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...

The Wnt canonical ligands elicit the activation of β-catenin transcriptional activity, a response dependent on, but not limited to, β-catenin stabilization through the inhibition of GSK3 activity. Two mechanisms have been proposed for this inhibition, one dependent on the binding and subsequent block of GSK3 to LRP5/6 Wnt coreceptor and another one on its sequestration into multivesicular bodies (MVBs). Here we report that internalization of the GSK3-containing Wnt-signalosome complex into MVBs is dependent on the dissociation of p120-catenin/cadherin from this complex. Disruption of cadherin-LRP5/6 interaction is controlled by cadherin phosphorylation and requires the previous separation of p120-catenin; thus, p120-catenin and cadherin mutants unable to dissociate from the complex block GSK3 sequestration into MVBs. These mutants substantially inhibit, but do not completely prevent, the β-catenin upregulation caused by Wnt3a. These results, besides elucidating how GSK3 is sequestered into MVBs, support this mechanism as cause of β-catenin stabilization by Wnt.

The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases β-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1.