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• W1968153823 abstract "Article23 August 2013free access Source Data WASH inhibits autophagy through suppression of Beclin 1 ubiquitination Pengyan Xia Pengyan Xia Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Shuo Wang Shuo Wang Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ying Du Ying Du Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhenao Zhao Zhenao Zhao State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Shi Lei Shi Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Sun Lei Sun Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guanling Huang Guanling Huang Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Buqing Ye Buqing Ye Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chong Li Chong Li Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhonghua Dai Zhonghua Dai Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ning Hou Ning Hou State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Xuan Cheng Xuan Cheng State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Qingyuan Sun Qingyuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Li Lei Li State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiao Yang Corresponding Author Xiao Yang State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Zusen Fan Corresponding Author Zusen Fan Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Pengyan Xia Pengyan Xia Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Shuo Wang Shuo Wang Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ying Du Ying Du Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhenao Zhao Zhenao Zhao State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Shi Lei Shi Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Sun Lei Sun Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guanling Huang Guanling Huang Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Buqing Ye Buqing Ye Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chong Li Chong Li Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zhonghua Dai Zhonghua Dai Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ning Hou Ning Hou State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Xuan Cheng Xuan Cheng State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Qingyuan Sun Qingyuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lei Li Lei Li State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiao Yang Corresponding Author Xiao Yang State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China Search for more papers by this author Zusen Fan Corresponding Author Zusen Fan Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Author Information Pengyan Xia1,‡, Shuo Wang1,‡, Ying Du1, Zhenao Zhao2, Lei Shi1, Lei Sun3, Guanling Huang1, Buqing Ye1, Chong Li1, Zhonghua Dai1, Ning Hou4, Xuan Cheng4, Qingyuan Sun2, Lei Li2, Xiao Yang 4 and Zusen Fan 1 1Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 2State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 3Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 4State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, China ‡These authors contributed equally to this work. *Corresponding authors. The State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing 100071, China. Tel.:+86 10 63895937; Fax:+86 10 63895937; E-mail: [email protected] CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. Tel.:+86 10 64888457; Fax:+86 10 64871293; E-mail: [email protected] The EMBO Journal (2013)32:2685-2696https://doi.org/10.1038/emboj.2013.189 There is a Have you seen? (October 2013) associated with this Article. 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 Autophagy degrades cytoplasmic proteins and organelles to recycle cellular components that are required for cell survival and tissue homeostasis. However, it is not clear how autophagy is regulated in mammalian cells. WASH (Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue) plays an essential role in endosomal sorting through facilitating tubule fission via Arp2/3 activation. Here, we demonstrate a novel function of WASH in modulation of autophagy. We show that WASH deficiency causes early embryonic lethality and extensive autophagy of mouse embryos. WASH inhibits vacuolar protein sorting (Vps)34 kinase activity and autophagy induction. We identified that WASH is a new interactor of Beclin 1. Beclin 1 is ubiquitinated at lysine 437 through lysine 63 linkage in cells undergoing autophagy. Ambra1 is an E3 ligase for lysine 63-linked ubiquitination of Beclin 1 that is required for starvation-induced autophagy. The lysine 437 ubiquitination of Beclin 1 enhances the association with Vps34 to promote Vps34 activity. WASH can suppress Beclin 1 ubiquitination to inactivate Vps34 activity leading to suppression of autophagy. Introduction Macroautophagy (herein referred to as autophagy) degrades cytoplasmic proteins and organelles to recycle cellular components that are required for cell survival and tissue homeostasis (Shintani and Klionsky, 2004; Lee et al, 2010; Bodemann et al, 2011; Mizushima and Komatsu, 2011). During autophagy induction, double-membrane vesicles called autophagosomes are produced to sequester intracellular cargos and fused with lysosomes to form autolysosomes for subsequent degradation. Many autophagy-related genes (Atg) have been identified and characterized in yeast and some of them are evolutionarily conserved (Nakatogawa et al, 2009; Egan et al, 2011). Deficiency of some Atg or Atg-related genes resulted in early embryonic lethality or death of neonates in mice (Yue et al, 2003; Kuma et al, 2004; Fimia et al, 2007). Beclin 1, a homologue of the Atg6/vacuolar protein sorting (Vps)30 protein in yeast, was first identified as a Bcl-2 interacting protein. Bcl-2 sequesters Beclin 1 from the core Beclin 1–Vps34 complex and inhibits autophagy (Pattingre et al, 2005). Beclin 1 is critical for localization of autophagic proteins to a preautophagosomal structure (PAS) through interaction with the class III phosphoinositide 3 kinase (PI3KC3/Vps34) (Weidberg et al, 2011). Vps34 can phosphorylate the D-3 position on the inositol ring of phosphatidylinositol to generate phosphatidylinositol-3-phosphate (PI3P), which is essential for recruiting other regulatory factors to the site of autophagosome formation (Miller et al, 2010). Beclin 1 association with Vps34 was reported to be regulated through their post-translational modifications or other protein partners (Fimia et al, 2007; Takahashi et al, 2007; Zalckvar et al, 2009; Furuya et al, 2010; Shi and Kehrl, 2010). However, it is unclear how Vps34 activity is regulated in the process of autophagy. WASH (Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue) is a recently identified member of the WASP family (Linardopoulou et al, 2007). WASH plays an essential role in endosome sorting through facilitating tubule fission via Arp2/3 activation (Derivery et al, 2009; Gomez and Billadeau, 2009). These reports showed that WASH is an endosomal protein that exists in the FAM21-containing multiprotein complex. However, its real in vivo roles have not been defined yet. Here, we demonstrate a novel function of WASH in modulation of autophagy. We found that WASH deficiency causes early embryonic lethality and extensive autophagy of mouse embryos. WASH is a negative regulator of autophagy through suppression of lysine 437 ubiquitination of Beclin 1 to inhibit Vps34 activity. Results WASH deficiency causes embryonic lethality and extensive autophagy To explore the in vivo roles of WASH, we generated WASH-conditional knockout (KO) mice (WASHflox/flox) with loxP sites flanking exon3 of the WASH gene (Figure 1A). WASH−/− mice were produced by crossing WASHflox/flox mice with Ella-Cre transgenic mice (Figure 1B). Surprisingly, no WASH−/− neonates were obtained from WASH+/−mice. Indeed, homozygous mutation of the WASH gene led to early embryonic lethality (Figure 1C). We observed that WASH was constitutively expressed in many tissues and various embryonic days of embryos (Supplementary Figure S1). We further found that WASH deficiency caused embryonic abnormality at embryonic day (E) 7.5 and the abnormal embryos were resorbed at E9.5. We found that E7.5 embryos of WASH−/− mice had no obvious three layers (endoderm, mesoderm, and ectoderm) (Figure 1D). Some cavities, such as ectoplacental cavity, exocoelomic cavity, and amniotic cavity, were not well organized. Interestingly, the E7.5 WASH−/−embryonic cells exhibited massive cell death that was not apoptotic cell death (Figure 1E). Surprisingly, these embryos presented substantial autophagosome-like structures by electron microscopy (Figure 1F, upper panel). Moreover, we observed that light chain 3 (LC3) was localized in the autophagosome-like structures by immuno-electron microscopy. To confirm this autophagic phenotype, we isolated E7.5 embryos and lysed them for immunoblotting. Microtubule-associated LC3 is an autophagy indicator by conversion of LC3-I into LC3-II (Kabeya et al, 2000), and p62 is an autophagy substrate. We found that WASH−/− embryos exhibited dramatically enhanced LC3 conversion and a reduced level of p62 (Figure 1F, lower panel). WASH was deleted in the E7.5 WASH−/− embryo which was confirmed by immunoblotting with anti-WASH antibody (Figure 1F, lower panel). We concluded that excessive autophagy caused by WASH deficiency leads to autophagic cell death of embryos, which is in agreement with autophagic cell death in previous reports (Yu et al, 2004; Elgendy et al, 2011). Collectively, WASH deficiency leads to early embryonic lethality and extensive autophagy of mouse embryos. Figure 1.WASH deficiency causes embryonic lethality and extensive autophagy. (A) Strategy to generate WASH-knockout (WASH−/−) mice. The coding exons of mWASH gene were shown as white boxes. The target vector with exon3 of mWASH gene was flanked with two loxP sites (arrow) and one neomycin resistance gene. The primers used for genotyping were KO primers for deficient genotypes, loxP primers for loxP site identification, and Southern blot probe for floxed mWASH gene. KO, knockout, m, mouse. (B) Detection of WASH gene targeting. The floxed WASH gene by gene targeting was analysed by Southern blotting (left panel) and PCR (middle panel). The genotypes of the offspring were analysed by PCR (right panel). (C) WASH deficiency is embryonic lethal. E11.5 of WASH deficiency showed a shrinked decidua. (D) Histological analysis of E7.5 WASH+/+ and WASH−/− embryos. Scale bar, 100 μm. (E) WASH-deficient embryos do not undergo apoptosis. TUNEL assay was performed for apoptosis detection. PI, propidium iodide. FITC, fluoresceinisothiocyanate. Scale bar, 100 μm. (F) E7.5 embryos of WASH−/− mice present extensive autophagy. Ultrastructure of isolated E7.5 embryos was stained with antibody against LC3 and visualized by immuno-electron microscopy. Black arrowhead indicates autophagosome. *, Cellular reference. Scale bar, 500 nm (upper panel). WASH+/+ or WASH−/− embryos were isolated, and LC3 conversion and p62 level were analysed by immunoblotting (lower panel). Bands were quantified and analysed by Image J and shown as means±s.d. ***P<0.001. Above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page. Source Data for Figure 1 [embj2013189-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint WASH inhibits autophagy To examine how WASH regulates autophagy, we generated WASH−/− mouse embryonic fibroblasts (MEFs) by expressing Cre recombinase in WASHflox/flox MEFs. WASH-deficient MEFs were treated with Earle's balanced salt solution (EBSS), an amino acid and growth factor-free solution mimicking a nutrient-deprived condition. As expected, WASH deficiency enhanced much more conversion of LC3-I into LC3-II and degradation of p62 with nutrient deprivation compared with wild-type (WT) (WASH+/+) MEFs (Figure 2A). The autophagic process in WASH−/− MEFs took place more rapidly than that of WASH+/+ MEFs. Notably, a lysosomal inhibitor bafilomycin A1 (BafA1) was able to block the degradation of LC3-II and p62 during autophagy (Figure 2A), suggesting that WASH−/− MEFs induce robust autophagy and autophagic flux. Similar results were obtained by immunofluorescence staining (Figure 2B). Expectedly, WASH−/− MEFs showed a lower level of polyubiquitinated proteins (Figure 2C), and BafA1 could block the autophagic process but not a proteasome inhibitor MG132. Additionally, WASH−/− MEFs showed much more autophagosomes visualized by immuno-electron microscopy (Figure 2D). We observed that different morphologies between the WASH KO embryos and MEFs. The severe enlarged autophagosomes in WASH KO embryos might be caused by overactivated autophagy, which might not appear in cultured MEFs. Taken together, WASH deficiency enhances autophagy induction. Figure 2.WASH inhibits autophagy. (A) WASH deficiency enhances autophagy induction. WASH+/+ or WASH−/− MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 (BafA1), and harvested for immunoblotting. Ratios of LC3-II/β-actin were calculated and shown at the right panel. (B) WASH knockout accelerates LC3 conversion by confocal microscopy. Endogenous LC3 puncta were visualized by staining with antibody against LC3 (left panel), and calculated as shown in the right panel. Nuclei were stained with DAPI. Scale bar, 10 μm. (C) Poly-ubiquitinated proteins are reduced in WASH−/− MEFs. WASH+/+ and WASH−/− MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132. Cells were harvested for immunoblotting with anti-poly-ubiquitin antibody (Enzo, clone FK1) that only recognizes poly-ubiquitinated ubiquitin chains. (D) Autophagosome-like structures in WASH+/+ and WASH−/− MEFs were visualized after starvation for 1 h by immuno-electron microscopy with antibody against LC3 (left panel). Black arrowhead indicates autophagosome. Scale bar, 500 nm. *, Cellular reference. Fifty cells were quantified from three independent experiments (right panel). Data were shown as means±s.d. *P<0.05 and **P<0.01. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013189-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint WASH is localized in autophagosomes WASH was colocalized with GFP-LC3-positive autophagosomes under starvation (Figure 3A), but not under normal culture conditions (CM). This colocalization was not merged with EEA1 with EBSS treatment (Figure 3B), an early endosome marker, which colocalizes with WASH in CM culture, suggesting that WASH exerts its autophagic role independently of its endosomal trafficking function. p16-Arc is a component of the Arp2/3 complex that is essential for endosomal sorting and FAM21 is required for the endosomal localization of WASH (Singh et al, 2003; Gomez and Billadeau, 2009). p16-Arc or FAM21 silencing did not influence autophagy induction compared with the control shRNA-treated (shCtrl) cells (Supplementary Figure S2A and B). The VCA domain of WASH is required for the endosomal sorting function (Derivery et al, 2009; Jia et al, 2010). Additionally, the VCA domain-truncated WASH (ΔVCA) mutant could still rescue the accelerated autophagy induction in WASH KO MEFs comparable to that of the full-length (FL) WASH restoration (Supplementary Figure S2C). These observations indicate that WASH suppresses autophagy independently of its endosomal sorting function. Atg4 is essential for formation of autophagosomes, and Atg4 mutant (C74A) that impairs LC3 lipidation disrupts the closure of autophagosomes (Fujita et al, 2008). Importantly, in Atg4 (C74A)-expressed cells, WASH colocalized with Atg5 (Figure 3C), suggesting that WASH is localized in forming autophagosomes. However, WASH did not colocalize with the lysosome marker LAMP1 during the autophagy process (Figure 3D), which suggests that WASH does not function in autolysosomes. p62 is an autophagic substrate that is associated with LC3 on isolated membranes and incorporated into autophagosomes (Mathew et al, 2009; Itakura and Mizushima, 2011). Expectedly, WASH colocalized with p62 under starvation (Figure 3E). To further confirm the precise localization of WASH, we performed immuno-electron microscopy. We observed that WASH resided in the unclosed and closed autophagosomes, but not in the autolysosomes (Figure 3F). Moreover, WASH was not localized in the autolysosomes that was verified by detection of fractionation of organelles (Figure 3G). These results suggest that WASH may function in regulation of autophagosome formation. Figure 3.WASH is localized in autophagosomes. (A) WASH colocalizes with GFP-LC3 upon starvation. HeLa cells stably expressing GFP-LC3 were stained with anti-WASH antibody after treatment with EBSS or culture medium (CM) for 1 h. (B) Autophagy-related WASH does not colocalize with EEA1. HeLa cells stably expressing GFP-LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH. (C) WASH localizes to unclosed autophagosomes. HeLa cells stably expressing GFP-Atg5 were transfected with Atg4B(C74A) mutant, followed by stimulation with EBSS for 1 h. Cells were stained with anti-WASH antibody (red). (D) WASH does not localize in autolysosomes. HeLa cells were treated with CM or EBSS in the presence or absence of 20 nM BafA1 at 37°C for 1 h. WASH and LAMP1 were visualized by staining with anti-WASH and anti-LAMP1 antibodies. (E) Colocalization of WASH and autophagy substrate p62 during autophagy. HeLa cells treated with or without EBSS were stained with anti-p62 and anti-WASH antibodies. For (A–E), scale bar, 10 μm. (F) Immuno-electron microscopy analysis of HeLa cells treated with EBSS for 1 h. Black arrowhead indicates WASH particle, red arrowhead indicates autophagosome, and black arrow denotes autolysosome. In all, 73±2% of autophagosomes contains WASH particles. Scale bar, 100 nm. (G) WASH is not localized in autolysosomes. Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies. The above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013189-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint WASH suppresses Vps34 activity We next wanted to test whether WASH inhibits autophagy through disturbing the Beclin 1–Vps34 complex. We found that anti-Beclin 1 antibody could precipitate much more Vps34 protein in WASH−/− MEFs than in WASH+/+MEFs, especially after EBSS treatment (Figure 4A). WASH deficiency did not change the stability of Beclin 1 even with EBSS treatment (Supplementary Figure S3). More importantly, WASH deficiency increased the Beclin 1-bound Vps34 kinase activity (Figure 4B). WIPI1 is a PI3P-binding protein that is involved in the formation of phagophores (Proikas-Cezanne et al, 2007). Notably, GFP-WIPI1 puncta were dramatically enhanced in WASH−/− MEFs in EBSS-induced autophagy (Figure 4C). Additionally, in WASH-silenced cells, Beclin 1 could precipitate more Vps34 protein compared with those of shCtrl cells. WASH depletion augmented Vps34 kinase activity (Figure 4D). By contrast, Flag–WASH-overexpressed MEFs declined the amount of co-purifying Vps34 protein level and dots of WIPI1 (Figure 4E and F). Collectively, WASH significantly inhibits Vps34 activity during the induction of autophagy. Figure 4.WASH inhibits Vps34 activity. (A) WASH deficiency enhances the interaction of Vps34 with Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and immunoprecipitated for Beclin 1. IP, immunoprecipitation. Ratio of Vps34/Beclin 1 was calculated by the Image J software and shown in the right panel. (B) WASH KO MEFs were treated with or without EBSS at 37°C for 1 h followed by kinase assays. Cells were lysed and incubated with anti-Beclin 1 antibody to precipitate the autophagy-related Vps34. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection. RLU, relative light unit. (C) WASH deficiency enhances Vps34 activity. WASH+/+ and WASH−/− MEFs stably expressing GFP-WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy (left panel). GFP-WIPI1 dots were calculated and shown in the right panel. Scale bar, 10 μm. (D) WASH-silenced HeLa cells were treated with or without EBSS at 37°C for 1 h, and detected as above. (E) WASH attenuates the interaction of Beclin 1 with Vps34. MEFs stably expressing vector or Flag–WASH were starved with EBSS for 1 h, followed by immunoprecipitation with anti-Beclin 1 antibody. Ratio of Vps34/Beclin 1 was shown in the right panel. (F) WASH overexpression reduces Vps34 activity. MEFs stably expressing GFP-WIPI1 were transfected with vector or Flag–WASH and then starved with EBSS for 1 h, followed by visualization with confocal microscopy (upper panel). GFP-WIPI1 dots were calculated and shown in the lower panel. Scale bar, 10 μm. Data are shown as means±s.d. **P<0.01 and ***P<0.001. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page. Source Data for Figure 4 [embj2013189-sup-0004-SourceData-S4.pdf] Download figure Download PowerPoint WASH interacts with Beclin 1 We screened a human spleen cDNA library to fish out WASH interactors using a yeast two-hybrid approach. Beclin 1 is a core component of Vps34 complex (Kihara et al, 2001; Funderburk et al, 2010). Interestingly, we identified that WASH was a novel interacting protein of Beclin 1 (Figure 5A). Bloc1s2, a known interactor of WASH (Monfregola et al, 2010), was used as a positive control in verification of the association via the yeast two-hybrid assay. Additionally, WASH did not interact with other components of the Vps34 or the Ulk1/Ulk2 complex (Supplementary Figure S4). The WASH–Beclin 1 interaction was verified in co-transfected human embryonic kidney epithelial 293T (HEK293T) cells by co-immunoprecipitation (co-IP) (Figure 5B and C). Using truncated WASH fragments, we defined that WASH (aa121–221) was necessary and sufficient for interaction with Beclin 1 (Figure 5D). Importantly, the deletion of aa121–221 (Δ121–221) of WASH failed to restore the accelerated autophagy process in WASH KO MEFs (Supplementary Figure S5A). However, the deletion of aa121–221 (Δ121–221) of WASH was able to rescue the enhanced EGFR degradation in WASH KO MEFs, while the WASH (ΔVCA) mutant had no such activity (Supplementary Figure S5B). These data confirmed that WASH exerts its autophagy regulation independently of its endosomal sorting role. Additionally, the central region of Beclin 1 (aa200–238) was mainly sufficient for association with WASH (Figure 5E). Moreover, recombinant MBP-tagged WASH (MBP–WASH) was able to pull down recombinant Beclin 1 (rBeclin 1) (Figure 5F), indicating that WASH directly binds to Beclin 1. Moreover, GST–Beclin 1 or anti-Beclin 1 antibody could precipitate endogenous WASH and vice versa (Figure 5G). We observed that the colocalization rate between WASH and Beclin 1 was declined under nutrient-deprived conditions compared with CM conditions by confocal microscopy (Figure 5H), indicating that reduced WASH binding appeared undergoing autophagy. Additionally, with EBSS treatment, the decreased association between WASH and Beclin 1 was confirmed by co-IP assays (Figure 5I). Figure 5.WASH associates with Beclin 1. (A) The interaction of WASH with Beclin 1 was validated by yeast two-hybrid assays. Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD) fused WASH and Gal4 activating domain (AD) fused Beclin 1. p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls. (B, C) WASH co-immunoprecipitates Beclin 1 in mammalian cells. Flag-tagged Beclin 1 and Myc-tagged WASH were co-transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection. IP, immunoprecipitation. (D) Mapping the interacting regions between WASH and Beclin 1. (E) The indicated Flag-tagged Beclin 1 constructs encoding different regions of Beclin 1 were co-transfected with full-length HA-tagged WASH (HA–WASH) into HEK293T cells followed by immunoprecipitation. (F) WASH directly interacts with Beclin 1. Recombinant MBP-tagged WASH (MBP–WASH) or Beclin 1 (rBeclin 1) was expressed in E. coli and then subjected to MBP pull-down assay. (G) GST–Beclin 1 precipitates WASH from HeLa cell lysates. GST-Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting. (H) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy. HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH. The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and WASH was calculated and shown as means±s.d. in the lower panel. Scale bar, 10 μm. (I) HeLa cells were treated with CM or EBSS for 1 h, followed by immunoprecipitation with antibodies against WASH (left panel) or Beclin 1 (right panel), and the immunoprecipitates were detected with the indicated antibodies.***P<0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page. Source Data for Figure 5 [e" @default.
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• W1968153823 title "WASH inhibits autophagy through suppression of Beclin 1 ubiquitination" @default.
• W1968153823 cites W1972336737 @default.
• W1968153823 cites W1977767269 @default.
• W1968153823 cites W1978405493 @default.
• W1968153823 cites W1979339081 @default.
• W1968153823 cites W1982307380 @default.
• W1968153823 cites W1983831622 @default.
• W1968153823 cites W1985124617 @default.
• W1968153823 cites W1991752238 @default.
• W1968153823 cites W1992659717 @default.
• W1968153823 cites W1994239968 @default.
• W1968153823 cites W1996747646 @default.
• W1968153823 cites W1998170956 @default.
• W1968153823 cites W1999477136 @default.
• W1968153823 cites W1999801453 @default.
• W1968153823 cites W2007970201 @default.
• W1968153823 cites W2009781189 @default.
• W1968153823 cites W2016040396 @default.
• W1968153823 cites W2017779665 @default.
• W1968153823 cites W2042884520 @default.
• W1968153823 cites W2052470191 @default.
• W1968153823 cites W2056663185 @default.
• W1968153823 cites W2064381459 @default.
• W1968153823 cites W2064746187 @default.
• W1968153823 cites W2065896797 @default.
• W1968153823 cites W2066800765 @default.
• W1968153823 cites W2068784898 @default.
• W1968153823 cites W2071519991 @default.
• W1968153823 cites W2078013685 @default.
• W1968153823 cites W2079849337 @default.
• W1968153823 cites W2081431643 @default.
• W1968153823 cites W2085442981 @default.
• W1968153823 cites W2092321143 @default.
• W1968153823 cites W2095806089 @default.
• W1968153823 cites W2103659405 @default.
• W1968153823 cites W2104095808 @default.
• W1968153823 cites W2104383425 @default.
• W1968153823 cites W2112329697 @default.
• W1968153823 cites W2116247281 @default.
• W1968153823 cites W2123777737 @default.
• W1968153823 cites W2125233904 @default.
• W1968153823 cites W2128558707 @default.
• W1968153823 cites W2135901758 @default.
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