease in β-catenin levels resulted from a reduction in β-catenin RNA, Northern blots from uninduced and dexamethasone-induced HT1080 cells were hybridized with cDNAs to β-catenin and plakoglobin (Fig. 4 A). Dexamethasone stimulation resulted in a dramatic increase in plakoglobin RNA levels (Fig. 4 A, compare lanes 2 with 3), but did not effect the level of β-catenin RNA (Fig. 4 A). This suggests that the decrease in β-catenin levels of plakoglobin overexpressing cells does not result from a loss of β-catenin mRNA.


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 RNA and stability of β-catenin  protein in cells overexpressing inducible plakoglobin. (A) RNA  was extracted from MDCK cells (lane 1), untransfected HT1080  cells (lane 4), and plakoglobin-transfected HT1080 cells before  (lane 3), and 12 h after stimulation with dexamethasone (lane 2).  Equal amounts of total cell RNA were analyzed by Northern blot  hybridization with plakoglobin (PG) and β-catenin (β-CAT)  cDNAs. The levels of 18 and 28S rRNA are shown for comparison. (B) Uninduced (−Dex) and cells induced for 12 h (+Dex)  were pulse labeled with [35S]methionine for 30 min, followed by  chase with fresh medium. Equal amounts of radioactive cellular  proteins were immunoprecipitated with anti–β-catenin antibody  and analyzed by SDS-PAGE. (C) The radioactivity in the β-catenin band in B and in an identical independent experiment was determined by a phosphorimager, and the values ±SD are presented  as percent of the values obtained after 30 min pulse labeling.
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Related In: Results  -  Collection

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Figure 4: Expression of β-catenin RNA and stability of β-catenin protein in cells overexpressing inducible plakoglobin. (A) RNA was extracted from MDCK cells (lane 1), untransfected HT1080 cells (lane 4), and plakoglobin-transfected HT1080 cells before (lane 3), and 12 h after stimulation with dexamethasone (lane 2). Equal amounts of total cell RNA were analyzed by Northern blot hybridization with plakoglobin (PG) and β-catenin (β-CAT) cDNAs. The levels of 18 and 28S rRNA are shown for comparison. (B) Uninduced (−Dex) and cells induced for 12 h (+Dex) were pulse labeled with [35S]methionine for 30 min, followed by chase with fresh medium. Equal amounts of radioactive cellular proteins were immunoprecipitated with anti–β-catenin antibody and analyzed by SDS-PAGE. (C) The radioactivity in the β-catenin band in B and in an identical independent experiment was determined by a phosphorimager, and the values ±SD are presented as percent of the values obtained after 30 min pulse labeling.
Mentions: To determine if the decrease in β-catenin levels resulted from a reduction in β-catenin RNA, Northern blots from uninduced and dexamethasone-induced HT1080 cells were hybridized with cDNAs to β-catenin and plakoglobin (Fig. 4 A). Dexamethasone stimulation resulted in a dramatic increase in plakoglobin RNA levels (Fig. 4 A, compare lanes 2 with 3), but did not effect the level of β-catenin RNA (Fig. 4 A). This suggests that the decrease in β-catenin levels of plakoglobin overexpressing cells does not result from a loss of β-catenin mRNA.

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