The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase ␤-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3␤. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3␤, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3␤. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 downregulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.The human SNAIL family of zinc finger transcription factors, composed of SNAIL1 and SNAIL2 (also called SNAIL and SLUG, respectively) plays a fundamental role in initiating epithelial-mesenchymal transition (EMT), 6 a key developmental program that results in the acquisition of mesenchymal characteristics by epithelial cells (1). EMT is required for essential processes, such as gastrulation and formation of neural crest cells, and is also relevant in pathological processes, such as fibrosis, cancer cell invasion, and hypoxia (1-4). Expression of SNAIL1 induces a more invasive phenotype, at least in part through its inhibition of E-cadherin gene expression (2). SNAIL1 represses transcription of E-cadherin by binding to three E-boxes present in the human E-cadherin promoter (5). Moreover, SNAIL1 has additional cellular functions independent of EMT, because it confers resistance to cell death (6 -8).SNAIL1 is a highly unstable protein and is very sensitive to proteasome inhibitors (9). SNAIL1 degradation by the proteasome requires its interaction with the E3 ubiquitin ligase ␤-TrCP1/FBXW1 and the subsequent ubiquitylation of SNAIL1 protein (9). ␤-TrCP1/FBXW1, like ␤-TrCP2/ FBXW11, recognizes the destruction motif DpSXXpS (where pS represents phosphoserine) and participates in the degradation of many substrates, including ␤-catenin (10, 11). Before its interaction with ␤-TrCP1/FBXW1, SNAIL1 degradation requires nuclear GSK-3␤ (glycogen synthase kinase-3␤) phosphorylation. This modification unmasks a nuclear export sequence (NES) and promotes SNAIL1 export from the nucleus (12). In the cytosol, SNAIL1 undergoes a second phosphorylation by GSK-3␤, which targets the prot...

p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as β-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for β-catenin, p120-catenin depletion prevents the increase in β-catenin transcriptional activity even in the absence of β-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of β-catenin transcriptional activity different of β-catenin stability.

There was an error published in J. Cell Sci. 125, 5288-5301.In Fig. 2B, panels labelled as the actin input and p120-catenin IP:p120-catenin panels were inadvertently assembled to show the same blots. Concerns were also raised about possible duplications and/or splices in Figs 4A, 4E, 5D, 6A and 6C. The authors were unable to obtain the original data in order to address these concerns and therefore repeated the experiments. The new data are shown in the figures below and have been verified by the corresponding author's institute as supporting the original conclusions of the study. There are no changes to the figure legends, which are accurate.The authors acknowledge the increased contribution of Beatriz Del Valle-Pérez in repeating the experiments and apologise to the readers for any confusion that these errors might have caused. Fig. 2. Wnt-induced Rac1 activation is dependent on Vav2 interaction with p120-catenin. Vav2 (A) and p120-catenin (B) were immunoprecipitated from 500 µg SW-480 whole-cell extracts treated with control or Wnt3a-conditioned medium for the times indicated. Protein complexes were analyzed by western blot (WB) with anti-p120-catenin, anti-Vav2, anti-E-cadherin and anti-Rac1. 10 µg of SW-480 whole-cell extracts were included as internal reference (input). The graphs on the right are autoradiograms from four different experiments that were quantified and the mean ± s.d. obtained after 2 hours of incubation with Wnt3a medium. Each value is presented relative to that obtained in cells treated with control medium and normalized with respect to the amount of immunoprecipitated Vav2 or p120-catenin. (C) SW-480 cells stably expressing scrambled or shRNA specific for Vav2 were treated with control or Wnt3a-conditioned medium for 2 hours. GST-PAK pull-down assays were performed and active Rac1 was determined by WB. Autoradiograms from five different experiments performed in triplicate were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted cells treated with control medium. (D) Cytosolic and nuclear lysates were obtained from control and Vav2-depleted SW-480 cells treated with control or Wnt3a-conditioned medium for 15 hours. β-catenin distribution between the two cell compartments was analyzed by WB. 2120© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016Science ( ) 129, 2120Science ( -2123Science ( doi:10.1242 Journal of Cell Science 480 (E,F) cells overexpressing either GFP-p120-catenin wt isoform 1, GFP-p120-catenin point mutants Y112E or Y217E or the empty vector phrGFP, treated when indicated with Wnt3a-conditioned medium for 15 hours. The nuclear fraction was separated from the cytosolic and membrane-associated fraction as detailed in Materials and Methods. β-catenin levels in each cellular fraction were analyzed by WB. Lamin-β1 was used as a nuclear marker and pyruvate kinase as a marker for the cytosolic-plus-membrane fraction. In the right panel of C, p120-catenin wt, Y112E and Y217E...

There was an error published in J. Cell Sci. 124, 2298-2309.Some of the blots presented in Fig. 4A were inadvertently duplicated in Fig. 6D. The blots in Fig. 4A are correct as presented. The correct Fig. 6 is presented below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study.The authors apologise to the readers for any confusion that this error might have caused. . Kaiso also interacts with β-catenin and its binding is regulated by p120-catenin. (A) 2 pmol of recombinant GST-Kaiso or GST as a control was incubated with 1 pmol of full-length β-catenin or two deletion mutants comprising armadillo repeats 1-12 (amino acids 120-683) or 1-6 (amino acids 120-420). Protein complexes were affinity purified with glutathione-Sepharose and analyzed by western blotting (WB) with anti-β-catenin antibody. Blots were re-analyzed with anti-GST antibody to ensure that a similar amount of fusion protein was present. (B) 2 pmol of recombinant GST-Kaiso, or GST as a control, was incubated with 2 pmol of p120-catenin (amino acids 102-911) (left-hand panel) or 2 pmol of full-length β-catenin (right-hand panel). Protein complexes were affinity purified with glutathione-Sepharose and analyzed by western blotting with the indicated antibodies. 'St' indicates the signal obtained with known amounts of recombinant proteins used as control. (C) Pull-down assays were performed by incubating 7 pmol of GST-Kaiso with 400 μg of whole-cell extracts from SW-480 cells transfected with pcDNA3-Tcf-4-HA. When indicated, 35 pmols of p120-catenin or β-catenin was added to the incubation medium. Protein complexes were affinity purified and analyzed by western blotting with the indicated antibodies. (D) SW-480 cells were infected with scrambled shRNA (Ctl) or shRNA specific for p120-catenin, and were treated with control or Wnt3a-conditioned medium for 6 hours. Kaiso was immunoprecipitated (IP) and immunocomplexes were analyzed by western blotting. (E) SW480 cells were treated with control or Wnt3a-conditioned medium for 6 hours. β-catenin was immunoprecipitated from SW-480 total cell extracts and the immunocomplex was analyzed by western blotting. In the input lane, 5% of each total cell extract used is shown. (F) Kaiso was immunoprecipitated with a specific antibody from total cell extracts of HT29-M6 cells treated with control or Wnt3a-conditioned medium for 6 hours. Associated proteins were analyzed by western blotting. All the data presented in this figure are representative of at least three independent experiments. Irr IgG, an irrelevant IgG used as control in the immunoprecipitation. 873