The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8статья из журнала
Аннотация: Article15 October 2019Open Access Source DataTransparent process The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8 Abhay Narayan Singh Abhay Narayan Singh Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Judith Oehler Judith Oehler Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Ignacio Torrecilla Ignacio Torrecilla Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Susan Kilgas Susan Kilgas Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Shudong Li Shudong Li Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Bruno Vaz Bruno Vaz Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Claire Guérillon Claire Guérillon Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author John Fielden John Fielden Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Esperanza Hernandez-Carralero Esperanza Hernandez-Carralero Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Search for more papers by this author Elisa Cabrera Elisa Cabrera Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Search for more papers by this author Iain DC Tullis Iain DC Tullis Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Mayura Meerang Mayura Meerang Institute of Pharmacology and Toxicology-Vetsuisse Faculty, University of Zurich, Zurich, Switzerland Search for more papers by this author Paul R Barber Paul R Barber Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Raimundo Freire Raimundo Freire Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Universidad Fernando Pessoa Canarias, Santa Maria de Guia, Spain Search for more papers by this author Jason Parsons Jason Parsons Department of Molecular and Clinical Cancer Medicine, Cancer Research Centre, University of Liverpool, Liverpool, UK Search for more papers by this author Borivoj Vojnovic Borivoj Vojnovic Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Anne E Kiltie Anne E Kiltie orcid.org/0000-0001-7208-2912 Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Niels Mailand Niels Mailand orcid.org/0000-0002-6623-709X Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Kristijan Ramadan Corresponding Author Kristijan Ramadan [email protected] orcid.org/0000-0001-5522-021X Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Abhay Narayan Singh Abhay Narayan Singh Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Judith Oehler Judith Oehler Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Ignacio Torrecilla Ignacio Torrecilla Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Susan Kilgas Susan Kilgas Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Shudong Li Shudong Li Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Bruno Vaz Bruno Vaz Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Claire Guérillon Claire Guérillon Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author John Fielden John Fielden Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Esperanza Hernandez-Carralero Esperanza Hernandez-Carralero Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Search for more papers by this author Elisa Cabrera Elisa Cabrera Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Search for more papers by this author Iain DC Tullis Iain DC Tullis Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Mayura Meerang Mayura Meerang Institute of Pharmacology and Toxicology-Vetsuisse Faculty, University of Zurich, Zurich, Switzerland Search for more papers by this author Paul R Barber Paul R Barber Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Raimundo Freire Raimundo Freire Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain Universidad Fernando Pessoa Canarias, Santa Maria de Guia, Spain Search for more papers by this author Jason Parsons Jason Parsons Department of Molecular and Clinical Cancer Medicine, Cancer Research Centre, University of Liverpool, Liverpool, UK Search for more papers by this author Borivoj Vojnovic Borivoj Vojnovic Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Anne E Kiltie Anne E Kiltie orcid.org/0000-0001-7208-2912 Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Niels Mailand Niels Mailand orcid.org/0000-0002-6623-709X Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Kristijan Ramadan Corresponding Author Kristijan Ramadan [email protected] orcid.org/0000-0001-5522-021X Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Abhay Narayan Singh1,‡, Judith Oehler1,8,‡, Ignacio Torrecilla1, Susan Kilgas1, Shudong Li1, Bruno Vaz1, Claire Guérillon2, John Fielden1, Esperanza Hernandez-Carralero3,4, Elisa Cabrera3,4, Iain DC Tullis1, Mayura Meerang5,9, Paul R Barber1, Raimundo Freire3,4,6, Jason Parsons7, Borivoj Vojnovic1, Anne E Kiltie1, Niels Mailand2 and Kristijan Ramadan *,1 1Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK 2Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark 3Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Spain 4Instituto de Tecnologías Biomédicas, Universidad de La Laguna, La Laguna, Spain 5Institute of Pharmacology and Toxicology-Vetsuisse Faculty, University of Zurich, Zurich, Switzerland 6Universidad Fernando Pessoa Canarias, Santa Maria de Guia, Spain 7Department of Molecular and Clinical Cancer Medicine, Cancer Research Centre, University of Liverpool, Liverpool, UK 8Present address: Department of Biochemistry, University of Oxford, Oxford, UK 9Present address: Department of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +44 01865 617 349; E-mail: [email protected] The EMBO Journal (2019)38:e102361https://doi.org/10.15252/embj.2019102361 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 E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers. Synopsis The RNF8 ubiquitin ligase is a key mediator of the ubiquitin-mediated DNA damage response and regulator of double strand break repair pathway choice. Here, these roles are shown to depend on homeostatic regulation of RNF8 by the ubiquitin-dependent segregase p97/VCP and the deubiquitinase Ataxin 3 (ATX3). A p97-ATX3 complex maintains proteasome-dependent RNF8 homeostasis under physiological condition. The p97-ATX3 complex extracts RNF8 from chromatin after its recruitment to DNA damage sites. p97-ATX3-mediated RNF8 extraction promotes timely RNF168 recruitment and utilization of non-homologous end joining (NHEJ) repair. Inactivation of the p97-ATX3 complex hyper-sensitizes BRCA2/homologous recombination-deficient cells to ionizing radiation. Introduction DNA double-strand breaks (DSBs) are the most deleterious DNA lesions and, if not accurately repaired, can lead to chromosomal aberrations, immunodeficiency, neurodegeneration, cancer, accelerated ageing and cell death (Friedberg et al, 2006; Jackson & Bartek, 2009; Panier & Durocher, 2013; Jeggo et al, 2016). Mammalian cells execute two main DSB repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR), promoted by 53BP1 and BRCA1/BRCA2/Rad51, respectively (Chapman et al, 2012; Aparicio et al, 2014; Hustedt & Durocher, 2016). The coordination of DSB repair is under the strict control of various post-translational modifications (PTMs). These include ubiquitination generated by two E3 ubiquitin ligases RNF8 and RNF168 which has emerged as an essential PTM for DSB repair pathway choice and cell survival (Kolas et al, 2007; Mailand et al, 2007; Doil et al, 2009; Pinato et al, 2009; Stewart et al, 2009; Gatti et al, 2015). RNF8 is the first E3 ubiquitin ligase recruited to sites of DSBs, where it ubiquitinates histone H1 with K63-ubiquitin-linked chains (K63-Ub) in cooperation with the E2 ubiquitin conjugating enzyme Ubc13 (Thorslund et al, 2015). RNF8-dependent H1/K63-Ub is essential to recruit the second E3 ubiquitin ligase RNF168. RNF168 further amplifies and spreads the ubiquitin signal onto the surrounding histones, H2A-type histones, that serve as docking sites for 53BP1 recruitment to chromatin to prevent excessive 5′-DNA end resection (the initial step of the HR pathway) and to promote NHEJ-mediated repair (Mattiroli et al, 2012; Chang et al, 2017; Dev et al, 2018; Noordermeer et al, 2018). BRCA1 and Rad51, the essential enzymes for HR, are also recruited to sites of DSBs in a ubiquitin-dependent manner; however, their recruitment is strictly dependent on RNF8 and not RNF168 (Kim et al, 2007; Sobhian et al, 2007; Wang & Elledge, 2007; Sy et al, 2011; Munoz et al, 2012; Nakada et al, 2012; Watanabe et al, 2013; Zong et al, 2019), supporting the notion that HR is not absolutely dependent on the RNF168-mediated ubiquitin signalling cascade. Separation of function between these two main DSB repair E3 ubiquitin ligases—RNF8 and RNF168—was further observed in RIDDLE syndrome patient cells bearing RNF168 mutations. These cells are radiosensitive due to their inability to recruit 53BP1 (NHEJ pathway) to sites of DSBs, while maintaining recruitment of BRCA1 and Rad51 (HR pathway) to the break sites (Stewart et al, 2007). This was also confirmed in the Rnf168-knockout mouse cells (Zong et al, 2019). Finally, structural and cell biological data further demonstrate an independent and direct role of RNF8 and its K63-Ub in BRCA1 recruitment to sites of DNA lesions (Hodge et al, 2016). Altogether this suggests that the RNF168/Ubiquitin/53BP1 cascade is essential to mediate NHEJ and is functionally distinguished from the RNF8/K63-Ub/BRCA1/Rad51-governed HR repair pathway. Thus, to have balanced DSB repair, the levels of RNF8 and RNF168 have to be tightly regulated. Unlike RNF168, whose protein expression is tightly regulated by two E3 ubiquitin ligases and a deubiquitinating enzyme (DUB), namely UBR5, TRIP12 and USP7, respectively (Gudjonsson et al, 2012; Zhu et al, 2015), how the homeostasis of RNF8 is regulated is not known. This is a key issue, not only for accurate DNA repair after genotoxic stress but also for genome stability under physiological conditions, since hyper-accumulation of RNF8 is directly linked to tumorigenesis and poor prognosis in breast cancer patients (Kuang et al, 2016; Lee et al, 2016a,b). Here, we present data demonstrating that RNF8 homeostasis is regulated by the ATPase p97 (p97)—also known as VCP unfoldase in humans—and its associated DUB Ataxin 3 (ATX3). The AAA+ ATPase p97 is a homo-hexameric barrel-like protein, which forms multiple complexes and sub-complexes with its associated cofactors that confer p97 specificity to various substrates and cellular pathways (Meyer & Weihl, 2014; Buchberger et al, 2015). These p97 complexes bind (via cofactors) and process (via p97 ATPase activity) ubiquitinated substrates by either unfolding or segregating them from different cellular locations including chromatin (Vaz et al, 2013; Dantuma et al, 2014). In turn, ubiquitinated substrates remodelled by the p97 system are either presented to the proteasome for their degradation or recycled by DUBs (Meyer et al, 2012). Thus, the p97 system plays a central role in maintaining protein homeostasis. Here, we report that p97 and ATX3 (Rao et al, 2017) form a constitutive physical complex with RNF8. The p97–ATX3 complex safeguards the soluble pool of RNF8 under physiological conditions. However, under genotoxic conditions, when RNF8 is rapidly recruited to sites of DNA lesions and orchestrates the DNA damage response, the p97–ATX3 complex facilitates RNF8 chromatin extraction to balance DSB repair pathway choice and improve cell survival to ionising radiation (IR). Our data reveal the p97–ATX3 complex as a crucial mediator of RNF8 regulation and identify a fundamental role of the p97–ATX3–RNF8 axis in promoting genome stability and resistance to IR. Results RNF8 is a ubiquitinated substrate of p97 To test the hypothesis that the p97 system regulates RNF8 homeostasis, we monitored the total pool of endogenous RNF8 in HeLa and HEK293 cells when either p97 was siRNA-depleted or its ATPase activity was blocked. The ATPase activity of p97 was blocked by either acute chemical inhibition (CB5083) or doxycycline (DOX)-inducible expression of the p97E578Q (p97EQ; dominant negative) variant. Inactivation of p97 by either approach led to increased total levels of endogenous RNF8 under physiological and IR-treated conditions (Fig 1A–F). Both chemically inhibited p97 and the p97EQ variant bind, but are unable to process, the substrates due to inactivation of p97 ATPase activity (Ye et al, 2003; Meerang et al, 2011; Fig 1G). Similarly, cycloheximide (CHX) chase experiments in p97-inactivated HEK293 cells, either by siRNA-mediated p97 depletion or by mild expression of p97EQ variant, demonstrated a marked delay in endogenous RNF8 degradation kinetics when compared to control cells (Figs 1H and I, and EV1A–C), suggesting that the rate of RNF8 degradation and its homeostasis are under the control of the p97 system and this effect is not cell type-specific. Figure 1. RNF8 is a ubiquitinated substrate of p97 under physiological and genotoxic conditions Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (A) (***P < 0.001; unpaired t-test, n = 3, mean + SEM). Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10 μM for 6 h) under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (C) (**P < 0.01, ***P < 0.001; unpaired t-test, n = 3, mean + SEM). Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-inducible mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (E) (**P < 0.01, ****P < 0.0001; unpaired t-test, n = 4, mean + SEM). Model representing the processing of ubiquitinated substrate by p97 ATPase activity. Inactivation of p97 ATPase activity leads to the accumulation of ubiquitinated substrate. Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion. Graph represents the quantifications of (H) (***P < 0.001, ****P < 0.0001; two-way ANOVA, n = 3, mean + SEM) and Western blot for efficacy of siRNA depletion of p97 (right). Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion. Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97-WT under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (K) (*P < 0.05; unpaired t-test, n = 2, mean + SEM). Source data are available online for this figure. Source Data for Figure 1 [embj2019102361-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. p97 regulates RNF8 turnover Western blot analysis showing increased RNF8 protein level in HeLa cells by CHX chase after siRNA-mediated p97 depletion. Quantification of (A) showing reduced RNF8 degradation rate after siRNA-mediated p97 depletion. Western blot analysis of CHX chase in HEK293 cells showing reduced RNF8 degradation rate after doxycycline-inducible mild expression of p97EQ (dominant negative ATPase inactive) variant. Western blot analysis of Flag-RNF8 denaturing-IP showing RNF8 K48-Ub, K63-Ub and total-Ub (HA) ubiquitination pattern in soluble pool (cytosol + nucleosol) and on chromatin under physiological conditions and after IR (10 Gy). Western blot analysis showing comparison of Ub-K48 and Ub-K63 antibodies against different amounts (0.25, 0.5 and 1.0 μg) of purified recombinant K48- or K63-Ub chains (Ub-2 to Ub-7). Western blot analysis showing distribution of different proteins under physiological conditions and after IR (10 Gy) in cytosol, nucleosol, loosely bound chromatin (LB Chro) and tightly bound chromatin (TB Chro) of HEK293 cells. Representative IF images showing presence of ATX3 in the nucleus of U2OS cells (scale bar: 10 μm). Nuclei are marked by white lines. Source data are available online for this figure. Download figure Download PowerPoint As p97 unfolds and extracts predominantly ubiquitinated proteins from different cellular locations, we next asked if RNF8 is modified by ubiquitination. HEK293 cells co-transfected with Flag-RNF8 and HA-Ubiquitin were mock-treated or exposed to IR. The cells were biochemically fractionated into total soluble (cytosol and nucleosol) and chromatin pools, and Flag-RNF8 was subsequently immunopurified under denaturing conditions from both fractions to analyse its ubiquitination status (Fig EV1D). Ubiquitination of RNF8 was analysed using antibodies recognising either total ubiquitination (i.e. HA antibody) or specific ubiquitin linkages with a similar affinity (K48-Ub and K63-Ub; Fig EV1E). RNF8 was strongly ubiquitinated under both physiological and genotoxic conditions, and the K48-Ub appeared to be the main ubiquitin signal on both soluble and chromatin-bound RNF8 (Fig EV1D). Furthermore, RNF8 was hyper-ubiquitinated with K48-Ub in p97-depleted cells when compared to control cells (Fig 1J), further supporting a role for the p97 system in processing ubiquitinated RNF8. To directly prove that RNF8 is a substrate of p97, we performed co-immunoprecipitation experiments and analysed the physical association between p97 and RNF8 in vivo. Flag-RNF8 and Strep-tagged p97 wild type (WT) or p97EQ were mildly co-expressed in HEK293 cells. The cells were then mock-treated or exposed to IR, and Strep-tagged p97 complexes were isolated from total cell extract over streptavidin beads under native and physiological salt (150 mM NaCl) conditions (Fig 1K and L). The p97-WT isolate precipitated equal amounts of RNF8 in untreated and IR-treated conditions. However, the physical association of RNF8 with the p97EQ variant was increased ~5-fold when compared to p97-WT, revealing it to be a trapped p97 substrate. Overall, these data suggest that the p97 ATPase forms a stable physical complex with the E3 ubiquitin ligase RNF8 under physiological and genotoxic conditions in vivo to prevent RNF8 hyper-accumulation. Homeostasis of RNF8 is controlled by auto-ubiquitination and the ubiquitin–proteasome system RNF8 is an E3 ubiquitin ligase that, in association with E2-conjugating enzymes Ubc13, Ubc8 and Ube2S, forms K63-Ub, K48-Ub or K11-Ub chains, respectively, on various substrates (Feng & Chen, 2012; Lok et al, 2012; Mallette et al, 2012; Zhang et al, 2013; Thorslund et al, 2015; Paul & Wang, 2017). As the K48-Ub is the main signal for proteasomal degradation, we asked whether RNF8 is also able to ubiquitinate itself (auto-ubiquitination) with K48-Ub and whether this serves as a signal for its own degradation. Flag-RNF8-WT or Flag-RNF8 bearing one inactivating amino acid mutation (C403S) in its E3-ligase active centre (RING finger mutated variant, RING*) (Mailand et al, 2007) was expressed and isolated under denaturing conditions. While RNF8-WT was heavily K48-polyubiquitinated and to a much lesser extent K63-polyubiquitinated, the inactive RNF8-RING* variant was almost completely devoid of ubiquitination (Fig 2A). Additionally, siRNA-mediated depletion of p97 further enhanced polyubiquitination of RNF8-WT, but not RNF8-RING*, suggesting that RNF8 is K48-auto-ubiquitinated and subsequently processed by p97. Figure 2. Homeostasis of RNF8 is controlled by auto-ubiquitination and the ubiquitin–proteasome system Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97). Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10 μM). Graph represents the quantifications of (B) (ns: not significant, P > 0.05, ****P < 0.0001; two-way ANOVA, n = 2, mean + SEM). Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10 μM for 6 h). Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8. Graph represents the quantifications of (E) (nsP > 0.05, ****P < 0.0001; two-way ANOVA, n = 3, mean + SEM). Source data are available online for this figure. Source Data for Figure 2 [embj2019102361-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint p97 is a central component in the ubiquitin-dependent proteasome degradation system, but recent data also suggest a role for p97 in the autophagy-dependent degradation of ubiquitinated substrates (Bug & Meyer, 2012). To investigate whether RNF8 homeostasis is under control of the ubiquitin–proteasome system, we monitored the rate of endogenous RNF8 degradation by cycloheximide (CHX) chase under physiological conditions (Fig 2B and C, and Appendix Fig S1A and B). The rate of RNF8 turnover/degradation was analysed in HeLa cells treated with either DMSO or the proteasome inhibitor MG132. Endogenous RNF8 degradation was strongly suppressed when the proteasome was inhibited (Fig 2B and C). Similar to p97-depleted cells (Fig 1J), inhibition of the proteasome strongly boosted the K48 ubiquitination of RNF8 under physiological conditions (Fig 2D). Additionally, the degradation kinetics of the Flag-RNF8-WT and Flag-RNF8-RING* variants were compared in U2OS cells where the endogenous RNF8 was depleted by RNF8 shRNA (Appendix Fig S1C). RNF8-WT was rapidly degraded, but the degradation of RNF8-RING* was completely blocked (Fig 2E and F). Altogether this suggests that RNF8 generates K48-Ub chains on itself and thus regulates its own turnover and homeostasis via p97 and the proteasome. Ataxin 3 is a nuclear p97-associated DUB that interacts with RNF8 The aforementioned results suggest that RNF8 is heavily auto-K48-polyubiqiutinated, triggering its degradation by the p97–proteasome system. This raises the question of how RNF8 is protected from premature/accelerated degradation. As p97 and the proteasome co-exist with several DUBs, we reasoned that a DUB associated with p97 and/or the proteasome might be involved in the regulation of RNF8 homeostasis. To test this hypothesis, we performed a SILAC-based quantitative p97-interactome mass spectrometry. We focused on RNF8 in the nuclear compartment due to its crucial role in DSB repair. Thus, the p97-Strep-tag proteome was isolated as previously described (Ritz et al, 2011) from nuclear fractions of HEK293 cells before and after exposure to 10 Gy IR (Fig 3A, Table EV1). As expected, p97 interacted with its two main cofactors, the Npl4–Ufd1 complex and p47 and its associated cofactor UBXD7, inside the nucleus. Interestingly, the p97-associated DUB ATX3 was the only DUB identified in the nuclear p97 proteome, which mildly increased in association after IR. The p97 proteome also contained many proteasome subunits, but not the proteasome-associated DUBs Rpn11/PSMD14, Ubp6/USP14 or UCH37/UCHL5 (Collins & Goldberg, 2017). We therefore decided to focus on ATX3. Figure 3. Ataxin 3 is a nuclear p97 associated DUB that interacts with RNF8 Schematic representation of a SILAC-based mass spectrometry approach (upper graph). The p97 interactome showing ATX3 as the only DUB in nucleus with increased interaction with p97 after IR (10 Gy; lower graph). Schematic representation of ATX3 domain structure. C14: cysteine catalytic centre at amino acid 14; UBS1 or 2: ubiquitin-binding site 1 or 2; UIM: ubiquitin-interacting motif; VBM: p97 binding motif; polyQ: polyglutamine region. Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy). Western blot analy
Год издания: 2019
Авторы: Abhay Narayan Singh, Judith Oehler, Ignacio Torrecilla, Susan Kilgas, Shudong Li, Bruno Vaz, Claire Guérillon, John Fielden, Esperanza Hernández-Carralero, Elisa Barrón‐Cabrera, Iain D. C. Tullis, Mayura Meerang, Paul R. Barber, Raimundo Freire, Jason L. Parsons, Borivoj Vojnovic, Anne E. Kiltie, Niels Mailand, Kristijan Ramadan
Издательство: Springer Nature
Источник: The EMBO Journal
Ключевые слова: DNA Repair Mechanisms, Cancer-related Molecular Pathways, Ubiquitin and proteasome pathways
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