Limits...
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.

Show MeSH

Related in: MedlinePlus

Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc-Xenopus β-catenin and GSK3β mutant (S/T>A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000). WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999). The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously (Funayama et al., 1995). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172558&req=5

fig3: Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc-Xenopus β-catenin and GSK3β mutant (S/T>A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000). WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999). The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously (Funayama et al., 1995). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).

Mentions: To further determine the regions of β-catenin control this binding selectivity, we also examined the binding properties of a series of β-catenin deletion mutants expressed in cells. Full-length NH2-terminal myc-, or COOH-terminal flag-tagged β-catenin exhibits preferential binding to TCF-GST relative to cad-GST when transfected into HEK293 cells incubated with Wnt3a-conditioned media (Fig. 3). Importantly, the level of exogenous β-catenin expression needed to be kept low in order to detect β-catenin binding selectivity (compare 0.2 μg with 2 μg plasmid), suggesting that the cellular machinery responsible for generating binding selectivity may be easily saturable. As a control, simply diluting the sample transfected with 2 μg plasmid by 10-fold, so that the β-catenin levels were similar to those extracts transfected with 0.2 μg plasmid, did not result in differential binding (unpublished data), indicating that binding selectivity is due to an active cellular process. A construct bearing a deletion of the COOH terminus (hβ-catΔC695) shows equivalent binding to both cadherin and TCF, consistent with a role for the COOH terminus in regulating binding selectivity. Curiously, however, deletion of both NH2- and COOH-terminal domains generates a protein that binds TCF significantly better than the cadherin, even at relatively high levels of expression (e.g., compare 3 μg plasmid for Xβ-cat arm 12 with WTX-β-cat 2 μg). It was recently proposed that the NH2-terminal region of β-catenin may be required for efficient cadherin binding, as a GST-β-catenin fusion protein missing the first 119 aa showed little cadherin binding activity in vitro (Castano et al., 2002). We also find that Δ89β-catenin binds poorly to the cadherin compared with TCF, even at our highest expression levels (Fig. 3). Thus, we suggest that the NH2-terminal region of β-catenin is required for cadherin but not TCF binding, which gives rise to apparent binding selectivity. When β-catenin is able to bind the cadherin (i.e., when the NH2 terminus is present), however, the COOH terminus is required for generating β-catenin binding selectivity.


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

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

Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc-Xenopus β-catenin and GSK3β mutant (S/T>A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000). WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999). The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously (Funayama et al., 1995). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc-Xenopus β-catenin and GSK3β mutant (S/T>A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000). WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999). The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously (Funayama et al., 1995). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).
Mentions: To further determine the regions of β-catenin control this binding selectivity, we also examined the binding properties of a series of β-catenin deletion mutants expressed in cells. Full-length NH2-terminal myc-, or COOH-terminal flag-tagged β-catenin exhibits preferential binding to TCF-GST relative to cad-GST when transfected into HEK293 cells incubated with Wnt3a-conditioned media (Fig. 3). Importantly, the level of exogenous β-catenin expression needed to be kept low in order to detect β-catenin binding selectivity (compare 0.2 μg with 2 μg plasmid), suggesting that the cellular machinery responsible for generating binding selectivity may be easily saturable. As a control, simply diluting the sample transfected with 2 μg plasmid by 10-fold, so that the β-catenin levels were similar to those extracts transfected with 0.2 μg plasmid, did not result in differential binding (unpublished data), indicating that binding selectivity is due to an active cellular process. A construct bearing a deletion of the COOH terminus (hβ-catΔC695) shows equivalent binding to both cadherin and TCF, consistent with a role for the COOH terminus in regulating binding selectivity. Curiously, however, deletion of both NH2- and COOH-terminal domains generates a protein that binds TCF significantly better than the cadherin, even at relatively high levels of expression (e.g., compare 3 μg plasmid for Xβ-cat arm 12 with WTX-β-cat 2 μg). It was recently proposed that the NH2-terminal region of β-catenin may be required for efficient cadherin binding, as a GST-β-catenin fusion protein missing the first 119 aa showed little cadherin binding activity in vitro (Castano et al., 2002). We also find that Δ89β-catenin binds poorly to the cadherin compared with TCF, even at our highest expression levels (Fig. 3). Thus, we suggest that the NH2-terminal region of β-catenin is required for cadherin but not TCF binding, which gives rise to apparent binding selectivity. When β-catenin is able to bind the cadherin (i.e., when the NH2 terminus is present), however, the COOH terminus is required for generating β-catenin binding selectivity.

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