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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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Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.
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fig4: Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.

Mentions: To better characterize the molecular forms of β-catenin that bind to cad-GST and TCF-GST fusion proteins, we examined cytosolic fractions by gel filtration chromatography (Fig. 4). We began our analysis with Xenopus embryos, because it was easier to obtain a large amount of cytosolic β-catenin from cell lysates (Fig. 4 A). One half of each column fraction was precipitated with TCA to show the total profile of β-catenin (Fig. 4 A, top blot); the other half was subjected to cadherin-GST affinity precipitation. β-Catenin eluted across a number of fractions extending from 66 kD (BSA standard, peak fraction 37) to 232 kD (catalase standard, peak fraction 30). The major peak corresponded to ∼100 kD (fraction 35), suggesting that most of the cytosolic β-catenin is monomeric. Importantly, only fractions from the higher molecular mass “shoulder” of the broad β-catenin profile were able to interact with the cadherin cytoplasmic domain (Fig. 4 A, middle blot), whereas TCF could also bind the lower molecular sized fractions corresponding to the major peak (Fig. 4 B below and not depicted). The high molecular weight cadherin-binding fractions cofractionated with α-catenin (Fig. 4 A, bottom blot), suggesting that they contain β-catenin–α-catenin dimers. A similar binding profile was observed with a cytosolic lysate from Wnt-expressing Rat1 cells (Fig. 4 B). However unlike in Xenopus, the cadherin-binding fractions did not perfectly cofractionate with the α-catenin-containing fractions (Fig. 4 B). This small difference in fractionation profiles may be due to the presence of α-catenin homodimers, which may fractionate differently than β-catenin–α-catenin dimers during gel filtration (Koslov et al., 1997). Nevertheless, the cadherin preferentially binds a form of β-catenin that cofractionates with α-catenin, rather than the smaller molecular size fractions, which can bind TCF. Furthermore, the preferential binding of β-catenin to TCF compared with the cadherin observed in Fig. 1 appears to be due to an ability of TCF, but not the cadherin, to bind the monomeric form of β-catenin.


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

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

Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.
Mentions: To better characterize the molecular forms of β-catenin that bind to cad-GST and TCF-GST fusion proteins, we examined cytosolic fractions by gel filtration chromatography (Fig. 4). We began our analysis with Xenopus embryos, because it was easier to obtain a large amount of cytosolic β-catenin from cell lysates (Fig. 4 A). One half of each column fraction was precipitated with TCA to show the total profile of β-catenin (Fig. 4 A, top blot); the other half was subjected to cadherin-GST affinity precipitation. β-Catenin eluted across a number of fractions extending from 66 kD (BSA standard, peak fraction 37) to 232 kD (catalase standard, peak fraction 30). The major peak corresponded to ∼100 kD (fraction 35), suggesting that most of the cytosolic β-catenin is monomeric. Importantly, only fractions from the higher molecular mass “shoulder” of the broad β-catenin profile were able to interact with the cadherin cytoplasmic domain (Fig. 4 A, middle blot), whereas TCF could also bind the lower molecular sized fractions corresponding to the major peak (Fig. 4 B below and not depicted). The high molecular weight cadherin-binding fractions cofractionated with α-catenin (Fig. 4 A, bottom blot), suggesting that they contain β-catenin–α-catenin dimers. A similar binding profile was observed with a cytosolic lysate from Wnt-expressing Rat1 cells (Fig. 4 B). However unlike in Xenopus, the cadherin-binding fractions did not perfectly cofractionate with the α-catenin-containing fractions (Fig. 4 B). This small difference in fractionation profiles may be due to the presence of α-catenin homodimers, which may fractionate differently than β-catenin–α-catenin dimers during gel filtration (Koslov et al., 1997). Nevertheless, the cadherin preferentially binds a form of β-catenin that cofractionates with α-catenin, rather than the smaller molecular size fractions, which can bind TCF. Furthermore, the preferential binding of β-catenin to TCF compared with the cadherin observed in Fig. 1 appears to be due to an ability of TCF, but not the cadherin, to bind the monomeric form 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