FAM 134B oligomerization drives endoplasmic reticulum membrane scission for ER ‐phagy
2020; Springer Nature; Volume: 39; Issue: 5 Linguagem: Inglês
10.15252/embj.2019102608
ISSN1460-2075
AutoresXiao Jiang, Xinyi Wang, Xianming Ding, Mengjie Du, Boran Li, Xialian Weng, Jingzi Zhang, Lin Li, Rui Tian, Qi Zhu, She Chen, Liang Wang, Wei Liu, Lei Fang, Dante Neculai, Qiming Sun,
Tópico(s)Cellular transport and secretion
ResumoArticle13 January 2020free access FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy Xiao Jiang orcid.org/0000-0002-5305-8148 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xinyi Wang orcid.org/0000-0001-8884-9030 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xianming Ding orcid.org/0000-0002-9256-2419 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Mengjie Du Department of Neurology of Second Affiliated Hospital, Institute of Neuroscience, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Boran Li orcid.org/0000-0003-3159-0421 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xialian Weng Department of Cell Biology, Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jingzi Zhang Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China Search for more papers by this author Lin Li National Institute of Biological Sciences, Beijing, China Search for more papers by this author Rui Tian Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Qi Zhu Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author She Chen National Institute of Biological Sciences, Beijing, China Search for more papers by this author Liang Wang orcid.org/0000-0001-9343-9730 Department of Neurology of Second Affiliated Hospital, Institute of Neuroscience, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Wei Liu orcid.org/0000-0002-8033-4718 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Lei Fang Corresponding Author [email protected] orcid.org/0000-0002-2582-4845 Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China Search for more papers by this author Dante Neculai Corresponding Author [email protected] orcid.org/0000-0001-8887-9168 Department of Cell Biology, Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Qiming Sun Corresponding Author [email protected] orcid.org/0000-0003-4988-9886 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xiao Jiang orcid.org/0000-0002-5305-8148 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xinyi Wang orcid.org/0000-0001-8884-9030 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xianming Ding orcid.org/0000-0002-9256-2419 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Mengjie Du Department of Neurology of Second Affiliated Hospital, Institute of Neuroscience, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Boran Li orcid.org/0000-0003-3159-0421 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Xialian Weng Department of Cell Biology, Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Jingzi Zhang Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China Search for more papers by this author Lin Li National Institute of Biological Sciences, Beijing, China Search for more papers by this author Rui Tian Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Qi Zhu Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author She Chen National Institute of Biological Sciences, Beijing, China Search for more papers by this author Liang Wang orcid.org/0000-0001-9343-9730 Department of Neurology of Second Affiliated Hospital, Institute of Neuroscience, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Wei Liu orcid.org/0000-0002-8033-4718 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Lei Fang Corresponding Author [email protected] orcid.org/0000-0002-2582-4845 Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China Search for more papers by this author Dante Neculai Corresponding Author [email protected] orcid.org/0000-0001-8887-9168 Department of Cell Biology, Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Qiming Sun Corresponding Author [email protected] orcid.org/0000-0003-4988-9886 Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Author Information Xiao Jiang1,‡, Xinyi Wang1,‡, Xianming Ding1,‡, Mengjie Du2, Boran Li1, Xialian Weng3, Jingzi Zhang4, Lin Li5, Rui Tian1, Qi Zhu1, She Chen5, Liang Wang2, Wei Liu1, Lei Fang *,4, Dante Neculai *,3 and Qiming Sun *,1 1Department of Biochemistry, Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China 2Department of Neurology of Second Affiliated Hospital, Institute of Neuroscience, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China 3Department of Cell Biology, Department of General Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 4Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China 5National Institute of Biological Sciences, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 25 83596845; E-mail: [email protected] *Corresponding author. Tel: +86 571 88981627; E-mail: [email protected] *Corresponding author. Tel: +86 571 88208505; E-mail: [email protected] EMBO J (2020)39:e102608https://doi.org/10.15252/embj.2019102608 See also: C De Leonibus et al (March 2020) 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 Degradation of endoplasmic reticulum (ER) by selective autophagy (ER-phagy) is crucial for ER homeostasis. However, it remains unclear how ER scission is regulated for subsequent autophagosomal sequestration and lysosomal degradation. Here, we show that oligomerization of ER-phagy receptor FAM134B (also referred to as reticulophagy regulator 1 or RETREG1) through its reticulon-homology domain is required for membrane fragmentation in vitro and ER-phagy in vivo. Under ER-stress conditions, activated CAMK2B phosphorylates the reticulon-homology domain of FAM134B, which enhances FAM134B oligomerization and activity in membrane fragmentation to accommodate high demand for ER-phagy. Unexpectedly, FAM134B G216R, a variant derived from a type II hereditary sensory and autonomic neuropathy (HSAN) patient, exhibits gain-of-function defects, such as hyperactive self-association and membrane scission, which results in excessive ER-phagy and sensory neuron death. Therefore, this study reveals a mechanism of ER membrane fragmentation in ER-phagy, along with a signaling pathway in regulating ER turnover, and suggests a potential implication of excessive selective autophagy in human diseases. Synopsis How endoplasmic reticulum (ER) membranes are fragmented for subsequent autophagic degradation (ER-phagy) is ill-defined. CAMK2B-dependent phosphorylation of ER-phagy receptor FAM134B promotes its oligomerization and membrane scission activity, a process deregulated in sensory neuropathy. ER-phagy receptor FAM134B oligomerizes through its reticulon-homology domain (RHD). FAM134B oligomerization is required for ER membrane scission prior to autophagosomal engulfment. ER stress triggers the activation of CAMK2B, which phosphorylates FAM134B to enhance ER membrane fragmentation and ER-phagy. An HSAN type-II patient-derived variant, FAM134B-G216R, forms higher-order oligomers and induces massive ER-phagy, which leads to sensory neuron death. Introduction The endoplasmic reticulum (ER) is the largest intracellular organelle, which constitutes a continuous intracellular network of sheet and tubular membrane structures (Shibata et al, 2006). ER plays essential roles in protein and lipid synthesis, calcium homeostasis, organelle communication and innate immunity (Shibata et al, 2009). The oscillation of ER size and shape in response to varying environmental cues is crucial to cell homeostasis (Walter & Ron, 2011). Elimination of redundant ER is mediated by a selective autophagy pathway, which is coined as ER-phagy (Bernales et al, 2006, 2007). Selective autophagy is a cellular quality control pathway through which a variety of autophagy cargoes are specifically engulfed by autophagosomes and delivered to lysosomes for degradation (Stolz et al, 2014; Farre & Subramani, 2016; Zaffagnini & Martens, 2016; Gatica et al, 2018). The specificity of this process is governed by autophagy receptors that simultaneously bind to cargoes and the LC3 family members on the expanding autophagosomal membranes (Khaminets et al, 2016; Gatica et al, 2018). Recent studies have greatly advanced the understanding of ER-phagy by identifying the key autophagy receptors (Khaminets et al, 2015; Mochida et al, 2015; Fumagalli et al, 2016; Grumati et al, 2017; Smith et al, 2017; An et al, 2019; Chen et al, 2019; Chino et al, 2019). One essential question that remains unsolved is how the ER membrane is fragmented into “bite size” for subsequent autophagosomal sequestration (Mochida et al, 2015; Nakatogawa & Mochida, 2015). Moreover, little is known about how environmental or intracellular signals are transduced to trigger ER-phagy in a time-dependent manner (Rubinsztein, 2015). Lastly, the cause and consequence of excessive ER-phagy are poorly understood (Rubinsztein, 2015). FAM134B-mediated ER-phagy appears to be a good model system to address above questions, because FAM134B induces liposome fragmentation in vitro and mediates ER-phagy in vivo (Khaminets et al, 2015), and more importantly, the dysfunction of FAM134B causes hereditary sensory and autonomic neuropathy type 2 (HSAN II; Kurth et al, 2009). In this study, we show that FAM134B oligomerization drives the fragmentation of ER prior to ER-phagy. ER stress subsequently triggers CAMK2B-mediated FAM134B phosphorylation, which further enhances FAM134B oligomerization, ER scission, and ER-phagy. To strengthen our model, we provide evidence that a type II HSAN patient-derived FAM134B variant, FAM134BG216R, appears to be a gain-of-function mutant, as it is hyperactive in oligomerization, ER fragmentation, and ER-phagy, which results in sensory neuron death. Results FAM134B forms oligomers that are required for ER fragmentation and ER-phagy We observed that FAM134B formed oligomers in vivo and that this process was further enhanced by Thapsigargin (Tg)-induced ER stress (Fig 1A) or starvation (Appendix Fig S1A). These oligomers were partially resistant to denaturing solutions containing SDS and DTT. In contrast, the oligomerization of another reticulon protein, Reticulon 4 (RTN4), was not altered under the same conditions (Appendix Fig S1B). Purified recombinant human FAM134B analysis by native PAGE (Fig 1B) and size-exclusion chromatography (Appendix Fig S1C) revealed that the molecular weight of FAM134B oligomers was approximately 450–700 kD. Mutational analysis uncovered that the amino acids ranging from 84 to 233, the internal reticulon domain (RTND) of FAM134B, were required and sufficient for its self-association and oligomerization (Appendix Fig S1D–F). To measure ER fragmentation activity in vitro, we established a liposome fragmentation assay (Appendix Fig S1G), and we observed that purified recombinant FAM134BWT or FAM134B84-233 (RTND) but not the RTND-deletion mutant FAM134BΔ84-233 was able to induce liposome fragmentation in a dose-dependent manner (Fig 1C and D, and Appendix Fig S1H). In contrast, recombinant proteins for RTN4 WT or its RTND failed to induce liposome fragmentation in vitro at the same conditions (Appendix Fig S1I–L). To measure ER membrane scission in vivo, we chose the U2OS cell line because it expresses low levels of endogenous FAM134B (Khaminets et al, 2015), and we used Bafilomycin A to block lysosomal activity, which resulted in accumulation of ER membrane fragments. We observed that the expression of FAM134BWT or FAM134B84-233 but not that of FAM134BΔ84-233 induced the formation of puncta that were positive for both FAM134B and BAP31, an ER marker (Fig 1E and F, and Appendix Fig S1M). Therefore, FAM134B84-233 appeared to be required and sufficient to induce ER membrane scission in vivo at overexpression conditions. To measure ER-phagy activity, we applied the mCherry·EGFP tandem tagging strategy and GFP-cleavage assays (Khaminets et al, 2015; Klionsky et al, 2016). When the ER membrane fragments are digested by autolysosomes, they will either appear as mCherry+EGFP- foci under confocal microscope because mCherry is more stable in an acidic environment or they will produce free GFP visualized by Western analysis because of the partial digestion of GFP-SEC61B, an ER sheet resident protein. We observed that although FAM134B84-233 could fragmentate ER membrane, it failed to induce ER-phagy and deliver the fragmentated ER into autolysosomes for degradation (Fig 1G–I) due to the lack of an LC3-interacting motif (LIR; Appendix Fig S1F). In contrast, the mutant, FAM134BΔ84-233, which contains LIR but not RTND, was not able to induce ER-phagy, because of the missing oligomerization and ER-binding activities (Fig 1G–I). By transmission electron microscopy (TEM) and immunoelectron microscopy analysis, we confirmed that the expression of exogenous FAM134B WT was able to fragmentate ER membrane without inducing apoptotic response (Appendix Fig S1N–P), and these results indicated that the puncta structures positive for exogenous FAM134B appeared not to be ER membrane blebs. Together, RTND mediates FAM134B oligomerization that is required for ER membrane fragmentation and ER-phagy. Figure 1. FAM134B forms oligomers that are required for ER membrane scission and ER-phagy A. Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild-type (WT) 293T cells were treated with 1 μM of Thapsigargin (Tg) for different time, and the cell lysates were prepared and analyzed by Western blot. B. Analysis of the oligomer size of recombinant FAM134B WT using native PAGE. C. In vitro liposome fragmentation assay. After the injection of recombinant proteins (100 μg/100 μl) into the chamber (500 μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. “ns” means no significance, one-way ANOVA; error bars indicate SEM (n = 3). D. Quantification of the time between protein addition and liposome fragmentation. ***P < 0.001, one-way ANOVA; error bars indicate SEM (n = 3). E, F. Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84–233), or EGFP-FAM134B (Δ84–233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For each group, at least 30 cells were counted. Scale bars, 10 μm. ***P < 0.001, “ns” means no significance, one-way ANOVA; error bars indicate SEM. G, H. Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84–233), or mCherry-EGFP-FAM134B (Δ84–233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n = 50); for mCherry-EGFP-FAM134B (84–233), n = 56; for mCherry-EGFP-FAM134B (Δ84–233), n = 66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM. I. Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84–233)-Flag, or FAM134B (Δ84–233)-Flag. Source data are available online for this figure. Source Data for Figure 1 [embj2019102608-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Phosphorylation enhances FAM134B oligomerization and its activity in membrane fragmentation and ER-phagy To understand how FAM134B oligomerization was increased under autophagy-stimulating or ER-stress conditions, we conducted mass spectrometry analysis, which resulted in the identification of serine 149, 151, and 153 (S149, S151, S153) as potential FAM134B phosphorylation sites (Fig 2A, Appendix Fig S2A and B). Notably, all of these three residues reside within RTND. Mimicking de-phosphorylation by mutating these serines (S) into alanines (A) individually reduced FAM134B self-interaction (Fig 2B). In contrast, imitating permanent phosphorylation by replacing the serines (S) with aspartate (D) enhanced FAM134B self-association (Fig 2B). Consistently, simultaneous substitution of all three serines with alanines (FAM134BSA) or aspartates (FAM134BSD) significantly decreased or increased FAM134B self-binding, respectively (Fig 2C and Appendix Fig S2C). Consistently, purified recombinant protein of FAM134BSD exhibited significantly higher activity in liposome fragmentation than FAM134BWT (Fig 2D and Appendix Fig S2D). Under overexpression conditions, FAM134B phosphorylation significantly increased its activity in membrane fragmentation and ER-phagy in cultured U2OS cells (Fig 2E; Appendix Fig S2E–H). To further solidify this conclusion, we developed a stringent rescue assay. To do this, we first established FAM134B knockout (KO) cell lines, and we then used the FAM134B KO cells to set up a serial of stable cell lines which individually express different forms of FAM134B proteins in an inducible manner (Appendix Fig S2I and J). By titrating the inducer (Doxycycline, DOX) concentration and the induction time, we were able to express exogenous FAM134B proteins close to the endogenous levels (Appendix Fig S2K). Under these optimized conditions, we further confirmed the important role of FAM134B phosphorylation in membrane fragmentation and ER-phagy (Fig 2F–I). These results demonstrate that phosphorylation regulates FAM134B's activities in oligomerization, ER scission, and ER-phagy. Figure 2. Phosphorylation enhances FAM134B oligomerization and its activity in membrane scission and ER-phagy A. Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151. B. Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP. C. Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP. D. Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM (n ≧ 3). E. Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag, or FAM134B (SD)-Flag. F, G. Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells were engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA), or EGFP-FAM134B (SD) at endogenous levels, and lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n = 25); for EGFP-FAM134B (WT), n = 27; for EGFP-FAM134B (SA), n = 29; for EGFP-FAM134B (SD), n = 25. Scale bars, 10 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM. H, I. Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells were engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A), or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n = 25); for mCherry-EGFP-FAM134B (SA), n = 23; for mCherry-EGFP-FAM134B (SD), n = 21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM. Source data are available online for this figure. Source Data for Figure 2 [embj2019102608-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint CAMK2B phosphorylates FAM134B at serine 151 to promote oligomerization, ER fragmentation, and ER-phagy To understand how upstream signals regulate FAM134B phosphorylation, we first selected a panel of candidate kinases based on bioinformatic analysis of the sequence of the phosphorylation sites (S149, S151, S153). These kinases were further screened by in vitro kinase assays and mass spectrometry analyses (Appendix Fig S2L), which resulted in the identification of CAMK2B as the candidate kinase phosphorylating FAM134B at S151 (Appendix Fig S3A). It is reasonable to postulate that ER stress leads to the elevation of cytoplasmic calcium levels, which subsequently activates CAMK2B to trigger ER-phagy through FAM134B. Indeed, Tg treatment enhanced the interaction between CAMK2B and calmodulin (Appendix Fig S3B), which is the calcium sensor and plays a key role in CAMK2B activation, and Tg treatment also increased the colocalization and the association of CAMK2B with ER membrane structures (Fig 3A and B; Appendix Fig S3C). CAMK2B interacted with FAM134B under physiological conditions (Appendix Fig S3D and E), and CAMK2B phosphorylates FAM134B at S151 in in vitro kinase assays, which were validated by Western blot (Fig 3C) using a specific phosphor-antibody recognizing phosphorylated S151 of human FAM134B (p-FAM134B-S151) and by radioautography (Appendix Fig S3F). In addition, we also showed that mutating S151 to alanine (S151A) totally abolished the phosphorylation signal (Fig 3C and Appendix Fig S3F). Furthermore, the CAMK2B activators Ionomycin and EB1089 enhanced FAM134B phosphorylation at S151 in a time- or a dose-dependent manner in different cell lines (Appendix Fig S3G–N). In contrast, treating cells with the CAMK2B inhibitor KN-93 or with CAMK2B-specific shRNA repressed S151 phosphorylation (Fig 3D and E). Indeed, CAMK2B was able to stimulate FAM134B-mediated liposome fragmentation in vitro (Fig 3F). The CAMK2B activators or inhibitor stimulated or repressed ER scission and ER-phagy in cultured cells (Fig 3G and H; Appendix Fig S3O–R). More importantly, modulating CAMK2B activity by small molecules was able to dramatically alter ER-phagy levels in FAM134B or CAMK2B WT cells but not in FAM134B KO or CAMK2B knockdown (KD) cells, which further demonstrated the importance of CAMK2B-FAM134B signaling axis in ER-phagy (Fig 3I–K; Appendix Fig S3S and T). Therefore, the CAMK2B-FAM134B axis relays upstream signals to ER-phagy machineries to maintain ER homeostasis. Figure 3. CAMK2B phosphorylates FAM134B at Ser151 to enhance ER fragmentation and ER-phagy A, B. Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted (n = 16); for Tg treatment, 18 cells were counted (n = 18). ***P < 0.001, one-way ANOVA; error bars indicate SEM. C. In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A purified from E. coli were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B. D. FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100 nM EB1089 for 1 h) or/and inhibitor (10 μM KN93 for 2 h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, and whole cell lysates were analyzed by phospho-FAM134B (S151) antibody. E. FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells. F. In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10 min, and the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM (n = 3). G, H. Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n = 53); for Ionomycin, n = 56; for EB1089, n = 54; for KN-93, n = 58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P < 0.001, one-way ANOVA; error bars indicate SEM. I. FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089, or inhibitor KN93 for 24 h, and Western blot was performed to analyze the proteins as indicated. J. CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot. K. GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, and 24 h posttransfection, cells were treated with drugs as indicated and analyzed for GFP cleavage by Western blot. Note: The levels of Flag-FAM134B, CAMK2B, GFP-SEC61B, and Tubulin were analyzed by Western blot using different membranes. Source data are available online for this figure. Source Data for Figure 3 [embj2019102608-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint The patient-derived variant FAM134BG216R is a gain-of-function mutant in oligomerization, membrane scission, and ER-phagy FAM134B mutations cause type II HSAN, which is attributed to cell death of sensory neurons. Most of these type II HSAN patient-derived FAM134B mutants possess frameshift mutations or premature stop codons, leading to loss-of-function defects (Kurth et al, 2009; Davidson et al, 2012; Murphy et al, 2012). However, there is one pathogenic FAM134B variant, FAM134G216R, with uncertain significance (Davidson et al, 2012). Because G216R resides in the RTND of FAM134B, we postulated that this variant may affect FAM134B oligomerization. Surprisingly, FAM134BG216R displayed dramatically enhanced self-interaction and oligomerization (Fig 4A), which was mitigated by mutating S149, 151, and 153 into alanines either simultaneously (SA-G216R) (Fig 4A and Appendix Fig S4A) or individually (S149A-G216R, S151A-G216R, S153A-G216R; Appendix Fig S4B). As expected, FAM134BG216R showed significantly higher activity in liposome fragmentation, which was partially neutralized by the S149A, S151A, and S153A mutations (Fig 4B and Appendix Fig S4C–F). Under overexpression conditions, FAM134BG216R was able to induce ER scission and ER-phagy in a more efficient manner than FAM134BWT and other mutants in cultured cells (Fig 4C–F and Appendix Fig S4G). The puncta structure
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