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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: We show that during Wnt signaling, a form of beta-catenin is generated that binds TCF but not the cadherin cytoplasmic domain.Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation.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.

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 COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin (Huber et al., 1997; Graham et al., 2000; Pokutta and Weis, 2000; Huber and Weis, 2001). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid (Cox et al., 1999) and recombinant protein assays (Piedra et al., 2001). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein. Recombinant β-catenin (1.5 μg) purified from baculovirus (Suh and Gumbiner, 2003) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2). A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH2-terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).
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fig2: The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin (Huber et al., 1997; Graham et al., 2000; Pokutta and Weis, 2000; Huber and Weis, 2001). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid (Cox et al., 1999) and recombinant protein assays (Piedra et al., 2001). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein. Recombinant β-catenin (1.5 μg) purified from baculovirus (Suh and Gumbiner, 2003) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2). A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH2-terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).

Mentions: Several findings influenced our investigation of a mechanism that could generate a form of β-catenin that binds selectively to TCF. The COOH terminus of β-catenin can interact with the armadillo repeat region of β-catenin (Cox et al., 1999; Piedra et al., 2001) and compete with β-catenin binding to the cadherin cytoplasmic domain in vitro (Castano et al., 2002). These observations raised the possibility that conformational changes in the COOH terminus of β-catenin, and in particular, a “closed” conformation, might be incompatible with cadherin binding. Because Wnt signaling alters β-catenin binding to the cadherin and TCF differently (Fig. 1), we sought to determine whether the COOH terminus of β-catenin might contribute to this binding selectivity. Indeed, although the COOH terminus of β-catenin can compete β-catenin binding to the cadherin, as demonstrated previously (Castano et al., 2002), its interaction with TCF is not competed (Fig. 2 C). Thus, if a COOH-terminal conformational change giving rise to a closed form of β-catenin were to occur during Wnt signaling, it could alter β-catenin ligand interactions selectively.


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

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

The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin (Huber et al., 1997; Graham et al., 2000; Pokutta and Weis, 2000; Huber and Weis, 2001). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid (Cox et al., 1999) and recombinant protein assays (Piedra et al., 2001). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein. Recombinant β-catenin (1.5 μg) purified from baculovirus (Suh and Gumbiner, 2003) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2). A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH2-terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).
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Related In: Results  -  Collection

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fig2: The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin (Huber et al., 1997; Graham et al., 2000; Pokutta and Weis, 2000; Huber and Weis, 2001). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid (Cox et al., 1999) and recombinant protein assays (Piedra et al., 2001). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein. Recombinant β-catenin (1.5 μg) purified from baculovirus (Suh and Gumbiner, 2003) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2). A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH2-terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).
Mentions: Several findings influenced our investigation of a mechanism that could generate a form of β-catenin that binds selectively to TCF. The COOH terminus of β-catenin can interact with the armadillo repeat region of β-catenin (Cox et al., 1999; Piedra et al., 2001) and compete with β-catenin binding to the cadherin cytoplasmic domain in vitro (Castano et al., 2002). These observations raised the possibility that conformational changes in the COOH terminus of β-catenin, and in particular, a “closed” conformation, might be incompatible with cadherin binding. Because Wnt signaling alters β-catenin binding to the cadherin and TCF differently (Fig. 1), we sought to determine whether the COOH terminus of β-catenin might contribute to this binding selectivity. Indeed, although the COOH terminus of β-catenin can compete β-catenin binding to the cadherin, as demonstrated previously (Castano et al., 2002), its interaction with TCF is not competed (Fig. 2 C). Thus, if a COOH-terminal conformational change giving rise to a closed form of β-catenin were to occur during Wnt signaling, it could alter β-catenin ligand interactions selectively.

Bottom Line: We show that during Wnt signaling, a form of beta-catenin is generated that binds TCF but not the cadherin cytoplasmic domain.Phosphorylation of the cadherin reverses the TCF binding selectivity, suggesting another potential layer of regulation.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.

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