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Phosphorylation Switches Protein Disulfide Isomerase Activity to Maintain Proteostasis and Attenuate ER Stress

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Phosphorylation Switches Protein Disulfide Isomerase Activity to Maintain Proteostasis and Attenuate ER Stress Article Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress Jiaojiao Yu1,2,†, Tao Li1,2,†, Yu Liu3, Xi Wang1, Jianchao Zhang1,2 ,Xi’e Wang1, Guizhi Shi4, Jizhong Lou2,5, Likun Wang1,2, Chih-chen Wang1,2 & Lei Wang1,2,* Abstract Introduction Accumulated unfolded proteins in the endoplasmic reticulum (ER) The endoplasmic reticulum (ER) is the main cellular organelle for trigger the unfolded protein response (UPR) to increase ER protein protein folding and secretion, calcium storage, and lipid synthesis. folding capacity. ER proteostasis and UPR signaling need to be regu- Numerous folding enzymes and molecular chaperones in the ER lated in a precise and timely manner. Here, we identify phosphory- guide the secretion of properly folded proteins while retaining lation of protein disulfide isomerase (PDI), one of the most misfolded proteins or targeting their degradation (Sitia & Braakman, abundant and critical folding catalysts in the ER, as an early event 2003). An accumulation of unfolded/misfolded proteins or alterna- during ER stress. The secretory pathway kinase Fam20C phosphory- tion of redox and Ca2+ states in the ER results in imbalanced lates Ser357 of PDI and responds rapidly to various ER stressors. proteostasis and ER stress (Balch et al, 2008; Walter & Ron, 2011; Phosphorylation of Ser357 induces an open conformation of PDI and Karagoz et al, 2019; Ushioda & Nagata, 2019). Chronic ER stress turns it from a “foldase” into a “holdase”, which is critical for plays a central role in various human pathologies, including cancer, preventing protein misfolding in the ER. Phosphorylated PDI also diabetes, cardiovascular diseases, and neurodegenerative diseases binds to the lumenal domain of IRE1a, a major UPR signal trans- (Oakes & Papa, 2015; Wang & Kaufman, 2016). ducer, and attenuates excessive IRE1a activity. Importantly, PDI- Endoplasmic reticulum proteostasis is governed by a dynamic S359A knock-in mice display enhanced IRE1a activation and liver signaling network, the unfolded protein response (UPR), which damage under acute ER stress. We conclude that the Fam20C-PDI consists of three distinct arms in mammals defined by ER transmem- axis constitutes a post-translational response to maintain ER brane sensors—IRE1a, PERK, and ATF6 (Walter & Ron, 2011). The proteostasis and plays a vital role in protecting against ER stress- output of UPR signaling is to induce the expression of genes involved induced cell death. in ER protein quality control and/or translational repression of global protein synthesis to counteract proteostatic perturbations in Keywords endoplasmic reticulum; Fam20C; IRE1a; phosphorylation; protein the ER. The IRE1a branch is the most conserved arm of the UPR. ER disulfide isomerase stress induces the dimerization/oligomerization of IRE1a and its Subject Categories Membranes & Trafficking; Post-translational Modifi- autophosphorylation, which leads to XBP1 mRNA splicing and cations & Proteolysis; Translation & Protein Quality generates the functional spliced XBP1 (XBP1s) transcription factor DOI 10.15252/embj.2019103841 | Received 28 October 2019 | Revised 7 regulating multifarious targets (Yoshida et al, 2001; Calfon et al, February 2020 | Accepted 11 February 2020 | Published online 9 March 2020 2002; Lee et al, 2003; Shoulders et al, 2013). Nevertheless, hyperac- The EMBO Journal (2020) 39:e103841 tivation of IRE1a under prolonged ER stress results in the transition from an adaptive UPR to a terminal proapoptotic program by surpass- See also: JPL Coelho & MJ Feige (May 2020) ing the oligomerization threshold that expands the RNase substrate repertoire to many other mRNAs or precursors of apoptosis-inhibitory microRNAs (Han et al, 2009; Hollien et al, 2009; Upton et al, 2012; Ghosh et al, 2014). Moreover, IRE1a can trigger cellular apoptosis through the TRAF2-ASK1-JNK pathway (Urano et al, 2000; Nishitoh 1 National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China 3 CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 4 Laboratory Animal Center of Institute of Biophysics, Chinese Academy of Sciences, Aviation General Hospital of Beijing, University of Chinese Academy of Sciences, Beijing,China 5 Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China *Corresponding author. Tel: +86 10 64888501; E-mail: [email protected] †These authors contributed equally to this work ª 2020 The Authors The EMBO Journal 39:e103841 | 2020 1 of 21 The EMBO Journal Jiaojiao Yu et al et al,2002).Therefore,IRE1a functions as a central adjustor of cell a higher amplitude of XBP1 mRNA splicing (Figs 1B and EV1A). As fate under ER stress, and its activity is precisely and timely regulated the duration of splicing was similar (Fig EV1A), Fam20C may partici- by multiple regulatory elements (Hetz et al,2006;Lisbonaet al, 2009; pate in the early response to ER stress. Ectopic expression of Fam20C Rodriguez et al, 2012; Eletto et al, 2014; Sepulveda et al, 2018). wild-type (WT) decreased the XBP1 mRNA splicing level in Tg- However, the inherent latency of the UPR limits its responsiveness treated HepG2 cells compared with its inactive D478A mutant (DA) to the fluctuation of protein status in the ER, particularly in dedicated (Fig 1C). Similar results were observed when HepG2 cells were secretory cells with heavy protein folding burden. The post-transla- treated with another ER stress inducer tunicamycin (Tm), which tional regulation of BiP, a key chaperone in the ER, emerges as a inhibits protein N-glycosylation (Fig 1D and E). Again, we showed rapid and an economic way to regulate protein folding capacity that Fam20C can also negatively regulate IRE1a activity in HeLa cells during the early stage of ER stress (Preissler & Ron, 2018). However, by using Fam20C knockout (KO) cells generated by CRISPR/Cas9 whether post-translational regulation of other preexisting compo- technology previously reported (Zhang et al, 2018; Fig EV1B and C). nents contributes to the early response to ER stress remains unclear. To explore the mechanism by which Fam20C controls IRE1a The reversible phosphorylation of proteins is central to the regula- signaling, we set to identify the Fam20C interactome by co-immuno- tion of most aspects of cell function. Fam20C, a secretory pathway precipitation (co-IP) and mass spectrum (MS) analysis (Fig EV1D protein kinase, which recognizes S-x-E/pS motifs within the substrate and E). Three independent experiments led to the identification of a proteins, generates the majority of the secreted phosphoproteome total of 173 ER and Golgi proteins, which were based on DAVID GO (Tagliabracci et al, 2012, 2015; Cui et al, 2015). Functional mutations term analysis (Dataset EV1 and Fig EV1F). Among these Fam20C- in Fam20C cause a rare and often lethal osteosclerotic bone dysplasia interacting proteins, five proteins, including PDI (P4HB), P4HA1, called Raine syndrome (Raine et al, 1989). Recently, the critical roles P4HA2, TRIM68, and FKBP9, showed statistically significant of Fam20C in fine-tuning ER redox homeostasis (Zhang et al, 2018) increased binding with Fam20C after Tg treatment, whereas two and Ca2+ homeostasis (Pollak et al, 2018) have been revealed. proteins, SERCA2 and ERGIC2, showed statistically significant However, it is still an open question whether Fam20C is involved in decreased binding (Fig 1F). The increased binding between PDI and ER proteostasis regulation and UPR signaling under ER stress. Fam20C under ER stress was further confirmed by co-IP and In this study, unbiased proteomics analysis allows the identifi- immunoblotting (Fig 1G). We thereafter focused on PDI because it is cation of protein disulfide isomerase (PDI) as a substrate of Fam20C the most enriched protein among the Fam20C interactome under ER kinase under ER stress. PDI, also known as collagen prolyl 4-hydro- stress and is critical for protein folding and quality control in the ER. xylase subunit b (P4HB), is one of the most abundant enzymes in the ER. PDI is a versatile protein acting as both a thiol-disulfide PDI Ser357 is a genuine phosphosite of Fam20C oxidoreductase and a molecular chaperone (Hatahet & Ruddock, 2009; Wang et al, 2015). We show that Fam20C phosphorylates To determine whether PDI is phosphorylated by Fam20C during ER serine 357 (Ser357) of PDI and responds rapidly to various ER stres- stress, HepG2 cells were treated with Tg, and endogenous PDI was sors. Importantly, phosphorylation of Ser357 in the x-linker region immunoprecipitated and subjected to MS/MS analysis (Fig EV2A). induces an open conformation of PDI and turns it from a “foldase” to Three phosphorylation sites (Ser357, Ser331, and Ser427) were iden- a “holdase”, which is critical for preventing protein misfolding in the tified upon Tg treatment (Figs 2A and EV4D and E). Since phospho- ER. Furthermore, we demonstrate that phosphorylated PDI directly rylation of Ser331 and Ser427 had little effect on the conformation interacts with IRE1a to attenuate its signaling amplitude, which is and activity of PDI (see below), in this study, we focused on the critical for protecting against ER stress-induced liver damage, as phosphorylation effect
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