4E-BP is a target of the GCN2–ATF4 pathway during <i>Drosophila</i> development and aging

Kang, Minji; Vasudevan, Deepika; Kang, Kwonyoon; Kim, Kyunggon; Park, Jung-Eun; Zhang, Nan; Zeng, Xiaomei; Neubert, Thomas A.; Marr, Michael T.; Ryoo, Hyung Don

doi:10.1083/jcb.201511073

Cited by 78 publications

(117 citation statements)

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“…To understand the regulatory roles of USP14 on cell cycle in vivo , we analyzed Drosophila eyes, which provides an excellent model for developmentally regulated cell proliferation [40]. First, to determine whether USP14 knockdown in vivo has any differences in the population of S and M phase cells, we examined the incorporation of bromodeoxyuridine (BrdU) and phospho-histone H3 (pH3) antibody labeling.…”

Section: Resultsmentioning

confidence: 99%

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Lee

Park

Yun

et al. 2018

Cell Physiol Biochem

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Background/Aims: The 26S proteasome is the key proteolytic complex for recognition and degradation of polyubiquitinated target substrates in eukaryotes. Among numerous proteasome-associated proteins, a deubiquitinating enzyme (DUB) USP14 has been identified as an endogenous inhibitor of the proteasome. Here, we explored the complex regulatory functions of USP14 that involve ubiquitin (Ub) homeostasis and substrate degradation in flies and mammals. Methods: USP14-null primary and immortalized mouse embryonic fibroblasts (MEFs) and USP14 knocked-down Drosophila were analyzed in this study. We measured proteasome and DUB activities using fluorogenic reporter substrates and adduct-forming probes. To examine the levels of ubiquitin, we performed immunoblotting and immunohistochemistry. Mass spectrometry (MS) was used to examine polyUb chain linkages and USP14-interacing proteins. Cell cycle was analyzed by flow cytometry, BrdU labeling, and phospho-histone H3 staining. Results: The homeostasis of Ub in USP14^–/–MEFs was markedly perturbed because of facilitated clearance of Ub. This phenomenon was recapitulated in muscles of USP14-deficient Drosophila with old ages. Absolute quantitation using MS also revealed that USP14^–/– MEFs contained significantly increased amounts of Ub, compared with wild-type. The key phenotype of USP14^–/– MEFs was their delayed proliferation originated from prolonged interphase possibly through aberrant degradation of cyclins A and B1. We found that knocking down USP14 in Drosophila resulted in delayed eye development associated with reduced mitotic activity. Conclusion: Our study identifies novel cellular functions of USP14 not only in cellular Ub hometostasis but also in cell cycle progression. USP14 was also essential for proper Drosophila eye development. These results strongly suggest that the USP14-mediated proteasome activity regulation may be directly related to various human diseases including cancer.

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Section: Resultsmentioning

confidence: 99%

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Lee

Park

Yun

et al. 2018

Cell Physiol Biochem

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“…There are two known transcriptional regulators of 4e-bp , the best characterized of which is the stress-responsive transcription factor, FOXO (Jünger et al, 2003; Puig et al, 2003). Recently, we reported that the eIF2α-kinase-responsive transcription factor, ATF4, regulates 4e-bp transcription in Drosophila through an intronic element in the 4e-bp locus (Kang et al, 2017; Yamaguchi et al, 2008). Based on this regulation, we developed the 4E-BP intron -dsRed reporter that faithfully reports ATF4 activity in Drosophila tissues (Kang et al, 2017) and this led us to examine the possible role of eIf2α kinases in 4e-bp induction during infection.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we reported that the eIF2α-kinase-responsive transcription factor, ATF4, regulates 4e-bp transcription in Drosophila through an intronic element in the 4e-bp locus (Kang et al, 2017; Yamaguchi et al, 2008). Based on this regulation, we developed the 4E-BP intron -dsRed reporter that faithfully reports ATF4 activity in Drosophila tissues (Kang et al, 2017) and this led us to examine the possible role of eIf2α kinases in 4e-bp induction during infection. We specifically tested if ATF4 is active in the context of infection, by orally infecting 3 rd instar larvae with the non-lethal gram-negative bacterial pathogen, Ecc15 .…”

Section: Resultsmentioning

confidence: 99%

“…It is worth noting that GCN2 engages two different translation inhibition mechanisms: 1) phospho-eIF2α, which induces ATF4 and, 2) 4e-bp , which is transcriptionally induced by ATF4 (Kang et al, 2017; Yamaguchi et al, 2008). We primarily focused on 4E-BP because the effect of phospho-eIF2α on translation may be temporary since ATF4 induces the expression of an eIF2α phosphatase subunit, GADD34, thereby stimulating eIF2α dephosphorylation.…”

Section: Discussionmentioning

confidence: 99%

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The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection

et al. 2017

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Summary Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor, ATF4, and its upstream kinase, GCN2. Loss of gcn2, atf4 or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5’ untranslated regions (UTRs) of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5’UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.

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“…This led to the idea that mRNAs that need to be efficiently translated under conditions associated with eIF2α phosphorylation should have the means to bypass the inhibition of translation that is imposed by the GCN2-ATF4-4E-BP pathway. Indeed, they found that transcripts induced by this pathway, including those that encode antimicrobial peptides and BiP, contain IRES elements that allow them to bypass the inhibition of translation imposed by 4E-BP induction (Kang et al, 2017;Vasudevan et al, 2017).…”

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confidence: 99%

Meeting Report – proteostasis in Ericeira

Adrain

Henis-Korenblit

Domingos

2018

Journal of Cell Science

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It was a sunny Ericeira, in Portugal, that received the participants of the EMBO Workshop on Proteostasis, from 17 to 21 November 2017. Most participants gave talks or presented posters concerning their most recent research results, and lively scientific discussions occurred against the backdrop of the beautiful Atlantic Ocean. Proteostasis is the portmanteau of the words protein and homeostasis, and it refers to the biological mechanisms controlling the biogenesis, folding, trafficking and degradation of proteins in cells. An imbalance in proteostasis can lead to the accumulation of misfolded proteins or excessive protein degradation, and is associated with many human diseases. A wide variety of research approaches are used to identify the mechanisms that regulate proteostasis, typically involving different model organisms (yeast, invertebrates or mammalian systems) and different methodologies (genetics, biochemistry, biophysics, structural biology, cell biology and organismal biology). Around 140 researchers in the proteostasis field met in the Hotel Vila Galé, Ericeira, Portugal for the EMBO Workshop in Proteostasis, organized by Pedro Domingos (ITQB-NOVA, Oeiras, Portugal) and Colin Adrain (IGC, Oeiras, Portugal). In this report, we attempt to review and integrate the ideas that emerged at the workshop. Owing to space restrictions, we could not cover all talks or posters and we apologize to the colleagues whose presentations could not be discussed.

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4E-BP is a target of the GCN2–ATF4 pathway during Drosophila development and aging

Cited by 78 publications

References 68 publications

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection

Meeting Report – proteostasis in Ericeira

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