Quantitative Ubiquitylome Analysis Reveals the Specificity of RNF111/Arkadia E3 Ubiquitin Ligase for its Degradative Substrates SKI and SKIL/SnoN in TGF-β Signaling Pathway

Abstract

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-β (TGF-β) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate 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 the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with 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 before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the 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.

The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia

“…UBXN7 stabilizes RNF111 in a RING and UAS dependent-manner UBXN7 and RNF111 are both nuclear proteins [5,23]; it is therefore possible that UBXN7 modulates the E3 ubiquitin ligase activity of RNF111 in the nucleus. We and others have shown previously that RNF111 is an E3 ubiquitin ligase that auto-ubiquitylates [22,33,34]. Here we showed that endogenous protein level of RNF111 detected by western-blot on U2OS cell lysates is increased in the presence of the proteasome inhibitor MG132 (Fig.…”

“…Such inactivation can be achieved by mutation of the first cysteine of the RING domain to an alanine (CA) that unfolds the first zinc-finger structure (Fig. 1a), and we have shown previously that this mutation abolishes RNF111 E3 ubiquitin ligase activity, SKIL ubiquitylation, and SMADdependent transcription [19,22]. We then compared the interactome of RNF111-WT and RNF111-CA mutant by performing GFP-Trap affinity purification coupled mass spectrometry on HEK-293 cells individually transfected with GFP-RNF111-WT, GFP-RNF111-CA, and GFP as a negative control (Fig.…”

“…3a, lanes 1 and 2), suggesting that RNF111 auto-ubiquitylation is associated with RNF111 degradation by the proteasome. To confirm that the RING domain of RN111 is responsible for its proteasomal degradation, we used two independent U2OS RNF111-RING-KO clones (clones #1 and #2) that we engineered previously with the CRISPR technology to express truncated forms of RNF111 corresponding to amino acids 1 to 432 which are therefore devoid of the C-terminal RING domain [22]. Note that western blot analysis confirms that endogenous RNF111 proteins indeed migrate at 170 kDa and not 107 kDa, and that the truncated forms of RNF111 migrate around 80 kDa and not 46 kDa as expected, which confirm that the anomalous RNF111 migration mainly lies in the N-terminal region (Fig.…”

“…TGF-β binding to its receptors TBRI and TBRII leads to SMAD2 and SMAD3 phosphorylation and their subsequent association with SMAD4 as heterotrimeric complexes that activate transcription of specific target genes [17,18]. Upon TGF-β signal, RNF111 binds phosphorylated SMAD2/3 (P-SMAD2/3) and fosters SMAD-dependent transcription by inducing ubiquitylation and proteasomal degradation of SKIL/SnoN and SKI, two highly related repressors of the TGF-β pathway [19][20][21][22]. Beside its important role in TGF-β signaling, RNF111 also has the ability to bind sumoylated proteins and acts as a SUMO-targeted ubiquitin ligase for promyelocytic leukemia (PML) in response to arsenite treatment and for xeroderma pigmentosum C (XPC) in response to UV [23,24].…”

SUMOylation-mediated PSME3-20 S proteasomal degradation of transcription factor CP2c is crucial for cell cycle progression

“…BRCA1 and SKI/SKIL, positive controls for STUbL-mediated protein degradation via RNF4 and RNF111, respectively, showed increased protein stability after the cell treatment with siRNF4 or siRNF111 (fig. S5) ( 41 , 48 ). In these experiments, CP2c protein levels were not augmented by siRNF4 and/or siRNF111 treatment ( Fig.…”