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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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Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT (Gottardi and Gumbiner, 2004). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.
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fig8: Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT (Gottardi and Gumbiner, 2004). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.

Mentions: We also asked whether modification of the cadherin could affect β-catenin binding selectivity. A previous study showed that the serine-rich, β-catenin binding region of the cadherin is phosphorylated in vivo (Stappert and Kemler, 1994), and this phosphorylation can enhance β-catenin binding to the cadherin (Lickert et al., 2000; Huber and Weis, 2001). We therefore wished to explore whether β-catenin binding selectivity for TCF in Wnt stimulated cells would be altered by cadherin phosphorylation in our binding assay. Phosphorylation of the cadherin greatly enhances binding to β-catenin compared with unphosphorylated cadherin (Fig. 8 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200402153/DC1). Indeed, phospho-cadherin is able to binding the same fraction of β-catenin that binds TCF (Fig. 8, B and C), suggesting that cadherin phosphorylation allows the monomeric, closed form of β-catenin to bind the cadherin. Thus, although Wnt signaling generates a form of β-catenin that exhibits preferential binding to TCF over the cadherin, this mechanism can be overridden by extensive phosphorylation of the cadherin.


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

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

Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT (Gottardi and Gumbiner, 2004). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.
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fig8: Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT (Gottardi and Gumbiner, 2004). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.
Mentions: We also asked whether modification of the cadherin could affect β-catenin binding selectivity. A previous study showed that the serine-rich, β-catenin binding region of the cadherin is phosphorylated in vivo (Stappert and Kemler, 1994), and this phosphorylation can enhance β-catenin binding to the cadherin (Lickert et al., 2000; Huber and Weis, 2001). We therefore wished to explore whether β-catenin binding selectivity for TCF in Wnt stimulated cells would be altered by cadherin phosphorylation in our binding assay. Phosphorylation of the cadherin greatly enhances binding to β-catenin compared with unphosphorylated cadherin (Fig. 8 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200402153/DC1). Indeed, phospho-cadherin is able to binding the same fraction of β-catenin that binds TCF (Fig. 8, B and C), suggesting that cadherin phosphorylation allows the monomeric, closed form of β-catenin to bind the cadherin. Thus, although Wnt signaling generates a form of β-catenin that exhibits preferential binding to TCF over the cadherin, this mechanism can be overridden by extensive phosphorylation of the cadherin.

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