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Distinct molecular forms of beta-catenin are targeted to adhesive or transcriptional complexes.

Gottardi CJ, Gumbiner BM - J. Cell Biol. (2004)

Bottom Line: The Wnt-stimulated, TCF-selective form is monomeric and is regulated by the COOH terminus of beta-catenin, which selectively competes cadherin binding through an intramolecular fold-back mechanism.Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation.In contrast, the main cadherin-binding form of beta-catenin is a beta-catenin-alpha-catenin dimer, indicating that there is a distinct molecular form of beta-catenin that can interact with both the cadherin and alpha-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. c-gottardi@northwestern.edu.

ABSTRACT
Beta-catenin plays essential roles in both cell-cell adhesion and Wnt signal transduction, but what precisely controls beta-catenin targeting to cadherin adhesive complexes, or T-cell factor (TCF)-transcriptional complexes is less well understood. We show that during Wnt signaling, a form of beta-catenin is generated that binds TCF but not the cadherin cytoplasmic domain. The Wnt-stimulated, TCF-selective form is monomeric and is regulated by the COOH terminus of beta-catenin, which selectively competes cadherin binding through an intramolecular fold-back mechanism. Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation. In contrast, the main cadherin-binding form of beta-catenin is a beta-catenin-alpha-catenin dimer, indicating that there is a distinct molecular form of beta-catenin that can interact with both the cadherin and alpha-catenin. We propose that participation of beta-catenin in adhesion or Wnt signaling is dictated by the regulation of distinct molecular forms of beta-catenin with different binding properties, rather than simple competition between cadherins and TCFs for a single constitutive form. This model explains how cells can control whether beta-catenin is used independently in cell adhesion and nuclear signaling, or competitively so that the two processes are coordinated and interrelated.

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β-Catenin binding selectivity as a function of APC mutant status or GSK inhibition by LiCl. (A) A cytosolic fraction was prepared from colon carcinoma cell lines containing wild-type (HCT116) or mutant (HT29 and DLD1) forms of APC. (B) Selective binding activity of β-catenin in response to short-term, but not long-term treatment with LiCl. HEK293T cells were treated with 10 mM LiCl for 3, 6, and 15 h, after which cytosolic fractions were affinity precipitated as described above.
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fig6: β-Catenin binding selectivity as a function of APC mutant status or GSK inhibition by LiCl. (A) A cytosolic fraction was prepared from colon carcinoma cell lines containing wild-type (HCT116) or mutant (HT29 and DLD1) forms of APC. (B) Selective binding activity of β-catenin in response to short-term, but not long-term treatment with LiCl. HEK293T cells were treated with 10 mM LiCl for 3, 6, and 15 h, after which cytosolic fractions were affinity precipitated as described above.

Mentions: To explore the signaling pathways that might control these forms of β-catenin, we sought to examine the role of the APC tumor suppressor gene product and glycogen synthase kinase (GSK)-3β, two key components in the Wnt pathway, as well as the role of known phosphorylations of β-catenin in response to Wnts. We examined several colon carcinoma cell lines that contain either wild-type or mutant forms of APC (Fig. 6 A). All of these cell lines manifest constitutive β-catenin signaling due to inactivating mutations in APC (HT29, DLD1), or activating mutations within the GSK-3β regulatory region of β-catenin (HCT116). No differences in β-catenin binding to cadherin- and TCF-GST fusion proteins were observed, suggesting that β-catenin binding selectivity is not simply due to inhibition of APC-mediated destruction of β-catenin. Interestingly, the role of GSK-3β is more complex. Short-term inhibition of GSK3β by lithium chloride (LiCl; under 4 h at 10 mM) mimics the Wnt effect on β-catenin, i.e., β-catenin preferentially binds TCF-GST greater than cad-GST (Fig. 6 B, lanes 1 and 2). Therefore, inhibition of GSK3β activity alone is sufficient to mimic the effect of Wnt on β-catenin binding activities. Curiously, however, long-term inhibition of GSK3β by LiCl (over 6 h at 10 mM) generates a pool of β-catenin that binds TCF and cadherin-GST proteins equally well (Fig. 6 B, lanes 3–6). Thus, more potent effects of LiCl do not mimic Wnt signaling, but instead result in the accumulation of high levels of β-catenin with no binding specificity, similar to tumor cells with APC mutations. One interpretation of these two types of effects is that GSK might have multiple targets besides the NH2 terminus of β-catenin (e.g., APC, Rubinfeld et al., 1996; or Axin, Jho et al., 1999). Alternatively, long-term incubation with LiCl could have pleiotropic effects on cell signaling pathways, or the cellular machinery that regulates β-catenin binding to TCF versus cadherin may be easily saturable, so that differential binding is not observed when β-catenin levels rise to unphysiological levels. This explanation is consistent with findings that total cytosolic levels of β-catenin appear to increase substantially with the duration of LiCl treatment (Fig. 6 B, compare lanes 2, 4, and 6), and because expression levels via transfection give similar results (Fig. 3).


Distinct molecular forms of beta-catenin are targeted to adhesive or transcriptional complexes.

Gottardi CJ, Gumbiner BM - J. Cell Biol. (2004)

β-Catenin binding selectivity as a function of APC mutant status or GSK inhibition by LiCl. (A) A cytosolic fraction was prepared from colon carcinoma cell lines containing wild-type (HCT116) or mutant (HT29 and DLD1) forms of APC. (B) Selective binding activity of β-catenin in response to short-term, but not long-term treatment with LiCl. HEK293T cells were treated with 10 mM LiCl for 3, 6, and 15 h, after which cytosolic fractions were affinity precipitated as described above.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172558&req=5

fig6: β-Catenin binding selectivity as a function of APC mutant status or GSK inhibition by LiCl. (A) A cytosolic fraction was prepared from colon carcinoma cell lines containing wild-type (HCT116) or mutant (HT29 and DLD1) forms of APC. (B) Selective binding activity of β-catenin in response to short-term, but not long-term treatment with LiCl. HEK293T cells were treated with 10 mM LiCl for 3, 6, and 15 h, after which cytosolic fractions were affinity precipitated as described above.
Mentions: To explore the signaling pathways that might control these forms of β-catenin, we sought to examine the role of the APC tumor suppressor gene product and glycogen synthase kinase (GSK)-3β, two key components in the Wnt pathway, as well as the role of known phosphorylations of β-catenin in response to Wnts. We examined several colon carcinoma cell lines that contain either wild-type or mutant forms of APC (Fig. 6 A). All of these cell lines manifest constitutive β-catenin signaling due to inactivating mutations in APC (HT29, DLD1), or activating mutations within the GSK-3β regulatory region of β-catenin (HCT116). No differences in β-catenin binding to cadherin- and TCF-GST fusion proteins were observed, suggesting that β-catenin binding selectivity is not simply due to inhibition of APC-mediated destruction of β-catenin. Interestingly, the role of GSK-3β is more complex. Short-term inhibition of GSK3β by lithium chloride (LiCl; under 4 h at 10 mM) mimics the Wnt effect on β-catenin, i.e., β-catenin preferentially binds TCF-GST greater than cad-GST (Fig. 6 B, lanes 1 and 2). Therefore, inhibition of GSK3β activity alone is sufficient to mimic the effect of Wnt on β-catenin binding activities. Curiously, however, long-term inhibition of GSK3β by LiCl (over 6 h at 10 mM) generates a pool of β-catenin that binds TCF and cadherin-GST proteins equally well (Fig. 6 B, lanes 3–6). Thus, more potent effects of LiCl do not mimic Wnt signaling, but instead result in the accumulation of high levels of β-catenin with no binding specificity, similar to tumor cells with APC mutations. One interpretation of these two types of effects is that GSK might have multiple targets besides the NH2 terminus of β-catenin (e.g., APC, Rubinfeld et al., 1996; or Axin, Jho et al., 1999). Alternatively, long-term incubation with LiCl could have pleiotropic effects on cell signaling pathways, or the cellular machinery that regulates β-catenin binding to TCF versus cadherin may be easily saturable, so that differential binding is not observed when β-catenin levels rise to unphysiological levels. This explanation is consistent with findings that total cytosolic levels of β-catenin appear to increase substantially with the duration of LiCl treatment (Fig. 6 B, compare lanes 2, 4, and 6), and because expression levels via transfection give similar results (Fig. 3).

Bottom Line: The Wnt-stimulated, TCF-selective form is monomeric and is regulated by the COOH terminus of beta-catenin, which selectively competes cadherin binding through an intramolecular fold-back mechanism.Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation.In contrast, the main cadherin-binding form of beta-catenin is a beta-catenin-alpha-catenin dimer, indicating that there is a distinct molecular form of beta-catenin that can interact with both the cadherin and alpha-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. c-gottardi@northwestern.edu.

ABSTRACT
Beta-catenin plays essential roles in both cell-cell adhesion and Wnt signal transduction, but what precisely controls beta-catenin targeting to cadherin adhesive complexes, or T-cell factor (TCF)-transcriptional complexes is less well understood. We show that during Wnt signaling, a form of beta-catenin is generated that binds TCF but not the cadherin cytoplasmic domain. The Wnt-stimulated, TCF-selective form is monomeric and is regulated by the COOH terminus of beta-catenin, which selectively competes cadherin binding through an intramolecular fold-back mechanism. Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation. In contrast, the main cadherin-binding form of beta-catenin is a beta-catenin-alpha-catenin dimer, indicating that there is a distinct molecular form of beta-catenin that can interact with both the cadherin and alpha-catenin. We propose that participation of beta-catenin in adhesion or Wnt signaling is dictated by the regulation of distinct molecular forms of beta-catenin with different binding properties, rather than simple competition between cadherins and TCFs for a single constitutive form. This model explains how cells can control whether beta-catenin is used independently in cell adhesion and nuclear signaling, or competitively so that the two processes are coordinated and interrelated.

Show MeSH
Related in: MedlinePlus