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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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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.
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Related In: Results  -  Collection

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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: 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