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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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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: 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