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Activity of the beta-catenin phosphodestruction complex at cell-cell contacts is enhanced by cadherin-based adhesion.

Maher MT, Flozak AS, Stocker AM, Chenn A, Gottardi CJ - J. Cell Biol. (2009)

Bottom Line: It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering beta-catenin (beta-cat) from T-cell factor family transcription factors.We show that axin, APC2, GSK-3beta and N-terminally phosphorylated forms of beta-cat can localize to cell-cell contacts in a complex that is molecularly distinct from the cadherin-catenin adhesive complex.Together, these data suggest that cadherin-based cell-cell adhesion limits Wnt signals by promoting the activity of a junction-localized beta-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering beta-catenin (beta-cat) from T-cell factor family transcription factors. Whether changes in intercellular adhesion can affect beta-cat signaling and the mechanism through which this occurs has remained unresolved. We show that axin, APC2, GSK-3beta and N-terminally phosphorylated forms of beta-cat can localize to cell-cell contacts in a complex that is molecularly distinct from the cadherin-catenin adhesive complex. Nonetheless, cadherins can promote the N-terminal phosphorylation of beta-cat, and cell-cell adhesion increases the turnover of cytosolic beta-cat. Together, these data suggest that cadherin-based cell-cell adhesion limits Wnt signals by promoting the activity of a junction-localized beta-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development.

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Density-dependent turnover of cytosolic β-cat. (A) Turnover of cytosolic (GST-ICAT bound) β-cat by CHX chase method from LiCl-treated subconfluent and confluent MDCK cells. Densitometry values were normalized to the t0 value and plotted as shown. The plot represents the mean of two independent experiments; a set of representative immunoblots is shown. Error bars represent standard deviation of duplicate experiments. The numbers below each lane reflect normalized densitometry values. Affinity ppt, affinity precipitated; WB, Western blot. (B) Lysates from subconfluent (SC) and confluent (C) cells immunoprecipitated for E-cad and blotted for β-cat. (C and D) MDCK cells stably expressing TOP-dGFP show enhanced β-cat signaling in cells along a colony edge (C) and a scratch wound (D). (E) Cadherins promote the phosphorylation and turnover of β-cat in adhesive cells. (F) Less adhesive cells (e.g., migrations during wound healing) are sensitized to Wnt signals. (G) Adhesion-based modulation of Wnt signaling may be important in stem cell maintenance and differentiation. β, β-cat; GSC, germline stem cell; LRP, low density lipoprotein receptor–related protein; P, phosphate group. Bars, 30 µm.
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fig5: Density-dependent turnover of cytosolic β-cat. (A) Turnover of cytosolic (GST-ICAT bound) β-cat by CHX chase method from LiCl-treated subconfluent and confluent MDCK cells. Densitometry values were normalized to the t0 value and plotted as shown. The plot represents the mean of two independent experiments; a set of representative immunoblots is shown. Error bars represent standard deviation of duplicate experiments. The numbers below each lane reflect normalized densitometry values. Affinity ppt, affinity precipitated; WB, Western blot. (B) Lysates from subconfluent (SC) and confluent (C) cells immunoprecipitated for E-cad and blotted for β-cat. (C and D) MDCK cells stably expressing TOP-dGFP show enhanced β-cat signaling in cells along a colony edge (C) and a scratch wound (D). (E) Cadherins promote the phosphorylation and turnover of β-cat in adhesive cells. (F) Less adhesive cells (e.g., migrations during wound healing) are sensitized to Wnt signals. (G) Adhesion-based modulation of Wnt signaling may be important in stem cell maintenance and differentiation. β, β-cat; GSC, germline stem cell; LRP, low density lipoprotein receptor–related protein; P, phosphate group. Bars, 30 µm.

Mentions: Our observation that Ca2+-dependent adhesion limits the accumulation of cytosolic β-cat independent of changes in the cadherin complex suggests that β-cat turnover may be enhanced by adhesion. To test this directly, we measured the turnover of cytosolic β-cat in the presence or absence of robust cell contacts in MDCK cells. The phospho forms of β-cat localize to cell–cell contacts in these cells and exhibit fractionation characteristics similar to E-cad–expressing SW480 cells (Fig. S2, A and B). As with NHEKs, treating MDCK cells with LiCl generated cytosolic β-cat, and this pool of β-cat was followed by affinity precipitation with GST-ICAT (Fig. S3 A) using the cycloheximide (CHX) chase method. Compared with low Ca2+ conditions, cytosolic β-cat disappears more rapidly in the presence of normal Ca2+ (Fig. S3 B). This difference in turnover does not appear to be caused by the low concentrations of Ca2+ itself because cells plated at subconfluency possess more cytosolic β-cat than confluent cells and exhibit slower turnover rates (Fig. 5 A). Critically, cadherin protein levels and the integrity of the cadherin–catenin complex are unchanged by density (Fig. 5, A and B) or low Ca2+ conditions (Fig. S3 C), as previously described (McCrea and Gumbiner, 1991). Ca2+-dependent adhesion also limits Wnt3a-dependent β-cat accumulation (Fig. S3 D), indicating that the observed effect is not LiCl specific. Altogether, these data suggest that cadherin-based adhesion may limit the extent and/or duration of Wnt/β-cat signals by enhancing the activity of a junction-localized phosphodestruction complex (Fig. 5 E). According to our model, when less adherent cells receive a Wnt signal such as during migrations that accompany wound healing, the ability of cadherins to activate the junction-localized degradation complex is somehow reduced, allowing the signaling pool of β-cat to persist (Fig. 5 F). Indeed, an MDCK clone that stably expresses a TCF optimal promoter (TOP)–destabilized GFP (dGFP; Dorsky et al., 2002) shows enhanced reporter activation in cells along the colony edge (Fig. 5 C) or scratch wounds (Fig. 5 D).


Activity of the beta-catenin phosphodestruction complex at cell-cell contacts is enhanced by cadherin-based adhesion.

Maher MT, Flozak AS, Stocker AM, Chenn A, Gottardi CJ - J. Cell Biol. (2009)

Density-dependent turnover of cytosolic β-cat. (A) Turnover of cytosolic (GST-ICAT bound) β-cat by CHX chase method from LiCl-treated subconfluent and confluent MDCK cells. Densitometry values were normalized to the t0 value and plotted as shown. The plot represents the mean of two independent experiments; a set of representative immunoblots is shown. Error bars represent standard deviation of duplicate experiments. The numbers below each lane reflect normalized densitometry values. Affinity ppt, affinity precipitated; WB, Western blot. (B) Lysates from subconfluent (SC) and confluent (C) cells immunoprecipitated for E-cad and blotted for β-cat. (C and D) MDCK cells stably expressing TOP-dGFP show enhanced β-cat signaling in cells along a colony edge (C) and a scratch wound (D). (E) Cadherins promote the phosphorylation and turnover of β-cat in adhesive cells. (F) Less adhesive cells (e.g., migrations during wound healing) are sensitized to Wnt signals. (G) Adhesion-based modulation of Wnt signaling may be important in stem cell maintenance and differentiation. β, β-cat; GSC, germline stem cell; LRP, low density lipoprotein receptor–related protein; P, phosphate group. Bars, 30 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2717642&req=5

fig5: Density-dependent turnover of cytosolic β-cat. (A) Turnover of cytosolic (GST-ICAT bound) β-cat by CHX chase method from LiCl-treated subconfluent and confluent MDCK cells. Densitometry values were normalized to the t0 value and plotted as shown. The plot represents the mean of two independent experiments; a set of representative immunoblots is shown. Error bars represent standard deviation of duplicate experiments. The numbers below each lane reflect normalized densitometry values. Affinity ppt, affinity precipitated; WB, Western blot. (B) Lysates from subconfluent (SC) and confluent (C) cells immunoprecipitated for E-cad and blotted for β-cat. (C and D) MDCK cells stably expressing TOP-dGFP show enhanced β-cat signaling in cells along a colony edge (C) and a scratch wound (D). (E) Cadherins promote the phosphorylation and turnover of β-cat in adhesive cells. (F) Less adhesive cells (e.g., migrations during wound healing) are sensitized to Wnt signals. (G) Adhesion-based modulation of Wnt signaling may be important in stem cell maintenance and differentiation. β, β-cat; GSC, germline stem cell; LRP, low density lipoprotein receptor–related protein; P, phosphate group. Bars, 30 µm.
Mentions: Our observation that Ca2+-dependent adhesion limits the accumulation of cytosolic β-cat independent of changes in the cadherin complex suggests that β-cat turnover may be enhanced by adhesion. To test this directly, we measured the turnover of cytosolic β-cat in the presence or absence of robust cell contacts in MDCK cells. The phospho forms of β-cat localize to cell–cell contacts in these cells and exhibit fractionation characteristics similar to E-cad–expressing SW480 cells (Fig. S2, A and B). As with NHEKs, treating MDCK cells with LiCl generated cytosolic β-cat, and this pool of β-cat was followed by affinity precipitation with GST-ICAT (Fig. S3 A) using the cycloheximide (CHX) chase method. Compared with low Ca2+ conditions, cytosolic β-cat disappears more rapidly in the presence of normal Ca2+ (Fig. S3 B). This difference in turnover does not appear to be caused by the low concentrations of Ca2+ itself because cells plated at subconfluency possess more cytosolic β-cat than confluent cells and exhibit slower turnover rates (Fig. 5 A). Critically, cadherin protein levels and the integrity of the cadherin–catenin complex are unchanged by density (Fig. 5, A and B) or low Ca2+ conditions (Fig. S3 C), as previously described (McCrea and Gumbiner, 1991). Ca2+-dependent adhesion also limits Wnt3a-dependent β-cat accumulation (Fig. S3 D), indicating that the observed effect is not LiCl specific. Altogether, these data suggest that cadherin-based adhesion may limit the extent and/or duration of Wnt/β-cat signals by enhancing the activity of a junction-localized phosphodestruction complex (Fig. 5 E). According to our model, when less adherent cells receive a Wnt signal such as during migrations that accompany wound healing, the ability of cadherins to activate the junction-localized degradation complex is somehow reduced, allowing the signaling pool of β-cat to persist (Fig. 5 F). Indeed, an MDCK clone that stably expresses a TCF optimal promoter (TOP)–destabilized GFP (dGFP; Dorsky et al., 2002) shows enhanced reporter activation in cells along the colony edge (Fig. 5 C) or scratch wounds (Fig. 5 D).

Bottom Line: It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering beta-catenin (beta-cat) from T-cell factor family transcription factors.We show that axin, APC2, GSK-3beta and N-terminally phosphorylated forms of beta-cat can localize to cell-cell contacts in a complex that is molecularly distinct from the cadherin-catenin adhesive complex.Together, these data suggest that cadherin-based cell-cell adhesion limits Wnt signals by promoting the activity of a junction-localized beta-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
It is well established that cadherin protein levels impact canonical Wnt signaling through binding and sequestering beta-catenin (beta-cat) from T-cell factor family transcription factors. Whether changes in intercellular adhesion can affect beta-cat signaling and the mechanism through which this occurs has remained unresolved. We show that axin, APC2, GSK-3beta and N-terminally phosphorylated forms of beta-cat can localize to cell-cell contacts in a complex that is molecularly distinct from the cadherin-catenin adhesive complex. Nonetheless, cadherins can promote the N-terminal phosphorylation of beta-cat, and cell-cell adhesion increases the turnover of cytosolic beta-cat. Together, these data suggest that cadherin-based cell-cell adhesion limits Wnt signals by promoting the activity of a junction-localized beta-cat phosphodestruction complex, which may be relevant to tissue morphogenesis and cell fate decisions during development.

Show MeSH
Related in: MedlinePlus