The Hypoxia-controlled FBXL14 Ubiquitin Ligase Targets SNAIL1 for Proteasome Degradation

Abstract

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...

Poly(ADP-ribose)-dependent regulation of Snail1 protein stability

“…This degradation of Snail1 by GSK-3b can be attenuated by Loxl2, a member of the lysyl oxidase gene family (Peinado et al, 2005), resulting in Snail1 stabilization and promotion of EMT. Recently, the hypoxia-controlled Fbxl-14 ubiquitin ligase has been shown to modulate Snail1 protein stability independently of its previous phosphorylation (Vinas-Castells et al, 2010).…”

Post-translational modifications of EMT transcriptional factors in cancer metastasis

“…Considering previous findings, we can speculate that phosphorylation of residues in different motif fulfill diverse functions to EMT transcriptional factors, which may due to their structure difference. Besides, ubiquitination of EMT transcriptional factors usually result in ubiquitin-dependent degradation, a pathway that was crucial for metastasis repression [33,44,47]. Overall, the molecular mechanism that Snail, Twist and ZEB could be dual-regulated by PTMs broaden our knowledge about function of EMT transcriptional factors during cancer metastasis.…”

Essential role of Fbxl14 ubiquitin ligase in regulation of vertebrate axis formation through modulating Mkp3 level

“…A recent study reports that the expression of Fbxl14 is markedly downregulated concomitantly with increased levels of its target Snail1 protein during hypoxia, which has been associated with tumor progression [7]. Mkp3, a novel target of Fbxl14 in this study, is involved in the development of cancer and has been suggested as a candidate tumor suppressor protein [9].…”

“…Although approximately 70 FBPs have been identified in humans [4,5], only a few of them have been well characterized. Fbxl14 (F-box and leucine-rich repeat protein 14) has been reported to regulate Snail2 protein during neural crest development of Xenopus laevis embryos [6] and to control Snail1 protein stability in mammalian cells [7]. There are two homologs of Fbxl14 in zebrafish, namely Fbxl14a and Fbxl14b.…”