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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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β-Catenin not phosphorylated at NH2-terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH2-terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH2-terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002).
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fig7: β-Catenin not phosphorylated at NH2-terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH2-terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH2-terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002).

Mentions: Several well-characterized NH2-terminal GSK phosphorylation sites are known to target β-catenin for degradation by an SCF-E3-ligase complex (Winston et al., 1999), and recent work has shown that Wnt signaling is specifically mediated through forms of β-catenin that remain unphosphorylated at these NH2-terminal sites S-33, -37, and T-41 (Staal et al., 2002). We therefore asked whether the state of phosphorylation at these sites is responsible for generating the form of β-catenin selective for TCF-binding. A pAb that recognizes NH2-terminal, unphosphorylated β-catenin was shown to stain cell nuclei, whereas cell–cell contact staining was conspicuously absent (Staal et al., 2002), raising the possibility that unphosphorylated forms of β-catenin might preferentially bind to TCF in the nucleus, and be unable to bind cadherins at the plasma membrane. Contrary to this suggestion, however, we find that β-catenin that is not phosphorylated at residues 33 and 37 can interact with the cad-GST in vitro (Fig. 7 A), associate with endogenous cadherin proteins in cell lysates (not depicted), and localize to sites of cell–cell contact (not the nucleus, as originally observed in Staal et al., 2002; Fig. 7 B). Moreover, cells transfected with a form of β-catenin that cannot be phosphorylated by GSK3β and casein kinase (CK1; S/T>A point mutants at residues 33, 37, 41 and 45) exhibit binding selectivity similar to cells transfected with wild-type β-catenin (Fig. 3). Thus, the preferential binding of β-catenin to TCF over cadherin is not simply due to NH2-terminal phosphorylation status.


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

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

β-Catenin not phosphorylated at NH2-terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH2-terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH2-terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002).
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Related In: Results  -  Collection

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

fig7: β-Catenin not phosphorylated at NH2-terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH2-terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH2-terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002).
Mentions: Several well-characterized NH2-terminal GSK phosphorylation sites are known to target β-catenin for degradation by an SCF-E3-ligase complex (Winston et al., 1999), and recent work has shown that Wnt signaling is specifically mediated through forms of β-catenin that remain unphosphorylated at these NH2-terminal sites S-33, -37, and T-41 (Staal et al., 2002). We therefore asked whether the state of phosphorylation at these sites is responsible for generating the form of β-catenin selective for TCF-binding. A pAb that recognizes NH2-terminal, unphosphorylated β-catenin was shown to stain cell nuclei, whereas cell–cell contact staining was conspicuously absent (Staal et al., 2002), raising the possibility that unphosphorylated forms of β-catenin might preferentially bind to TCF in the nucleus, and be unable to bind cadherins at the plasma membrane. Contrary to this suggestion, however, we find that β-catenin that is not phosphorylated at residues 33 and 37 can interact with the cad-GST in vitro (Fig. 7 A), associate with endogenous cadherin proteins in cell lysates (not depicted), and localize to sites of cell–cell contact (not the nucleus, as originally observed in Staal et al., 2002; Fig. 7 B). Moreover, cells transfected with a form of β-catenin that cannot be phosphorylated by GSK3β and casein kinase (CK1; S/T>A point mutants at residues 33, 37, 41 and 45) exhibit binding selectivity similar to cells transfected with wild-type β-catenin (Fig. 3). Thus, the preferential binding of β-catenin to TCF over cadherin is not simply due to NH2-terminal phosphorylation status.

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