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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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The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [35S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [35S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH2-terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.
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fig5: The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [35S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [35S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH2-terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.

Mentions: The higher molecular size fractions of β-catenin could be due to its association with α-catenin or other possible proteins. To see what proteins associate with β-catenin and bind to cad-GST and TCF-GST fusion proteins, we performed the binding assay using cytosol prepared from [35S]methionine and cysteine-labeling cells. Metabolic labeling of Wnt ± expressing cells reveals α-catenin as the major binding partner of cytosolic β-catenin (Fig. 5 A, lanes 6 and 7). No other major bands were detected between the 10–200 kD molecular mass region by [35S]methionine/cysteine labeling or Coomassie staining (unpublished data). The cadherin-GST appears to affinity precipitate β-catenin and α-catenin bands at a ratio ∼1:1, whereas the TCF-GST precipitates much more β-catenin than α-catenin (band ratios of ∼3:1; Fig. 5 A, lanes 8 and 9). Given that α-catenin and β-catenin contain nearly identical numbers of cysteine and methionine residues (40 and 41, respectively), and that the labeling time is long (13 h) compared with the half lives of both proteins (7 and 5 h, respectively; unpublished data), it appears that the cadherin binds a stoichiometric complex of β-catenin–α-catenin. We conclude, therefore, that the cadherin preferentially binds β-catenin that is associated with α-catenin, whereas TCF can bind monomeric β-catenin in addition to β-catenin–α-catenin dimers.


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

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

The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [35S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [35S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH2-terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.
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fig5: The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [35S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [35S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH2-terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.
Mentions: The higher molecular size fractions of β-catenin could be due to its association with α-catenin or other possible proteins. To see what proteins associate with β-catenin and bind to cad-GST and TCF-GST fusion proteins, we performed the binding assay using cytosol prepared from [35S]methionine and cysteine-labeling cells. Metabolic labeling of Wnt ± expressing cells reveals α-catenin as the major binding partner of cytosolic β-catenin (Fig. 5 A, lanes 6 and 7). No other major bands were detected between the 10–200 kD molecular mass region by [35S]methionine/cysteine labeling or Coomassie staining (unpublished data). The cadherin-GST appears to affinity precipitate β-catenin and α-catenin bands at a ratio ∼1:1, whereas the TCF-GST precipitates much more β-catenin than α-catenin (band ratios of ∼3:1; Fig. 5 A, lanes 8 and 9). Given that α-catenin and β-catenin contain nearly identical numbers of cysteine and methionine residues (40 and 41, respectively), and that the labeling time is long (13 h) compared with the half lives of both proteins (7 and 5 h, respectively; unpublished data), it appears that the cadherin binds a stoichiometric complex of β-catenin–α-catenin. We conclude, therefore, that the cadherin preferentially binds β-catenin that is associated with α-catenin, whereas TCF can bind monomeric β-catenin in addition to β-catenin–α-catenin dimers.

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