1. Volume 53, Issue 3, Pages 444-457 (February 2014)
Multivesicular GSK3 Sequestration upon Wnt Signaling Is Controlled by p120- Catenin/Cadherin Interaction with LRP5/6 
Meritxell Vinyoles, Beatriz Del Valle-Pérez, Josué Curto, Rosa Viñas-Castells, Lorena Alba-Castellón, Antonio García de Herreros, Mireia Duñach 
Molecular Cell 
Volume 53, Issue 3, Pages (February 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 53, 444-457DOI: (10.1016/j.molcel.2013.12.010)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Wnt Internalizes the LRP5/6 Complex but Not p120-Catenin or N-Cadherin
(A) HEK293 cells were transfected with CA-LRP or with an empty vector and treated with control or Wnt3a-conditioned medium for 4 hr. Then a protease protection assay was performed as described in Experimental Procedures. The presence of the indicated proteins was determined by western blot (WB) in the total extract and in the Digitonin (PK) or the Triton-solubilized fractions (PK-TX-100) after Proteinase K digestion. Blots were normalized using anti-EEA1, which localizes exclusively into early endosomes.
(B) HEK293 cells were stimulated with control or Wnt3a-conditioned medium for the indicated times, and a protease protection assay was performed as in (A).
(C) PSer1490 LRP5/6 phosphorylation, GSK3β internalization into MVBs (presence of GSK3β in the PK-resistant fraction), and β-catenin protein levels were measured in HEK293 and HeLa cells at different times of Wnt3a treatment. Autoradiograms from four different experiments were quantified and represented for each time point. Each value is presented relative to that obtained in cells treated with control medium.
(D) β-catenin transcriptional activity was determined using the TOP reporter plasmid in HEK293 cells transfected with CA-LRP or treated with Wnt3a-conditional medium for the indicated times before cell lysis.
(E) RNA was isolated from HEK293 cells transfected with CA-LRP or treated with Wnt3a for the indicated times. Expression of AXIN2, CCND1, TCF7, and EPHB3 was assessed by quantitative RT-PCR. The figure shows the average ± range of the results of at least three experiments.
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4. Figure 2
N-Cadherin or p120-Catenin Depletion Differentially Affects GSK3β Internalization and LRP5/6 Localization upon Wnt Activation
(A) HEK293 cells were depleted of p120-catenin using a specific p120 shRNA and transfected with either an empty vector or CA-LRP and stimulated with Wnt3a-conditioned medium for 4 hr. Cells were permeabilized with RB buffer supplemented with Digitonin (65 μg/ml), fixed, and analyzed by immunofluorescence with the indicated antibodies. Nuclear staining was performed with DAPI. The bar corresponds to 4 μm. No signal was obtained when the same analysis was performed in the absence of primary antibody. A representative WB showing the extent of p120-catenin downregulation is presented in the right panel.
(B–F) HEK293 cells were depleted of N-cadherin by using specific shRNA or a scrambled shRNA as a control.
(B) At 48 hr postdepletion, cells were transfected with E-cadherin or an empty vector and CA-LRP when indicated. After 24 hr, cells were stimulated with control or Wnt3a-conditioned medium for 4 hr, and a protease protection assay was performed.
(C and D) LRP5/6 or Frizzled were immunoprecipitated from total cell extracts of N-cadherin-depleted HEK293 cells transfected with CA-LRP or treated with Wnt3a-conditioned medium for 15 min. Associated proteins were analyzed by WB.
(E) Surface proteins were biotinylated in control and N-cadherin-depleted HEK293 cells. A pull-down assay was performed with streptavidin-agarose beads and biotinylated LRP5/6, and N-cadherin was analyzed by WB.
(F) Control and N-cadherin-depleted intact HEK293 cells were treated with Proteinase K for 10 min. Total cell extracts were prepared and surface N-cadherin, and LRP5/6 levels were detected by WB.
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5. Figure 3
Dissociation of p120-Catenin from N-Cadherin after Wnt Stimulation Is Required for GSK3β Internalization into Vesicles upon Wnt Signaling
(A) Control or p120-catenin-depleted HEK293 cells were cotransfected with either GFP-p120-catenin wild-type (WT), S268,269A GFP-p120-catenin, or GFP. After 24 hr, cells were stimulated with control or Wnt3a-conditioned medium for 4 hr, and internalization of GSK3β, Dvl-2, and Axin into MVBs was determined as above.
(B) Control and p120-catenin-depleted HEK293 cells expressing WT or S268,269A p120-catenin and GFP-CA-LRP in all conditions were permeabilized with RB plus Digitonin, fixed, and analyzed by immunofluorescence. The bar corresponds to 4 μm.
(C) p120-catenin-depleted HEK293 cells expressing the indicated proteins were treated with control or Wnt3a-conditioned medium for 2 hr. Lysates were immunoprecipitated with anti-LRP5/6 antibody, and immunocomplexes were analyzed by WB.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Caveolin Is Not Needed for the Early Events in Wnt Signaling but Is Required for Signalosome Internalization upon Wnt Activation
(A and B) WT or Cav1−/− MEFs were stimulated with control or Wnt3a-conditioned medium for 2 hr (A) or 30 min (B). Lysates were immunoprecipitated with anti-Caveolin (A) or anti-LRP5/6 (B) antibodies, and protein complexes were analyzed by WB. Note that the LRP5/6 interaction was detected 2 hr after Wnt stimulation.
(C) MEFs were stimulated with control or Wnt3a-conditioned medium for 4 hr, and a protease protection assay was performed.
(D) p120-catenin-depleted HEK293 cells expressing either WT, S268,269A GFP-p120-catenin, or GFP were treated with control or Wnt3a-conditioned medium for 2 hr. Lysates were immunoprecipitated with an anti-Caveolin antibody, and associated proteins were analyzed by WB.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
An E-Cadherin Mutant Unable to Dissociate from LRP5/6 Prevents GSK3β Sequestration into Vesicles upon Wnt Treatment
(A) E-cadherin binding assay was performed with cytosolic E-cadherin domain either WT or a mutant with all Ser residues replaced by Ala (Ser-Ala) and GST–cytosolic LRP or GST as a control. E-cadherin proteins were phosphorylated with recombinant CK1 kinase when indicated (+). Control and phosphorylated cytosolic WT and Ser-Ala E-cadherin were included as internal references in the WB.
(B) WT and Ser-Ala GST–cyto-E-cadherin (0.1 pmol) were phosphorylated with CK1 kinase domain (10 mU). Phosphorylation was analyzed by autoradiography.
(C) Pull-down assays were performed by incubating GST-cytosolic LRP with HEK293 cells extracts expressing WT or Ser-Ala E-cadherin and stimulated with control or Wnt3a-conditioned medium for 2 hr.
(D and E) N-cadherin-depleted HEK293 cells expressing WT or Ser-Ala E-cadherin were treated with control or Wnt3a-conditioned medium for 2 hr. Lysates were immunoprecipitated with anti-LRP5/6 (D) or anti-p120-catenin (E) antibodies. Associated proteins were analyzed by WB.
(F) A protease protection assay was performed from N-cadherin-depleted HEK293 cells expressing WT or Ser-Ala E-cadherin and treated with control or Wnt3a-conditioned medium for 4 hr.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Prevention of GSK3β Internalization into MVBs Decreases but Does Not Abolish β-Catenin Accumulation in Response to Wnt3a or CA-LRP
(A and B) Control and p120-catenin-depleted HEK293 overexpressing WT, S268,269A, or 350 end GFP-p120-catenin (and CA-LRP when mentioned) were stimulated with control or Wnt3a-conditioned medium for the indicated times (A) or for 6 hr (B). β-catenin accumulation was analyzed by WB.
(C) Autoradiograms from four different experiments performed in (A) and (B) were quantified, and the mean ±SD was obtained for each condition.
(D) N-cadherin-depleted HEK293 cells expressing WT or Ser-Ala E-cadherin (and CA-LRP when indicated) were stimulated with control or Wnt3a-conditioned medium for 6 hr, and β-catenin accumulation was analyzed by WB.
(E) Autoradiograms from three different experiments performed in (D) were quantified and represented as in (C).
(F) WT or Cav1−/− MEFs were stimulated with control or Wnt3a-conditioned medium for 6 hr, and β-catenin accumulation was analyzed by WB.
(G) Autoradiograms from three different experiments performed in (F) were quantified and represented as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model of the Involvement of p120-Catenin and E-Cadherin in the Wnt Pathway
In nonstimulated cells (A), p120-catenin is bound to phosphorylated, inactive CK1ε through its N-terminal regulatory domain and to cadherin through the armadillo domain. Cadherin is associated to the Wnt coreceptor LRP5/6. Upon binding of Wnt ligand and the formation of the LRP5/6-Fz complex, CK1ε is activated by removal of inhibitory phosphates in its C-terminal tail and phosphorylates Dvl-2 and/or LRP5/6, stabilizing the interaction between these two proteins; binding of Dvl-2 enables receptor complex clustering, LRP5/6 phosphorylation on Thr1479, and the recruitment of Axin to the complex (B). Axin-associated CK1α, GSK3α, and GSK3β also bind to the Wnt-receptor complex, contributing to LRP5/6 phosphorylation and inhibiting GSK3. Moreover, CK1α phosphorylates p120-catenin on Ser268 and Ser269, releasing this protein from the signalosome and facilitating the subsequent phosphorylation of cadherin and the disruption of this cadherin interaction with LRP5/6 (C). Cadherin-unbound, clustered LRP5/6 complexes are then internalized to MVBs, promoting a more extensive inhibition of GSK3 (D). Released p120-catenin enhances Vav2 activity toward Rac1, which is necessary for an efficient translocation of stabilized β-catenin to the nucleus and inactivates the transcriptional repressor Kaiso, required for a complete β-catenin transcriptional response.
Molecular Cell  , DOI: ( /j.molcel )
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