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• W3140857766 abstract "Article30 March 2021Open Access Transparent process USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling Nicole Giebel Nicole Giebel orcid.org/0000-0003-3110-4673 Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Anchel de Jaime-Soguero Anchel de Jaime-Soguero Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Ana García del Arco Ana García del Arco Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Jonathan J M Landry Jonathan J M Landry orcid.org/0000-0003-2262-9099 Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Marlene Tietje Marlene Tietje Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany Search for more papers by this author Laura Villacorta Laura Villacorta Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Vladimir Benes Vladimir Benes Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Vanesa Fernández-Sáiz Vanesa Fernández-Sáiz Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany Search for more papers by this author Sergio P Acebrón Corresponding Author Sergio P Acebrón [email protected] orcid.org/0000-0002-7694-2497 Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Nicole Giebel Nicole Giebel orcid.org/0000-0003-3110-4673 Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Anchel de Jaime-Soguero Anchel de Jaime-Soguero Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Ana García del Arco Ana García del Arco Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Jonathan J M Landry Jonathan J M Landry orcid.org/0000-0003-2262-9099 Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Marlene Tietje Marlene Tietje Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany Search for more papers by this author Laura Villacorta Laura Villacorta Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Vladimir Benes Vladimir Benes Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany Search for more papers by this author Vanesa Fernández-Sáiz Vanesa Fernández-Sáiz Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany Search for more papers by this author Sergio P Acebrón Corresponding Author Sergio P Acebrón [email protected] orcid.org/0000-0002-7694-2497 Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany Search for more papers by this author Author Information Nicole Giebel1,†, Anchel Jaime-Soguero1,†, Ana García del Arco1, Jonathan J M Landry2, Marlene Tietje3,4, Laura Villacorta2, Vladimir Benes2, Vanesa Fernández-Sáiz3,4 and Sergio P Acebrón *,1 1Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany 2Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 3Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany 4TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany †These authors contributed equally to this work *Corresponding author. Tel: +49 6221 545257; E-mail: [email protected] EMBO Reports (2021)22:e51415https://doi.org/10.15252/embr.202051415 See also: G Colozza & B-K Koo (May 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R-spondin induces auto-ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R-spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface. SYNOPSIS Clearance of the Wnt receptors by RNF43 and ZNRF3 has emerged as the main mechanism modulating Wnt/β-catenin signalling in adult stem cells. To ensure stem cell renewal, RNF43 and ZNRF3 are ubiquitinated and removed from the plasma membrane via R-spondin and LGR4 - a process that is counteracted by USP42. The deubiquitinase USP42 forms a tug-of-war with R-spondin and LGR4 to control the plasma membrane residence of ZNRF3 and RNF43 USP42 is a negative regulator of Wnt/β-catenin signalling that promotes clearance of the Wnt receptors USP42 functions as a roadblock for Wnt-driven growth and EMT in colon cancer cells Loss of USP42 renders intestinal organoids hypersensitive to paracrine Wnt signalling Introduction The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and tissue homeostasis (Niehrs, 2010; Clevers et al, 2014). In particular, Wnt/β-catenin signalling governs stem cell maintenance in many tissues, and its misregulation is a common cause of tumour initiation, most notably in colorectal cancer (Nusse & Clevers, 2017; Bugter et al, 2020). The stability of the Wnt receptors LRP6 and Frizzled (FZD) has emerged as the main mechanism modulating Wnt/β-catenin signalling in adult stem cells (de Lau et al, 2014; Leung et al, 2018; Fenderico et al, 2019). The RING-type E3 ubiquitin-ligases ZNRF3 and RNF43 bind Dishevelled (DVL) and induce endocytosis and degradation of the Wnt receptors (Hao et al, 2012; Koo et al, 2012; Jiang et al, 2015). The R-spondin family of secreted proteins (RSPO1–4) form a ternary complex with LGR4/5/6 and RNF43/ZNRF3 (Kazanskaya et al, 2004; Kazanskaya et al, 2008; Carmon et al, 2011; de Lau et al, 2011; Glinka et al, 2011; Zebisch & Jones, 2015), which induces RNF43/ZNRF3 auto-ubiquitination and clearance from the plasma membrane (Hao et al, 2012; Koo et al, 2012; Hao et al, 2016). Hence, R-spondin promotes the stabilisation of LRP6 and FZD at the cell surface, boosting the responsiveness to Wnt ligands (Kazanskaya et al, 2004; de Lau et al, 2011; Glinka et al, 2011; Koo et al, 2012). During development, ZNRF3/RNF43 are required for embryonic patterning, sex determination, as well as limb morphogenesis (Harris et al, 2018; Szenker-Ravi et al, 2018; Chang et al, 2020; Lee et al, 2020). In adults, activation of Wnt signalling induces the expression of ZNRF3/RNF43 in stem cells, which form a negative feedback loop that prevents their unscheduled proliferation (Koo et al, 2012; Koo et al, 2015). This mechanism has been proved to be critical for the homeostasis of the intestinal tract and the adrenal gland (Koo et al, 2012; Basham et al, 2019), as well as for the growth and metabolic zonation of the liver (Planas-Paz et al, 2016). Loss of ZNRF3/RNF43 function is prevalent across different types of cancer (Hao et al, 2016; Bugter et al, 2020). For instance, truncating mutations in ZNRF3/RNF43 and activating translocations of R-spondin occur in 20% and 10% of colorectal tumours, respectively (Seshagiri et al, 2012; Giannakis et al, 2014; Bond et al, 2016). Loss of ZNRF3/RNF43 activity leads to extensive cellular proliferation and metaplasia (Koo et al, 2012; Koo et al, 2015) and has been associated with epithelial-to-mesenchymal transition (EMT) in various Wnt-associated tumours (Hao et al, 2016; Murillo-Garzon & Kypta, 2017), including in colorectal cancer (Gujral et al, 2014; Wang et al, 2016). In contrast to previously reported mutations in downstream Wnt regulators such as β-catenin or APC, alterations in the R-spondin/LGR/RNF43/ZNRF3 axis render tumour dependency on Wnt ligands (Jiang et al, 2013; Koo et al, 2015; Han et al, 2017). This growth factor addiction is currently being exploited in clinical trials by using small molecule inhibitors against Porcupine (PORCN) (Jung & Park, 2020), which is required for Wnt secretion (Jiang et al, 2013; Koo et al, 2015). Protein ubiquitination is a dynamic post-translational modification that results from a tug-of-war between matching pairs of E3 ligases and deubiquitinating enzymes (DUBs) (Fraile et al, 2012; MacGurn et al, 2012). In transmembrane proteins, ubiquitination directs quality control, trafficking and removal from the plasma membrane (MacGurn et al, 2012). In particular, the E3 ligases ZNRF3/RNF43 catalyse their ubiquitination and plasma membrane clearance upon interaction with R-spondin and LGR4–6 (Hao et al, 2012), but the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unidentified. Here, we show that USP42 protects ZNRF3/RNF43 from R-spondin- and ubiquitin-dependent clearance at the plasma membrane. Mechanistically, USP42 interacts with the Dishevelled interacting region (DIR) of ZNRF3 and stalls the ZNRF3/LGR/RSPO complex. Accordingly, USP42 promotes the turnover of the Wnt receptors LRP6 and FZD and inhibits Wnt/β-catenin signalling. Furthermore, we show that USP42 functions as a roadblock for cell proliferation, survival and EMT in colon cancer cells by inhibiting paracrine Wnt signalling. Finally, we report that genetic ablation of Usp42 confers Wnt hypersensitivity to mouse small intestinal organoids. Our work reveals how ZNRF3 and RNF43 are stabilised at the plasma membrane and provides mechanistical insights on the modulation of the stem cell factors LGR4 and R-spondin. Results USP42 inhibits Wnt signalling by deubiquitinating ZNRF3 R-spondin proteins promote the auto-ubiquitination and membrane clearance of the E3 ligase ZNRF3/RNF43 (Hao et al, 2012; MacGurn et al, 2012), which is a key event for Wnt signalling modulation (Hao et al, 2012; Koo et al, 2012; Koo et al, 2015; Zebisch & Jones, 2015). However, the DUB responsible for ZNRF3/RNF43 deubiquitination and membrane stabilisation remains unknown (Fig 1A). Figure 1. USP42 deubiquitinates ZNRF3 and inhibits Wnt/β-catenin signalling A. Scheme of the R-spondin complex with ZNRF3 and LGR, which promotes auto-ubiquitination and clearance of ZNRF3. The deubiquitinase (DUB) that stabilises ZNRF3 remains unknown. B. Example of a TOPflash reporter assay used to screen for DUBs regulating Wnt signalling. Cells were transfected in triplicates with the single siRNAs against the indicated DUBs or the positive control ZNRF3 and stimulated with control or Wnt3a conditioned media. C. Scheme showing the predicted protein organisation of USP42, including the catalytic cysteine of the ubiquitin-specific protease (USP) domain, and the predicted monopartite nuclear localisation signals (NLS) as displayed by NLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). D. Representative immunofluorescence microscopy images showing endogenous USP42 (green) in HEK293T cells transfected with the indicated siRNAs from n = 3 independent experiments. Scale bar = 10 μm. E–H. TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1–4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs. In (E), 3 independent siRNAs or a siRNA pool against USP42 were used. In (F), Parental wt HEK293T and ZNRF3-/-/RNF43-/- HEK293T cells were used. I, J. Ubiquitination of ZNRF3 in HEK293T cells transfected with ubiquitin-HA (Ub-HA). Cells were co-transfected with siUSP42 (I) or GFP-USP42 (J). Cells were treated for 6 h with 10 μM MG132 and 5 nM Bafilomycin A1 to prevent ZNRF3-Ub degradation. Ubiquitinated ZNRF3 was isolated by immunoprecipitation (IP) under denaturing conditions and analysed in Western blots with anti-HA antibodies. K. Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1. L, M. TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1–4 conditioned media, or by transfection with the indicated constructs. HEK293T, Parental HEK293T and USP42-/- HEK293T cells were transfected with wt USP42, the catalytically inactive mutant USP42C120A, ZNRF3 or ZNRF3/RNF43. Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as *P < 0.05, **P < 0.01, ***P < 0.001, or n.s.: not significant. Download figure Download PowerPoint To identify a potential DUB regulating ZNRF3/RNF43, we performed a DUB small interfering RNA (siRNA) screen in HEK293T cells using the Wnt reporter assay (TOPflash) as a readout and identified USP42 among the candidates (Fig 1B and [Extended version] EV1A). USP42 encodes a protein of 145 kDa with no clear structural features except its deubiquitinase (USP) domain, and a predicted monopartite nuclear localisation signal (NLS) (Fig 1C). USP42 localises to the cytoplasm, plasma membrane and nucleus (Fig 1D). Nuclear USP42 has been shown to deubiquitinate and stabilise H2B, as well as p53 in response to genotoxic stress (Hock et al, 2011; Hock et al, 2014). Knockdown of USP42 in wt HEK293T cells by three independent siRNAs increased Wnt3a-induced TOPflash activity by > 2-fold (Fig 1B and E). Critically, knockdown of USP42 did not upregulate Wnt reporter activity in ZNRF3/RNF43 double knockout HEK293T cells (Fig 1F), indicating a requirement of the E3 ligases for USP42 activity in Wnt signalling. To further assess USP42 function in Wnt signalling, we performed additional epistasis experiments. Knockdown of USP42 cooperated with the four R-spondin proteins (RSPO1–4) in Wnt reporter assays (Fig 1G), and boosted signalling induced by the ligand-receptor complex (Wnt1/LRP6/FZD8), but not by β-catenin (Fig 1H). Taking together, these data indicate that USP42 is a novel negative regulator of R-spondin and Wnt/β-catenin signalling, functioning at the receptor level through RNF43/ZNRF3. We next examined whether USP42 regulates ZNRF3 and RNF43 ubiquitination. We immunoprecipitated ZNRF3 under denaturing conditions and found that knockdown of USP42 increased its poly-ubiquitination (Fig 1I). Conversely, ectopic expression of USP42 erased the ubiquitin chains from ZNRF3 and RNF43 (Figs 1J and EV1B, IPs) and resulted in the accumulation of non-ubiquitinated ZNRF3 or RNF43 (Figs 1K and EV1B, inputs). To examine whether USP42 interacts with ZNRF3/RNF43, we performed co-immunoprecipitation (co-IP) experiments. GFP-USP42 co-precipitated ZNRF3, but not the component of the β-destruction complex AXIN1 (Fig 1K). On the other hand, the positive control GFP-DVL1 co-precipitated both ZNRF3 and AXIN1 (Fig 1K), as previously described (Bilic et al, 2007; Jiang et al, 2015). Additional co-IP experiments revealed that USP42 also interacted with RNF43 (Fig EV1C). Click here to expand this figure. Figure EV1. USP42 functions in Wnt signalling A. Relative USP42 expression in HEK293T cells transfected with the indicated siRNAs. B. Ubiquitination of RNF43 in HEK293T cells co-transfected with ubiquitin-HA (Ub-HA) and GFP-USP42. Cells were treated for 6 h with 10 μM MG132 and 5 nM Bafilomycin A1 to prevent RNF43-Ub degradation. Ubiquitinated RNF43 was isolated by immunoprecipitation (IP) under denaturing conditions and analysed in Western blots with anti-HA antibodies. C. Representative co-immunoprecipitation experiments in HEK293T cells that were transfected as indicated. D. TOPflash reporter assay in HEK293T cells upon overexpression of USP42 wt, the catalytically inactive mutant USP42C120A, or the negative control USP39 in combination with the indicated constructs or treatments. E. Representative immunofluorescence microscopy images showing endogenous USP42 (green) in HEK293T cells electroporated with eSpCas9 (parental) or eSpCas9-USP42 gRNA (USP42 KO) and selected as indicated in the methods. Scale bar = 10 μm. Data information: Data are displayed as mean ± SD and show one representative of n ≥ 4 independent experiments with three biological replicates. Statistical significance was calculated by Student’s t-test (A) or one-way ANOVA analyses with Tukey correction (D) and defined as *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint As in the case of ZNRF3, ectopic expression of USP42 in HEK293T cells inhibited the Wnt reporter signal induced by any of the four R-spondin proteins (RSPO1–4) (Fig 1L). Importantly, expression of the catalytically inactive mutant USP42C120A did not inhibit Wnt reporter activity (Fig 1L). Furthermore, expression of USP42, but not USP42C120A inhibited Wnt reporter assays induced by Wnt3a or by components of the LRP6 signalosome (Wnt1/LRP6/FZD8 and Dvl1), but not by β-catenin (Fig EV1D). Finally, we generated USP42 KO HEK293T cells (Fig EV1E), which displayed higher TOPflash activation than the parental cells upon Wnt3a treatment (Fig 1M). Importantly, USP42 KO-induced Wnt activity was blocked by ectopic expression of RNF43/ZNRF3 or USP42, but not by USP42C120A (Fig 1M). Taking these results together, we conclude that USP42 functions in Wnt signalling by deubiquitinating ZNRF3 and RNF43. Cytoplasmic USP42 binds the Dishevelled interacting region of ZNRF3 We generated various USP42 and ZNRF3 deletion constructs to identify which domains participate in their functional interaction (Fig 2A). Deletion of the predicted NLS or the C-terminal domain of USP42 increased their cytoplasmic distribution compared to wild-type (wt) USP42 in HEK293T cells (Fig 2B). USP42∆NLS and USP42∆C co-precipitated ZNRF3 (Fig 2C) and inhibited Wnt signalling in reporter assays (Fig 2D) in a similar way as wt USP42. USP42∆N and USP42C120A neither co-precipitated ZNRF3 nor reduced Wnt reporter assay activity (Fig 2C and D). Additional co-IP experiments with truncated ZNRF3 constructs revealed that USP42 requires the ZNRF3 Dishevelled interacting region (DIR) for its functional interaction (Fig 2E). Interestingly, ectopic expression of USP42 did not affect DVL2 binding to ZNRF3 (Fig 2F), suggesting that they do not compete for binding within the large ZNRF3 DIR region (346–528 aa) (Jiang et al, 2015). We conclude that cytoplasmic and catalytically active USP42 interacts with the ZNRF3 DIR (Fig 2G), which is emerging as a key modulatory domain of the E3 ligase (Jiang et al, 2015; Chang et al, 2020; Spit et al, 2020; Tsukiyama et al, 2020). Figure 2. Cytoplasmic USP42 functionally interacts with the Dishevelled interacting region of ZNRF3 A. Scheme showing the GFP-USP42 and ZNRF3-HA constructs generated for this study. Note that ZNRF3ΔC did not express properly, possibly due to misfolding at the ER. B. Immunofluorescence microscopy showing HEK293T cells transfected with USP42 constructs from (A). Note that depletion of the putative NLS or the whole C-terminal domain containing the NLS leads to increased cytoplasmic expression of USP42. Representative images are shown. Scale bar = 10 μm. C. Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). Representative blots of n ≥ 3 independent experiments are shown. D. TOPflash reporter assay in HEK293T cells upon overexpression of the USP42 constructs shown in (A) or an empty vector. Cells were stimulated with control or Wnt3a and RSPO3 conditioned medium. Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as **P < 0.01, ***P < 0.001, or n.s.: not significant. E, F. Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F), cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown. G. Scheme showing the proposed interacting regions of USP42 and ZNRF3. Download figure Download PowerPoint USP42 antagonises R-spondin and promotes Wnt receptor turnover ZNRF3/RNF43 auto-ubiquitination promotes its turnover from the membrane (Hao et al, 2012; Hao et al, 2016). To assess whether USP42 stabilises ZNRF3/RNF43 directly at the plasma membrane, we performed cell surface protein biotinylation assays (Fig 3A, scheme). Expression of USP42 reduced the ubiquitination of ZNRF3 and RNF43 at the plasma membrane (Fig 3A, 2nd IP, lanes 3–4 and lanes 7–8, respectively). Accordingly, USP42 strongly increased ZNRF3 and RNF43 residence at the plasma membrane (Fig 3A, 1st IP, lanes 3–4 and lanes 7–8, respectively). Next, we investigated whether USP42 also protects ZNRF3/RNF43 from R-spondin. In the presence of LGR4, RSPO1 reduced the protein levels of mature ZNRF3 (Fig 3B, input: lanes 1,3, upper bands). Importantly, RSPO1 stimulated the interaction between ZNRF3 and USP42ΔNLS (Fig 3B, IP: lanes 2,4), which rescued RSPO1-dependent clearance of ZNRF3 (Fig 3B, input: lanes 3,4). Figure 3. USP42 antagonises R-spondin/LGR and promotes FZD and LRP6 protein turnover A. Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pulldown, and CUL1 for the total input of cell lysate. B, C. Co-immunoprecipitation experiments in HEK293T cells transfected with the indicated constructs and treated for 6 h with Bafilomycin A1. Cells were co-treated with RSPO1 (B) or RSPO3 (C) conditioned medium. In (B), co-immunoprecipitated cytoplasmic USP42 (USP42ΔNLS) protein levels were quantified relative to immunoprecipitated ZNRF3. ZNRF3 levels in the input were quantified relative to tubulin. In (C), co-precipitated LGR4 protein levels were quantified relative to immunoprecipitated ZNRF3ΔRing. Representative blots of n = 3 independent experiments are shown. D. Scheme showing the proposed function for USP42 towards ZNRF3. E. Immunofluorescence microscopy of HEK293T cells transfected with SNAP-FZD5 and the indicated constructs. Cells were incubated with SNAP-surface-549 for 15 min and chased for another 10 minutes prior fixation. Representative images from one out of two independent experiments are shown. The white arrows show SNAP-FZD5 undergoing plasma membrane clearance. Scale bar = 20 μm. F, G. Western blots of lysates from HEK293T cells transfected with the indicated constructs. The asterisk marks an unspecific band. Representative blots from at least three independent experiments are shown. H. Western blots of lysates from HEK293T cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 4 independent experiments are shown. Download figure Download PowerPoint To obtain further mechanistical insights on the ZNRF3 regulation by USP42, we analysed the formation the ternary complex between ZNRF3, R-spondin and LGR4. As previously described (Hao et al, 2012), catalytically inactive ZNRF3 (ZNRF3∆Ring) co-precipitated LGR4, and this interaction was enhanced in the presence of RSPO3 (Fig 3C). Interestingly, USP42 interaction with ZNRF3 increased ternary complex formation (Fig 3C). These results suggest that ZNRF3 ubiquitination functions as release signal from R-spondin/LGR4, which allows USP42 to stall the ternary complex. This is supported by the fact that full-length ZNRF3, which displays high levels of auto-ubiquitination, did not form a stable complex with LGR4 (not shown and (Hao et al, 2012)). Furthermore, our results showing that USP42 interacts with catalytically inactive ZNRF3 (ZNRF3∆Ring) (Figs 2E and 3C) support the possibility of additional E3 ligases contributing to ZNRF3 ubiquitination. In that respect, recent evidence pointed towards β-TrCP as a contributing E3 ligase for ZNRF3 turnover (Ci et al, 2018). Taking together, these results indicate that USP42 forms a tug-of-war with R-spondin and LGR4 to control ZNRF3/RNF43 ubiquitination and plasma membrane residence (Fig 3D). Given the prominent role of ZNRF3 and RNF43 at the plasma membrane promoting Wnt receptor turnover, we next examined whether USP42 impacts FZD and LRP6 protein levels (Fig 3D). Ectopic expression of USP42 or ZNRF3 in HEK293T cells increased FZD5 clearance from the plasma membrane (Fig 3E) and reduced total FZD5 protein levels (Fig 3F). Furthermore, USP42 cooperated with ZNRF3 in clearing FZD8 (Fig 3G). Finally, knockdown of USP42 in HEK293T increased endogenous LRP6 protein levels, both in basal conditions and upon RSPO3 treatment (Fig 3H). We conclude that USP42 assists ZNRF3/RNF43 to promote Wnt receptor turnover. USP42 is a roadblock for paracrine Wnt signalling in colorectal cancer cells We noticed that USP42 mRNA is often overexpressed in colorectal cancer (Fig 4A) (Cancer Genome Atlas, 2012). Furthermore, USP42 protein levels are elevated in several colon cancer cell lines, including HCT116 and RKO (The Protein Expression Atlas). Interestingly, colon cancer cells rely on paracrine Wnt signalling for their maintenance, even in the presence of downstream mutations in APC and β-catenin (Voloshanenko et al, 2013). Hence, we decided to explore whether USP42 control of the Wnt receptors modulates paracrine Wnt signalling in colon cancer cells. Figure 4. USP42 inhibits paracrine Wnt signalling in colorectal cancer cells A. USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, 2012) (n = 524). Information was retrieved from the cBioPortal in 2017, and updated as displayed in 03/2020. B. FACS analyses of cell surface LRP6 protein levels in HCT116 cells upon knockdown of USP42. Cells were untreated (left panel) or treated with RSPO3 for 12 h (right panel). The grey dotted line in both panels shows background signal upon staining with control IgGs. Representative panels from one out of n = 4 independent experiments are shown. C. Western blots of lysates from HCT116 cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 3 independent experiments are shown. D. TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. E, F. qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments. Data information: Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as **P < 0.01, ***P < 0.001, or n.s.: not significant. Download figure Download PowerPoint First, we analysed whether USP42 regulates the Wnt receptors in HCT116 cells. Knockdown of USP42 in HCT116 cells increased the surface levels of endogenous LRP6, including upon R-spondin treatment (Fig 4B and C). Accordingly, knockdown of USP42 in HCT116 cells promoted paracrine Wnt signalling activity in TOPflash reporter assays (Figs 4D and EV2A) and boosted Wnt3a-dependent activation of the pathway (Fig 4D). Furthermore, siUSP42 upregulated the expression of the stem cell Wnt target gene LGR5 in HCT116 cells (Fig 4E) and cooperated with Wnt3a to upregulate classical Wnt target AXIN2 (Fig 4F). Click here to expand this figure. Figure EV2. USP42 regulates Wnt and p53 signalling in HCT116" @default.
• W3140857766 created "2021-04-13" @default.
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• W3140857766 title "USP42 protects ZNRF3/RNF43 from R‐spondin‐dependent clearance and inhibits Wnt signalling" @default.
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• W3140857766 doi "https://doi.org/10.15252/embr.202051415" @default.
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