ATM ‐ CHK 2‐Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress

Article18 March 2020free access Source DataTransparent process ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress Qi-Qiang Guo Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Shan-Shan Wang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Shan-Shan Zhang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Hong-De Xu Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Xiao-Man Li Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Yi Guan Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Fei Yi Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Ting-Ting Zhou Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Bo Jiang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Ning Bai Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Meng-Tao Ma Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Zhuo Wang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Yan-Ling Feng Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Wen-Dong Guo Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Xuan Wu Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Gui-Feng Zhao Department of Experimental Oncology and Animal Center, Key Laboratory of Research and Application of Animal Models for Environmental and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang, China Search for more papers by this author Guang-Jian Fan orcid.org/0000-0003-0684-1380 Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Sheng-Ping Zhang Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Chuan-Gui Wang Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Long-Yue Cao Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology), Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Brian P O'Rourke Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Shi-Hui Liu Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Ping-Yuan Wang Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Shuai Han Corresponding Author [email protected] orcid.org/0000-0002-0398-2877 Department of Neurosurgery, The First Hospital of China medical University, Shenyang, China Search for more papers by this author Xiao-Yu Song Corresponding Author [email protected] orcid.org/0000-0002-6606-327X Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Liu Cao Corresponding Author [email protected] orcid.org/0000-0001-6471-1993 Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Qi-Qiang Guo Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Shan-Shan Wang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Shan-Shan Zhang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Hong-De Xu Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Xiao-Man Li Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Yi Guan Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Fei Yi Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Ting-Ting Zhou Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Bo Jiang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Ning Bai Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Meng-Tao Ma Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Zhuo Wang Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Yan-Ling Feng Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Wen-Dong Guo Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Xuan Wu Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Gui-Feng Zhao Department of Experimental Oncology and Animal Center, Key Laboratory of Research and Application of Animal Models for Environmental and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang, China Search for more papers by this author Guang-Jian Fan orcid.org/0000-0003-0684-1380 Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Sheng-Ping Zhang Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Chuan-Gui Wang Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Long-Yue Cao Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology), Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Brian P O'Rourke Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Shi-Hui Liu Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Ping-Yuan Wang Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Shuai Han Corresponding Author [email protected] orcid.org/0000-0002-0398-2877 Department of Neurosurgery, The First Hospital of China medical University, Shenyang, China Search for more papers by this author Xiao-Yu Song Corresponding Author [email protected] orcid.org/0000-0002-6606-327X Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Liu Cao Corresponding Author [email protected] orcid.org/0000-0001-6471-1993 Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China Search for more papers by this author Author Information Qi-Qiang Guo1, Shan-Shan Wang1, Shan-Shan Zhang1, Hong-De Xu1, Xiao-Man Li1, Yi Guan1, Fei Yi1, Ting-Ting Zhou1, Bo Jiang1, Ning Bai1, Meng-Tao Ma1, Zhuo Wang1, Yan-Ling Feng1, Wen-Dong Guo1, Xuan Wu1, Gui-Feng Zhao2, Guang-Jian Fan3, Sheng-Ping Zhang3, Chuan-Gui Wang3, Long-Yue Cao4, Brian P O'Rourke5, Shi-Hui Liu6, Ping-Yuan Wang7, Shuai Han *,8, Xiao-Yu Song *,1 and Liu Cao *,1 1Institute of Translational Medicine, Key Laboratory of Cell Biology of Ministry of Public Health, Key Laboratory of Medical Cell Biology of Ministry of Education, Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, China Medical University, Shenyang, China 2Department of Experimental Oncology and Animal Center, Key Laboratory of Research and Application of Animal Models for Environmental and Metabolic Diseases, Shengjing Hospital of China Medical University, Shenyang, China 3Institute of Translational Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 4Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology), Albert Einstein College of Medicine, Bronx, NY, USA 5Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA 6Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 7Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 8Department of Neurosurgery, The First Hospital of China medical University, Shenyang, China *Corresponding author. Tel: +8613840569777; E-mail: [email protected] *Corresponding author. Tel: +8618900910183; E-mail: [email protected] *Corresponding author. Tel: +8618900911888; E-mail: [email protected] EMBO J (2020)39:e103111https://doi.org/10.15252/embj.2019103111 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 homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia-telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1-Bcl-2 autophagy-regulatory complex formation in a ROS-dependent fashion. We further demonstrate that CHK2-mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2−/− mice display aggravated infarct phenotypes and reduced Beclin 1 p-Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2-induced autophagy in cell survival. Taken together, these results indicate that the ROS-ATM-CHK2-Beclin 1-autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress-induced tissue damage. Synopsis Whether hypoxia and nutrient starvation are coupled to cellular autophagy remains unclear. Here, DNA damage response kinases ATM and CHK2 are shown to trigger autophagy in response to reactive oxygen species (ROS) accumulation, suggesting a novel physiological adaptation pathway toward metabolic stress. Depletion of CHK2 or ATM impairs oxidative stress-induced autophagy in MEFs. CHK2 binds and phosphorylates Beclin1 at Ser90/Ser93, suppressing Beclin1-Bcl-2 autophagy regulatory complex formation. CHK2-induced autophagy limits intracellular ROS levels by clearing damaged mitochondria. CHK2-induced autophagy protects against cell death and tissue damage following cerebral ischemia. Introduction Nutrient deprivation and metabolic fluctuations cause oxidative stress due to increased reactive oxygen species (ROS) production coupled with an impaired antioxidant system, leading to redox state imbalance and cell death (Finkel & Holbrook, 2000; D'Autreaux & Toledano, 2007; Holmstrom & Finkel, 2014). Therefore, mechanisms of suppressing excessive ROS and maintaining cell and tissue homeostasis that are independent of the classic antioxidant systems are evolutionarily advantageous. We previously showed that cells deficient in essential autophagy genes have increased basal levels of ROS (Lee et al, 2012). Indeed, autophagy is an essential cellular process that plays a crucial role in recycling cellular components and damaged organelles to eliminate sources of ROS in response to diverse stress conditions (Kroemer et al, 2010; Scherz-Shouval & Elazar, 2011; Filomeni et al, 2015). Growing evidence indicates that ROS is one of the main intracellular signal transducers crucial for sustaining autophagy progression. For instance, the essential autophagy Cys protease ATG4 appears to be a direct target of oxidants generated during starvation. The redox-dependent inactivation of ATG4 leads in turn to increased autophagosome formation (Scherz-Shouval et al, 2007). Starvation-induced ROS has also been shown to oxidize high mobility group box 1, triggering its translocation from the nucleus to the cytoplasm where it interacts with Beclin 1, causing disassociation of Beclin 1 from Bcl-2 and induction of autophagy (Tang et al, 2010). ROS can also activate the lysosomal Ca2+ channel, mucolipin-1 (MCOLN1), triggering the release of Ca2+ into cytosol that results in calcineurin-dependent translocation of transcription factor EB and autophagy induction (Zhang et al, 2016b). However, other pathways presumably exist to couple redox status with autophagic flux and thereby help cells maintain homeostasis during nutrient deprivation or metabolic fluctuations. The ATM/CHK2 protein kinases have a intriguing connection to autophagy. The ATM kinases are activated by DNA double-strand breaks through the Mre11-Rad50-Nbs1 DNA repair complex. ATM kinases orchestrate signaling cascades that initiate the DDR and coordinate many processes including DNA repair, regulation of cell cycle checkpoints, and ultimately the induction of programmed cell death (Matsuoka et al, 1998; Lee et al, 2000; Yang et al, 2002; Bakkenist & Kastan, 2003; Lukas et al, 2003; Roos & Kaina, 2006). However, growing evidence indicates that ATM/CHK2 activation induced by H2O2 can also occur in the absence of DNA damage (Guo et al, 2010), indicating that ATM is an important sensor of ROS in human cells. Other studies suggest that ATM activity is required for ROS-induced autophagy. For instance, in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy (Alexander et al, 2010). A recent study showed that ATM signaling activates ULK1 and inhibits mTORC1 to induce autophagy of peroxisomes (pexophagy) in response to ROS (Zhang et al, 2015). However, the direct crosstalk between the DDR signaling and the autophagic machinery remains unclear. A key factor in autophagy induction is the Beclin 1 protein. Beclin 1 was the first autophagy-related protein identified in mammals (Liang et al, 1998), and BECN is an essential gene required during normal embryonic development (Yue et al, 2003). Beclin 1 also protects against the development of degenerative diseases (Shibata et al, 2006; Pickford et al, 2008; Lipinski et al, 2010; Ashkenazi et al, 2017) and functions as a tumor suppressor (Liang et al, 1999). Beclin 1 regulates both the initiation and the maturation of the autophagosome, by forming distinct PI3K complexes together with the core lipid kinase VPS34 and the regulatory component VPS15. Binding of either ATG14L or UVRAG to the complex promotes the initiation of autophagy and the formation of autophagosomes (Itakura et al, 2008; Liang et al, 2008). Following binding of RUBICON or Bcl-2 to the complex, VPS34 kinase activity is inhibited and autophagic flux is reduced (Liang et al, 1998; Matsunaga et al, 2009; Zhong et al, 2009). Post-transcriptional modifications of Beclin 1, in particular phosphorylation, play an important role in the regulation of autophagy by affecting VPS34 kinase activity and its interaction with various autophagy-regulatory proteins (Hill et al, 2019). For instance, ULK1, AMPK, MAPKAPK2/3, DAPK, CaMKII, and PGK1 can phosphorylate Beclin 1 to promote autophagy (Zalckvar et al, 2009; Kim et al, 2013; Russell et al, 2013; Wei et al, 2015; Fujiwara et al, 2016; Zhang et al, 2016a; Li et al, 2017; Qian et al, 2017). In contrast, AKT1, EGFR, FAK, HER2, and MST1 phosphorylate Beclin 1 to inhibit autophagy (Wang et al, 2012; Maejima et al, 2013; Wei et al, 2013; Cheng et al, 2017; Vega-Rubin-de-Celis et al, 2018). Beclin 1 phosphorylation is also precisely regulated by diverse signals including glucose starvation, serum starvation, amino acid deprivation, glutamine deprivation, ionomycin treatment, and hypoxia (Kim et al, 2013; Russell et al, 2013; Wei et al, 2013; Zhang et al, 2016a; Li et al, 2017; Qian et al, 2017). However, the redox-dependent regulation of the Beclin 1-interacting complex has not been characterized. In this study, we report an unexpected function of CHK2-mediated autophagy in limiting ROS levels during nutrient deprivation and metabolic fluctuations to maintain cell and tissue homeostasis. In response to ROS stimulation, CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, promoting autophagy via Beclin 1 release from Bcl-2 sequestration. Our findings thereby establish a novel and critical role for the ATM/CHK2/Beclin 1 axis in protecting cells from oxidative stress and delineate a pathway whereby ROS functions as a signaling molecule to modulate autophagic flux. Results CHK2 is involved in oxidative stress-induced autophagy To investigate the link between oxidative stress and autophagy, we explored candidate pathways that are involved in the ROS-mediated genotoxic stress under autophagic conditions. We found a potential correlation between CHK2 activation and autophagy biomarkers under starvation conditions with or without addition of N-acetyl cysteine (NAC), a scavenger of free radicals. Starvation caused the expected increase in autophagy, as demonstrated by the decrease in the autophagy substrate p62 and increase in the conversion of the nonlipidated form (LC3-I) to the phosphatidyl ethanolamine-conjugated form (LC3-II) of LC3. This nutrient stress was also accompanied by increased phosphorylation of CHK2 at Thr68, indicating the activation of CHK2. Interestingly, the ROS scavenger NAC could largely inhibit the activation of CHK2, as well as the upregulation of autophagy (Figs 1A and EV1A). These results demonstrate that starvation-induced ROS production is upstream of both endogenous CHK2 activation and autophagy induction. Figure 1. CHK2 is involved in oxidative stress-induced autophagy Western blot detection of p-CHK2 Thr68, CHK2, p62, and LC3 in H1299 cells in normal medium (HBSS “−”) or after 1 h HBSS starvation (HBSS “+”) pretreated with or without NAC (3 mM). Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500 μM) treatment. Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500 μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid. Autophagic flux is shown by representative confocal microscopic images of 293 cells stably expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500 μM) treatment for 3 h. Scale bar, 10 μm. Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500 μM) treatment for 1 h (n = 30). Data are presented as mean ± s.e.m. from three independent experiments; **P < 0.01 (Student's t-test). Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 h HBSS starvation or treated with H2O2 (500 μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures. Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 h HBSS starvation or treated with H2O2 (500 μM). Data are presented as mean ± s.e.m. from three independent experiments; *P < 0.05; **P < 0.01 (Student's t-test). Source data are available online for this figure. Source Data for Figure 1 [embj2019103111-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CHK2 is involved in oxidative stress-induced autophagy A. Intracellular ROS levels detected in H1299 cells treated with HBSS starvation. B. Western blot detection of p62 in H1299