Quantitative ubiquitylome analysis reveals specificity of RNF111/Arkadia E3 ubiquitin ligase for its degradative substrates SKI and SKIL/SnoN in TGF-β signaling pathway

Victor Laigle (1) , Florent Dingli (1) , Sadek Amhaz (1) , Tiphaine Perron (1) , Mouna Chouchène (2) , Sabrina Colasse (2) , Isabelle Petit (1) , Patrick Poullet (1) , Damarys Loew (1) , Céline Prunier (2) , Laurence Levy (2)

1 Institut Curie [Paris]
2 CRSA - Centre de Recherche Saint-Antoine

RNF111/Arkadia is an E3 ubiquitin ligase that activates the TGF-β pathway by degrading transcriptional repressors SKIL/SnoN and SKI, and truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim we carried out label free quantitative proteomics after enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared to U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins prior to proteomics analysis by mass spectrometry, the diGly remnant peptide immunoprecipitation with a K-ε-GG antibody (diGly) and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody (pan UB) that recognizes all ubiquitin chains and monoubiquitylation. While we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the pan UB method also constitutes a powerful approach since it enabled detection of 52 potential RNF111 substrates including SKI, SKIL and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation.

Domaines: Biochimie, Biologie Moléculaire

Victor Laigle, Florent Dingli, Sadek Amhaz, Tiphaine Perron, Mouna Chouchène, et al.. Quantitative ubiquitylome analysis reveals specificity of RNF111/Arkadia E3 ubiquitin ligase for its degradative substrates SKI and SKIL/SnoN in TGF-β signaling pathway. Molecular and Cellular Proteomics, 2021, pp.100173. ⟨10.1016/j.mcpro.2021.100173⟩. ⟨hal-03419151⟩