The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways
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ABSTRACT:
ORGANISM(S): Homo sapiens (human)
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PROVIDER: S-BSST1635 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.
| S-EPMC10724265 | biostudies-literature
Project description:Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and is critical to maintaining cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyzed mTOR-mediated phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy. An unbiased screen of kinase inhibitors revealed that CK2 is essential for ER-phagy driven by FAM134B and FAM134C after inhibition of mTOR. Using superresolution microscopy, we showed that CK2 activity is essential for the formation of high-density groups of FAM134B and FAM134C. Continually, the FAM134B and FAM134C proteins that carry point mutations of selected serine residues within their reticulon homology domain are unable to form high-density clusters. Furthermore, we provide evidence that ubiquitination regulates ER-phagy receptors and that dense clustering of FAM134B and FAM134C requires events upstream of ubiquitination. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of phosphosites prevents Torin1-induced ER-phagy flux as well as ubiquitination of FAM134 proteins, and consistently treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Therefore, we propose that CK2 dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.
2023-11-16 | PXD043003 | Pride
Project description:Selective autophagy of the ER (ERphagy) is an important regulator of ER remodeling and critical to maintain cellular homeostasis upon environmental changes. ERphagy receptors link the ER with autophagic membrane thus regulating ERphagy flux. We recently showed that members of the FAM134 family play overlapping and distinct roles during stress-induced ERphagy. Yet the mechanisms on how they are activated remain largely unknown. In this study we analyzed mTOR-mediated dynamic phosphorylation of FAM134 as a trigger of FAM134-driven ERphagy. An unbiased screen of kinase inhibitors revealed CK2 to be essential for FAM134B- and FAM134C-driven ERphagy upon mTOR inhibition. Identified dynamic phosphorylation sites on FAM134C in cells were fitting with predicted CK2 targeting sites, indicating a direct regulatory role of CK2 in FAM134-driven ERphagy. Using super-resolution microscopy, we showed that activity of CK2 is essential for the formation of high-density clusters of FAM134B and FAM134C. Consistently, FAM134B and FAM134C proteins carrying point mutations of selected Serin residues, within their reticulon homology domain, are unable to form high-density clusters. In addition, we provide evidence that the ubiquitination machinery is required for ERphagy and that FAM134B and FAM134C clustering is activated by phospho-dependent ubiquitination. Treatment with CK2 inhibitor SGC-CK2-1 prevents Torin1-induced ERphagy flux as well as ubiquitination of FAM134 proteins and consistently, treatment with E1 inhibitor suppresses Torin1-induced ERphagy flux. Therefore, we propose CK2 dependent phosphorylation of ERphagy receptors precedes ubiquitin-dependent ERphagy flux activation.
2022-08-17 | PXD036103 | Pride
Project description:The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.
| S-EPMC10247366 | biostudies-literature
Project description:Estrogen receptor-alpha (ER?) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ER? protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ER? protein and ESR1, which is currently unexplained. Cellular ER? protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ER?) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ER? transcriptional function. Here, we demonstrate that Pin1 can increase ER? protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ER? ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ER?. Quantitative in situ assessments of ER? protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ER? protein but not to ESR1 levels. These data show that ER? protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ER? protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ER?-positive BC.
| S-EPMC3815749 | biostudies-literature
Project description:Selective degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is initiated by ER-phagy receptors, which facilitate the incorporation of ER fragments into autophagosomes. FAM134 reticulon family proteins (FAM134A, FAM134B, and FAM134C) are ER-phagy receptors with structural similarities and nonredundant functions. Whether they respond differentially to the stimulation of ER-phagy is unknown. Here, we describe an activation mechanism unique to FAM134C during starvation. In fed conditions, FAM134C is phosphorylated by casein kinase 2 (CK2) at critical residues flanking the LIR domain. Phosphorylation of these residues negatively affects binding affinity to the autophagy proteins LC3. During starvation, mTORC1 inhibition limits FAM134C phosphorylation by CK2, hence promoting receptor activation and ER-phagy. Using a novel tool to study ER-phagy in vivo and FAM134C knockout mice, we demonstrated the physiological relevance of FAM134C phosphorylation during starvation-induced ER-phagy in liver lipid metabolism. These data provide a mechanistic insight into ER-phagy regulation and an example of autophagy selectivity during starvation.
| S-EPMC9432840 | biostudies-literature
Project description:RNA splicing and protein degradation systems allow the functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. Importantly, disruptive cryptic variants evade mRNA surveillance pathways and are translated into misfolded proteins. These spurious isoforms disrupt proteostasis causing cytosolic protein aggregates and ER stress. Proteotoxic exons activate unfolded protein response, causing CHOP-mediated cell death. In response to impaired splicing, eukaryotic cells enhance ubiquitination and ER-phagy to alleviate the pathogenic accumulation of proteotoxic isoforms. Our findings show how cryptic splicing-induced proteotoxicity can be mitigated, and provide insight into the molecular pathogenesis of spliceosome-associated diseases such as retinitis pigmentosa.
2024-09-25 | GSE272836 | GEO
Project description:Acute kidney injury (AKI) constitutes a prevalent clinical syndrome characterized by elevated morbidity and mortality rates, emerging as a significant public health issue. This study investigates the interplay between endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and ER-associated degradation (ER-phagy) in the pathogenesis of AKI. We employed four distinct murine models of AKI-induced by contrast media, ischemia-reperfusion injury, cisplatin, and folic acid-to elucidate the relationship between ER-phagy, ER stress, and apoptosis. Our findings reveal a marked decrease in ER-phagy coinciding with an accumulation of damaged ER, elevated ER stress, and increased apoptosis across all AKI models. Importantly, overexpression of DDRGK1 in HK-2 cells enhanced ER-phagy levels, ameliorating contrast-induced ER stress and apoptosis. These findings unveil a novel protective mechanism in AKI, wherein DDRGK1-UFL1-mediated ER-phagy mitigates ER stress and apoptosis in renal tubular epithelial cells. Our results thereby contribute to understanding the molecular underpinnings of AKI and offer potential therapeutic targets for its treatment.
| S-EPMC10794694 | biostudies-literature
Project description:Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
| S-EPMC9772183 | biostudies-literature
Project description: Not available
| S-BSST1434 | biostudies-other