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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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Triton X-100 solubility of β-catenin in cells expressing  full-length and mutant plakoglobin. HT1080 cells transfected with  full-length plakoglobin (A), or with the COOH-terminal deletion  mutant ΔC161 (B) were stimulated to express plakoglobin by  dexamethasone, and at various times after induction the levels of  plakoglobin and β-catenin were determined in the Triton X-100– soluble and –insoluble fractions as described in Fig. 7.
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Figure 8: Triton X-100 solubility of β-catenin in cells expressing full-length and mutant plakoglobin. HT1080 cells transfected with full-length plakoglobin (A), or with the COOH-terminal deletion mutant ΔC161 (B) were stimulated to express plakoglobin by dexamethasone, and at various times after induction the levels of plakoglobin and β-catenin were determined in the Triton X-100– soluble and –insoluble fractions as described in Fig. 7.

Mentions: We have also determined the distribution of β-catenin between the Triton X-100–soluble and –insoluble fractions in HT1080 cells expressing full-length plakoglobin and in COOH-terminal deleted plakoglobin expressing cells (ΔC161) where the truncated plakoglobin could not confer a decrease in β-catenin levels (see Fig. 9). The results summarized in Fig. 8 show that β-catenin levels were reduced in both Triton X-100–soluble and –insoluble fractions upon plakoglobin induction, and a lower molecular mass product of β-catenin (probably a degraded form) was apparent at later times after plakoglobin induction (Fig. 8 A, lanes 7–9). In ΔC161-expressing cells, no significant changes in β-catenin levels and detergent solubility were apparent when the mutant plakoglobin (mainly found in the soluble fraction) was induced (Fig. 8 B).


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)

Triton X-100 solubility of β-catenin in cells expressing  full-length and mutant plakoglobin. HT1080 cells transfected with  full-length plakoglobin (A), or with the COOH-terminal deletion  mutant ΔC161 (B) were stimulated to express plakoglobin by  dexamethasone, and at various times after induction the levels of  plakoglobin and β-catenin were determined in the Triton X-100– soluble and –insoluble fractions as described in Fig. 7.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Triton X-100 solubility of β-catenin in cells expressing full-length and mutant plakoglobin. HT1080 cells transfected with full-length plakoglobin (A), or with the COOH-terminal deletion mutant ΔC161 (B) were stimulated to express plakoglobin by dexamethasone, and at various times after induction the levels of plakoglobin and β-catenin were determined in the Triton X-100– soluble and –insoluble fractions as described in Fig. 7.
Mentions: We have also determined the distribution of β-catenin between the Triton X-100–soluble and –insoluble fractions in HT1080 cells expressing full-length plakoglobin and in COOH-terminal deleted plakoglobin expressing cells (ΔC161) where the truncated plakoglobin could not confer a decrease in β-catenin levels (see Fig. 9). The results summarized in Fig. 8 show that β-catenin levels were reduced in both Triton X-100–soluble and –insoluble fractions upon plakoglobin induction, and a lower molecular mass product of β-catenin (probably a degraded form) was apparent at later times after plakoglobin induction (Fig. 8 A, lanes 7–9). In ΔC161-expressing cells, no significant changes in β-catenin levels and detergent solubility were apparent when the mutant plakoglobin (mainly found in the soluble fraction) was induced (Fig. 8 B).

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