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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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Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin (Suh and Gumbiner, 2003) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.
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fig1: Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin (Suh and Gumbiner, 2003) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.

Mentions: We sought to determine the binding properties of β-catenin generated by Wnt signaling. Using an in vitro pull-down assay, we find that cytosolic β-catenin from cells stably expressing Wnt1 shows preferential binding to TCF-GST compared with a cadherin cytoplasmic domain-GST fusion protein (Fig. 1 A, lanes 1–4). The selective binding activity of β-catenin does not require stable, long-term expression of Wnt in cells, as similar binding properties are observed in human embryonic kidney (HEK) 293T cells incubated with Wnt3a-conditioned media for short periods (Fig. 1 A, lanes 7 and 8). Importantly, this difference in binding is not observed in the untreated or parental cell lines (Fig. 1, lanes 5, 6, 9, and 10), with recombinant β-catenin purified from SF9 cells (Fig. 1 B), or in a cell line that contains elevated levels of β-catenin due to loss of the APC tumor suppressor (Fig. 1, lanes 11 and 12). These findings demonstrate that the differential binding activity of β-catenin is actually induced by Wnts, and is not simply due to binding differences between cad-GST and TCF-GST recombinant proteins, nor to the accumulation of high levels of cytosolic β-catenin. Thus, Wnts may activate β-catenin signaling by generating a molecular form of β-catenin that selectively binds to the downstream transcription factor, TCF, as well as by raising the overall cytosolic levels of β-catenin.


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

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

Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin (Suh and Gumbiner, 2003) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172558&req=5

fig1: Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin (Suh and Gumbiner, 2003) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.
Mentions: We sought to determine the binding properties of β-catenin generated by Wnt signaling. Using an in vitro pull-down assay, we find that cytosolic β-catenin from cells stably expressing Wnt1 shows preferential binding to TCF-GST compared with a cadherin cytoplasmic domain-GST fusion protein (Fig. 1 A, lanes 1–4). The selective binding activity of β-catenin does not require stable, long-term expression of Wnt in cells, as similar binding properties are observed in human embryonic kidney (HEK) 293T cells incubated with Wnt3a-conditioned media for short periods (Fig. 1 A, lanes 7 and 8). Importantly, this difference in binding is not observed in the untreated or parental cell lines (Fig. 1, lanes 5, 6, 9, and 10), with recombinant β-catenin purified from SF9 cells (Fig. 1 B), or in a cell line that contains elevated levels of β-catenin due to loss of the APC tumor suppressor (Fig. 1, lanes 11 and 12). These findings demonstrate that the differential binding activity of β-catenin is actually induced by Wnts, and is not simply due to binding differences between cad-GST and TCF-GST recombinant proteins, nor to the accumulation of high levels of cytosolic β-catenin. Thus, Wnts may activate β-catenin signaling by generating a molecular form of β-catenin that selectively binds to the downstream transcription factor, TCF, as well as by raising the overall cytosolic levels of β-catenin.

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