Nuclear ubiquitination by FBXL5 modulates Snail1 DNA binding and stability

Abstract

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.

A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death

“…Alternatively, Snail1-F-Luc (Snail1-firefly luciferase fusion protein) and R-Luc (Renilla luciferase) were expressed with a pLEX-Snail-F-Luc and pMSCV-R-Luc vectors (a kind gift from Y. Kang, Princeton University) and used to measure Snail1 stability as previously described (40). Other transfections were carried out using Lipofectamine and plasmid pcDNA4-NICD-Myc (a kind gift from A. Bigas, IMIM), pcDNA3-␤-TrCP1-Flag, pcDNA3-Fbxl14-Myc, or pcDNA3-Fbxl5-Myc were previously described (41,42). For immunoprecipitation experiments of endogenous Akt1/2, total cell extracts were obtained with lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.5% Triton X-100, and protease inhibitors) and precleared with protein Amagnetic beads (Millipore) for 1 h at 4°C.…”

“…To obtain cell extracts with differentiated cytoplasmic and nucleoplasmic fractions, cells were scrapped in ice-cold buffer A (10 mM HEPES-KOH [pH 7.8], 1.5 mM MgCl 2 , 10 mM KCl, and protease inhibitors) and kept on ice for 10 min (42). A 1/30 volume of the lysate of 10% Triton X-100 was added, and the tubes were vortex mixed for 20 s. Centrifugation of the sample for 1 min at 11,000 rpm separated out the cytoplasmic extract.…”

“…Lentiviral expression in different cell lines was used to stably knock down Akt1 and Akt2 using shRNAs cloned in pLKO.1-puro vector (Mission; Sigma) (42). For viral infection, HEK 293T cells were seeded in p100 plates with 10 ml of medium at high confluence and transfected by mixing 1.5 ml of 150 mM NaCl and 78 l of PEI with a total of 20 g of DNA (50% was the shRNA or an equally distributed quantity of different shRNAs targeting the same gene, 10% was the pCMV-VSV-G vector, 30% was the pMDLg/pRRE vector, and 10% was the pRSV rev vector).…”

The regulation of snail: on the ubiquitin edge

“…Snail's ubiquitination and degradation are controlled by several F-box ligases, including β-TRCP1/Fbxw1, Fbxl14, Fbxl5, Fbxo11, and Fbxo45 [16][17][18][19][20][21][22] ( Figure 1). β-TRCP1/Fbxw1 is one of the best-characterized Snail E3 ligase.…”

“…Fbxl5 is localized predominantly in the nucleus. Interestingly, Fbxl5 interacts with the Snail C-terminus, the region involved in binding to the E-box sequence while it polyubiquitinates Snail on its N-terminal residues K85 and K146 as well as on its C-terminal residue K234 [18] . In addition, Fbxl5-induced Snail down-regulation is impaired by post-translational modifications that prevent nuclear export, such as Lats2 phosphorylation [23] .…”