AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis
2012; Springer Nature; Volume: 31; Issue: 20 Linguagem: Inglês
10.1038/emboj.2012.236
ISSN1460-2075
AutoresKatja Höpker, Henning Hagmann, Safiya Khurshid, Shuhua Chen, Pia Hasskamp, Tamina Seeger‐Nukpezah, Katharina Schilberg, Lukas C. Heukamp, Tobias Lamkemeyer, Martin L. Sos, Roman K. Thomas, Drew M. Lowery, Frederik Roels, Matthias Fischer, Max C. Liebau, Ulrike Resch, Tülay Kisner, Fabian Röther, Malte P. Bartram, Roman Ulrich Müller, Francesca Fabretti, Peter Kurschat, Björn Schumacher, Matthias Gaestel, René H. Medema, Michael B. Yaffe, Bernhard Schermer, Hans Christian Reinhardt, Thomas Benzing,
Tópico(s)DNA Repair Mechanisms
ResumoArticle21 August 2012free access Source Data AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis Katja Höpker Katja Höpker Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Henning Hagmann Henning Hagmann Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Safiya Khurshid Safiya Khurshid Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Shuhua Chen Shuhua Chen Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Pia Hasskamp Pia Hasskamp Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Tamina Seeger-Nukpezah Tamina Seeger-Nukpezah Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Katharina Schilberg Katharina Schilberg Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Lukas Heukamp Lukas Heukamp Institute of Pathology, University of Cologne, Cologne, Germany Search for more papers by this author Tobias Lamkemeyer Tobias Lamkemeyer Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Martin L Sos Martin L Sos Max Planck Institute for Neurological Research with Klaus-Joachim-Züls-Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany Department of Translational Genomics, University of Cologne, Cologne, Germany Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Search for more papers by this author Roman K Thomas Roman K Thomas Institute of Pathology, University of Cologne, Cologne, Germany Max Planck Institute for Neurological Research with Klaus-Joachim-Züls-Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany Department of Translational Genomics, University of Cologne, Cologne, Germany Search for more papers by this author Drew Lowery Drew Lowery Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA, USA Search for more papers by this author Frederik Roels Frederik Roels Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Matthias Fischer Matthias Fischer Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Max C Liebau Max C Liebau Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Ulrike Resch Ulrike Resch Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Tülay Kisner Tülay Kisner Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Fabian Röther Fabian Röther Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Malte P Bartram Malte P Bartram Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Roman Ulrich Müller Roman Ulrich Müller Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Francesca Fabretti Francesca Fabretti Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Peter Kurschat Peter Kurschat Department of Dermatology, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Björn Schumacher Björn Schumacher Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, Germany Search for more papers by this author Matthias Gaestel Matthias Gaestel Institute of Physiological Chemistry, Hannover Medical University, Hannover, Germany Search for more papers by this author René H Medema René H Medema Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Michael B Yaffe Michael B Yaffe Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA, USA Search for more papers by this author Bernhard Schermer Bernhard Schermer Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, Germany Search for more papers by this author H Christian Reinhardt Corresponding Author H Christian Reinhardt Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Department I of Internal Medicine, University Hospital of Cologne, Cologne, GermanyThere is a Have you seen? (October 2012) associated with this Article. Search for more papers by this author Thomas Benzing Corresponding Author Thomas Benzing Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, GermanyThere is a Have you seen? (October 2012) associated with this Article. Search for more papers by this author Katja Höpker Katja Höpker Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Henning Hagmann Henning Hagmann Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Safiya Khurshid Safiya Khurshid Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Shuhua Chen Shuhua Chen Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Pia Hasskamp Pia Hasskamp Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Tamina Seeger-Nukpezah Tamina Seeger-Nukpezah Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Katharina Schilberg Katharina Schilberg Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Lukas Heukamp Lukas Heukamp Institute of Pathology, University of Cologne, Cologne, Germany Search for more papers by this author Tobias Lamkemeyer Tobias Lamkemeyer Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Martin L Sos Martin L Sos Max Planck Institute for Neurological Research with Klaus-Joachim-Züls-Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany Department of Translational Genomics, University of Cologne, Cologne, Germany Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA Search for more papers by this author Roman K Thomas Roman K Thomas Institute of Pathology, University of Cologne, Cologne, Germany Max Planck Institute for Neurological Research with Klaus-Joachim-Züls-Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany Department of Translational Genomics, University of Cologne, Cologne, Germany Search for more papers by this author Drew Lowery Drew Lowery Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA, USA Search for more papers by this author Frederik Roels Frederik Roels Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Matthias Fischer Matthias Fischer Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Max C Liebau Max C Liebau Department of Pediatrics, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Ulrike Resch Ulrike Resch Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Tülay Kisner Tülay Kisner Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Fabian Röther Fabian Röther Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Malte P Bartram Malte P Bartram Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Roman Ulrich Müller Roman Ulrich Müller Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Francesca Fabretti Francesca Fabretti Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Peter Kurschat Peter Kurschat Department of Dermatology, University Hospital of Cologne, Cologne, Germany Search for more papers by this author Björn Schumacher Björn Schumacher Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, Germany Search for more papers by this author Matthias Gaestel Matthias Gaestel Institute of Physiological Chemistry, Hannover Medical University, Hannover, Germany Search for more papers by this author René H Medema René H Medema Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Michael B Yaffe Michael B Yaffe Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA, USA Search for more papers by this author Bernhard Schermer Bernhard Schermer Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, Germany Search for more papers by this author H Christian Reinhardt Corresponding Author H Christian Reinhardt Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Department I of Internal Medicine, University Hospital of Cologne, Cologne, GermanyThere is a Have you seen? (October 2012) associated with this Article. Search for more papers by this author Thomas Benzing Corresponding Author Thomas Benzing Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Systems Biology of Aging, University of Cologne, Cologne, GermanyThere is a Have you seen? (October 2012) associated with this Article. Search for more papers by this author Author Information Katja Höpker1,‡, Henning Hagmann1,‡, Safiya Khurshid1,2, Shuhua Chen2,3, Pia Hasskamp1, Tamina Seeger-Nukpezah1, Katharina Schilberg1, Lukas Heukamp4, Tobias Lamkemeyer2, Martin L Sos5,6,7, Roman K Thomas4,5,6, Drew Lowery8, Frederik Roels9, Matthias Fischer9,10, Max C Liebau9, Ulrike Resch2, Tülay Kisner1, Fabian Röther1, Malte P Bartram1, Roman Ulrich Müller1,2, Francesca Fabretti1, Peter Kurschat11, Björn Schumacher2,12, Matthias Gaestel13, René H Medema14, Michael B Yaffe8, Bernhard Schermer1,2,12, H Christian Reinhardt 2,3 and Thomas Benzing 1,2,10,12 1Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany 2Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 3Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany 4Institute of Pathology, University of Cologne, Cologne, Germany 5Max Planck Institute for Neurological Research with Klaus-Joachim-Züls-Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany 6Department of Translational Genomics, University of Cologne, Cologne, Germany 7Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA 8Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA, USA 9Department of Pediatrics, University Hospital of Cologne, Cologne, Germany 10Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 11Department of Dermatology, University Hospital of Cologne, Cologne, Germany 12Systems Biology of Aging, University of Cologne, Cologne, Germany 13Institute of Physiological Chemistry, Hannover Medical University, Hannover, Germany 14Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands ‡These authors contributed equally to this work *Corresponding authors: Department II of Internal Medicine, Center for Molecular Medicine Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University Hospital of Cologne, Koeln 50937, Germany. Tel.:+49 2214784480; Fax:+49 2214785959; E-mail: [email protected] I of Internal Medicine, University Hospital of Cologne, Koeln 50937, Germany. Tel.:+49 22147896701; Fax:+49 22147897835; E-mail: [email protected] The EMBO Journal (2012)31:3961-3975https://doi.org/10.1038/emboj.2012.236 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 Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens. Introduction In response to DNA damage, cells activate a complex signalling network to prevent further cell-cycle progression. Activation of this signalling cascade, which is collectively referred to as the DNA damage response (DDR), provides time for DNA repair, recruits repair machinery to the sites of genotoxic damage, or, if the lesions are beyond repair capacity, leads to the activation of additional pathways mediating apoptosis (Jackson and Bartek, 2009). The DDR has traditionally been divided into two major kinase branches operating through the upstream kinases ATM and ATR together with their respective effector kinases Chk2 and Chk1 (Jackson and Bartek, 2009). Moreover, p38 and its downstream substrate mitogen-activated protein kinase-activated protein kinase-2 (MK2) has recently been identified as a third checkpoint effector kinase complex operating downstream of ATM and ATR and parallel to Chk1 (Manke et al, 2005; Raman et al, 2007; Reinhardt et al, 2007, 2010). p38 and MK2 are components of a general stress kinase pathway that acts in response to a variety of stimuli, including inflammatory signals, reactive oxygen species, heat shock, hyperosmolar stress in addition to DNA damage (Kyriakis and Avruch, 2001). One of the major downstream targets of the DDR network is the transcription factor p53. DNA damage-induced phosphorylation of p53, through ATM, Chk2, MK2 and others, at amino-terminal sites close to the MDM2-binding region is thought to reduce ubiquitin-dependent degradation allowing accumulation in the nucleus, where p53 contributes to two distinct cellular responses (Toledo and Wahl, 2006): p53 promotes cell death in response to genotoxic stress through the induction of pro-apoptotic target genes such as PUMA, BAX and BAK. In contrast, p53-mediated transactivation of cell-cycle-arresting target genes, such as CDKN1A, GADD45A or RPRM, serves a protective function by allowing time to repair genotoxic lesions. The molecular cues that dictate the cellular decision between a protective p53-mediated cell-cycle arrest or p53-driven apoptosis have remained largely unclear (Reinhardt and Schumacher, 2012). Numerous regulatory mechanisms, involving selective DNA binding of p53 to its target gene promoters, selective transactivation of p53-bound target genes and differential stability of p53-dependent transcripts, have been reported to modulate these p53-dependent cell fate decisions, which are essential to repress the uncontrolled proliferation of incipient cancer cells carrying severely damaged genomic material (Oda et al, 2000b; Seoane et al, 2002; Wei et al, 2006; Das et al, 2007; Espinosa, 2008). Here, we identify the apoptosis-antagonizing transcription factor (AATF) as a critical regulator of p53-driven apoptosis. AATF had previously been shown to be a substrate of the canonical DDR kinases ATM/ATR and Chk2 (Bruno et al, 2006) and to exert anti-apoptotic activity through promoting expression of XIAP in response to genotoxic stress (Bruno et al, 2008). We now demonstrate that AATF negatively regulates p53-driven apoptosis by preventing efficient DNA damage-induced transactivation of the pro-apoptotic p53 target genes PUMA, BAX and BAK. Moreover, we show that AATF resides in cytoplasmic protein complexes with the cytoskeletal protein myosin-regulatory light chain-3 (MRLC3) in resting cells. Following DNA damage, this interaction is lost through MK2-dependent AATF phosphorylation resulting in nuclear translocation of AATF and the specific engagement of the PUMA, BAX and BAK promoters to repress p53-dependent transcription of these proapoptotic genes. Interestingly, AATF neither binds to the promoters, nor regulates the expression of the cell-cycle-regulating p53 target genes CDKN1A, GADD45α or RPRM. Furthermore, our data show that AATF amplification in tumours results in worse prognosis of neuroblastoma patients. We propose that AATF functions to repress the pro-apoptotic arm of the p53 response to promote a primarily growth-arresting cellular response to genotoxic insults. These data suggest AATF to be an attractive drug target since AATF depletion resulted in substantially increased sensitivity to frontline chemotherapeutic drugs. Results A phospho-proteomic screen links MRLC3 to DNA damage kinase signalling To identify new protein complexes that are regulated through DNA damage kinase signalling, we employed a phosphopeptide library versus protein expression library screening approach. We were interested in protein complexes that were disrupted in a phosphorylation-dependent manner, such as the p53:MDM2 complex, which dissociates upon p53 phosphorylation by numerous DDR kinases (Lavin and Gueven, 2006; Toledo and Wahl, 2006). We used a library of partially degenerate peptides resembling the substrate motif phosphorylated by the DDR kinases Chk1/2 and MK2, corresponding to the sequence [L/I/F]-X-[R/K]-[Q/X]-X-S/T-X-X-X, where X represents any amino acid except cysteine. Both phospho- and non-phospho versions of this library, hereafter denoted ϕ-X-R-X-X-pT and ϕ-X-R-X-X-T, were generated. Streptavidin bead-immobilized peptide libraries were used as bait in an interaction screen against a protein library produced by in vitro transcription/translation (Elia et al, 2003; Manke et al, 2003). We screened a total of ∼200 000 cDNAs arrayed in 2000 pools containing 100 individual, in vitro-translated and 35S-labelled cDNAs. Most in vitro-translated proteins did not bind to either library (Figure 1A). However, we identified 14 distinct pools, in which a 17-kDa protein displayed preferential binding to the ϕ-X-R-X-X-T library (Figure 1A). Progressive subdivision of these pools identified the cytoskeletal protein MRLC3 as the clone that preferentially associated with the ϕ-X-R-X-X-T library (Figure 1A and B). To further validate this interaction, we expressed FLAG-tagged MRLC3 in HEK293T cells and performed in vitro pull down experiments using the streptavidin-immobilized ϕ-X-R-X-X-T and ϕ-X-R-X-X-pT libraries as bait. As shown in Supplementary Figure 1A, MRLC3 displayed robust binding to the ϕ-X-R-X-X-T, but essentially no binding to the ϕ-X-R-X-X-pT library, suggesting that Thr-phosphorylation within the checkpoint kinase motif disrupts the interaction with MRLC3. Figure 1.Identification of a phosphorylation-sensitive protein complex consisting of AATF and MRLC3. (A) An oriented (pSer/pThr) phosphopeptide library, biased towards the basophilic phosphorylation motif of Chk1/2 and MK2, was immobilized on streptavidin beads. The phospho ϕXRXXpT and non-phosphorylated ϕXRXXT peptide libraries were screened for interaction against in vitro translated, 35S-Met-labelled proteins. (B) Identification of MRLC3 as a non-phospho binder occurred in pool 16B11 and through progressive subdivision to a single clone. (C) Yeast two-hybrid screening revealed AATF as an interactor of MRLC3. We further characterized this interaction through co-immunoprecipitation (co-IP), performed in the presence or absence of 1 μM okadaic acid (OA). FLAG.MRLC3 was immunoprecipitated from HEK293T cells co-expressing V5.AATF. FLAG.GFP served as a control. Lane 3 shows an interaction of FLAG.MRLC3 with V5.AATF, which was abolished by OA-mediated Ser/Thr phosphatase inhibition 1 h prior to lysis (lane 4). (D) The MRLC3:AATF complex is sensitive to UV-C-induced DNA damage. FLAG.MRLC3 and V5.AATF-expressing HEK293T cells were UV-C irradiated (20 J/m2) 30 min prior to lysis and IP with anti-FLAG beads. FLAG.GFP served as a negative control. While V5.AATF co-precipitated with FLAG.MRLC3 in the absence of UV-C, the interaction was abrogated in the presence of DNA damage. (E) Reversal of the co-IP experiment is shown in (D). Anti-FLAG IP reveals AATF.FLAG:V5.MRLC3 complexes that display strong sensitivity to UV-C-induced DNA damage. FLAG.GFP served as a negative control. (F) Endogenous AATF:MRLC complexes display UV-C sensitivity. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to UV-C (20 J/m2) 30 min prior to lysis and IP. GFP IP served as a negative control (lanes 1 and 2). While substantial amounts of MRLC co-immunoprecipitated with AATF (lane 3), this interaction was abolished by UV-C-induced DNA damage (lane 4). (G) Endogenous AATF:MRLC3 complexes are sensitive to the topoisomerase-II inhibitor doxorubicin. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to doxorubicin (1 μM) 1 h prior to IP. GFP antibody served as a negative control (lanes 1 and 2). Doxorubicin (lane 4) disrupted the interaction between AATF and MRLC (lane 3). Figure source data can be found with the Supplementary data. Download figure Download PowerPoint We next investigated the interactome of MRLC3 using yeast two-hybrid screening. These experiments identified AATF as a likely MRLC3-interacting protein. To confirm this interaction in mammalian cells, we performed co-immunoprecipitation experiments in HEK293T cells co-expressing V5.AATF and FLAG.MRLC3 or FLAG.GFP, as a control. While AATF could readily be detected in the FLAG.MRLC3 precipitates, it was undetectable in the FLAG.GFP precipitations, thus validating the interaction between AATF and MRLC3 (Supplementary Figure 1B). Since MRLC3 was identified as a protein with strong selective binding to peptides corresponding to the non-phosphorylated forms of checkpoint kinase substrate motifs, but not to these same peptides following phosphorylation, we asked whether the AATF:MRLC3 interaction could be disrupted by phosphatase inhibition. In agreement with the results of the phospho-proteomic screen, treatment of V5.AATF and FLAG.MRLC3-expressing cells with the Ser/Thr phosphatase inhibitor okadaic acid, abrogated the AATF:MRLC3 interaction (Figure 1C). We then went on to investigate whether the phosphorylation-sensitive interaction between AATF and MRLC3 is regulated by checkpoint kinases in response to genotoxic stress and performed co-immunoprecipitation experiments before and after DNA damage. As we had observed before, V5.AATF co-precipitated with FLAG.MRLC3 in mock-treated cells. In contrast, this interaction was abolished when cells were pre-treated with UV-C, indicating that genotoxic stress negatively regulates MRLC3:AATF complex formation (Figure 1D). Identical co-precipitation behaviour was observed when the FLAG and V5 tags were swapped (Figure 1E). Disruption of the MLRC3:AATF complex was also observed following treatment of cells with doxorubicin, indicating that the complex is sensitive to multiple types of genotoxic stress (Supplementary Figure 1C). To ask whether endogenous AATF and MRLC3 form similar DNA damage-sensitive complexes, we immunoprecipitated AATF from HCT116 cells and used immunoblotting to detect co-precipitating MRLC3. These experiments confirmed the existence of a physiological interaction between AATF and MRLC3 in resting cells (Figure 1F, lane 3). As expected, application of UV-C or addition of doxorubicin prior to cell lysis abolished this endogenous interaction (Figure 1F and G), recapitulating the effects seen with overexpressed proteins. These data demonstrate that AATF and MRLC3 form a phosphorylation-sensitive protein complex, which is disrupted in response to genotoxic stress, likely mediated through the activity of a basophilic checkpoint kinase. MRLC3 sequesters AATF in the cytoplasm While MRLC3 is believed to reside predominantly in the cytoplasm, the subcellular localization of AATF is less well understood (Watanabe et al, 2007). Furthermore, it remains unclear whether AATF or MRLC3 dynamically shuttle between distinct subcellular compartments upon disruption of the AATF:MRLC3 complex. We directly investigated the spatial dynamics of MRLC3 and AATF in mouse embryonic fibroblasts (MEFs), using biochemical separation of nuclear and cytoplasmic fractions through hypotonic lysis. As shown in Figure 2A, MRLC3 was found exclusively in the cytoplasm and its subcellular distribution was not affected by UV-C-induced genotoxic stress. In marked contrast, AATF showed a DNA damage-dependent dynamic re-localization between cytoplasm and nucleus. While only minuscule amounts of endogenous AATF were detectable in the nuclei of resting cell
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