scholarly journals Crosstalk between eIF2α and eEF2 phosphorylation pathways optimizes translational arrest in response to oxidative stress

2019 ◽  
Author(s):  
Marisa Sanchez ◽  
Yingying Lin ◽  
Chih-Cheng Yang ◽  
Philip McQuary ◽  
Alexandre Rosa Campos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Integrated Control ◽  
Elongation Factor ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
List Type ◽  
Translation Arrest ◽  
Transcriptional Reprogramming ◽  
Eif2α Phosphorylation ◽  
Translational Arrest

SUMMARYThe cellular stress response triggers a cascade of events leading to transcriptional reprogramming and a transient inhibition of global protein synthesis, which is thought to be mediated by phosphorylation of eukaryotic initiation factor-2α (eIF2α). Using mouse embryonic fibroblasts (MEFs) and the fission yeast S. pombe, we report here that rapid translational arrest and cell survival in response to hydrogen peroxide-induced oxidative stress do not rely on eIF2α kinases and eIF2α phosphorylation. Rather H2O2 induces a block in elongation through phosphorylation of eukaryotic elongation factor 2 (eEF2). Kinetic and dose-response analyses uncovered crosstalk between the eIF2α and eEF2 phosphorylation pathways, indicating that, in MEFs, eEF2 phosphorylation initiates the acute shutdown in translation, which is then maintained by eIF2α phosphorylation. Our results challenge the common conception that eIF2α phosphorylation is the primary trigger of translational arrest in response to oxidative stress and point to integrated control that may facilitate the survival of cancer cells.HIGHLIGHTSOxidative stress-induced translation arrest is independent of eIF2α phosphorylationOxidative stress blocks translation elongationOxidative stress triggers eEF2 kinase activationeEF2K KO cells are hypersensitive to oxidative stress

scholarly journals Reovirus Induces and Benefits from an Integrated Cellular Stress Response

2006 ◽  
Vol 80 (4) ◽  
pp. 2019-2033 ◽  
Author(s):  
Jennifer A. Smith ◽  
Stephen C. Schmechel ◽  
Arvind Raghavan ◽  
Michelle Abelson ◽  
Cavan Reilly ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Stress Response ◽  
Stress Responses ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Protein Levels ◽  
Eif2α Phosphorylation ◽  
Infected Cells ◽  
Reovirus Replication

ABSTRACT Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58IPK, the cellular inhibitor of the eukaryotic initiation factor 2α (eIF2α) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2α phosphorylation and unphosphorylated eIF2α is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2α phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2α kinases and phosphorylatable eIF2α. When eIF2α is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2α phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2α phosphorylation facilitates reovirus replication in two ways—first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

scholarly journals Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions

2016 ◽  
Vol 90 (16) ◽  
pp. 7268-7284 ◽  
Author(s):  
Emily V. Nelson ◽  
Kristina M. Schmidt ◽  
Laure R. Deflubé ◽  
Sultan Doğanay ◽  
Logan Banadyga ◽  
...  
Keyword(s):  
Stress Response ◽  
Ebola Virus ◽  
Cellular Stress ◽  
Stress Granule ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
Ebov Infection ◽  
Infected Cells ◽  
Translational Arrest ◽  
Viral Inclusions

ABSTRACTA hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions.IMPORTANCEAlthough progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

scholarly journals UV damage induces G3BP1-dependent stress granule formation that is not driven by translation arrest via mTOR inhibition

2020 ◽  
Author(s):  
Shan Ying ◽  
Denys A. Khaperskyy
Keyword(s):  
Translation Initiation Factor ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
Major Type ◽  
Uv Damage ◽  
Mtor Inhibition ◽  
Granule Formation ◽  
Translation Arrest ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACTTranslation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not cause dissociation of the 48S preinitiation complexes. The catalytic activity of the general control non-derepressible 2 (GCN2) kinase contributes to UV-induced SG formation, which is independent of the GCN2-mediated phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules specifically requires the Ras-GTPase-Activating Protein SH3-Domain-Binding Protein 1 (G3BP1). Our work reveals that in UV-treated cells the mechanisms of translation arrest and SG formation may be unlinked, resulting in condensation of ribonucleoproteins that do not represent the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.

scholarly journals Fission Yeast Mitogen-Activated Protein Kinase Sty1 Interacts with Translation Factors

2007 ◽  
Vol 7 (2) ◽  
pp. 328-338 ◽  
Author(s):  
Eva Asp ◽  
Daniel Nilsson ◽  
Per Sunnerhagen
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Elongation Factor ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Nitrogen Deprivation ◽  
Hyperosmotic Shock ◽  
Mitogen Activated Protein

ABSTRACT Signaling by stress-activated mitogen-activated protein kinase (MAPK) pathways influences translation efficiency in mammalian cells and budding yeast. We have investigated the stress-activated MAPK from fission yeast, Sty1, and its downstream protein kinase, Mkp1/Srk1, for physically associated proteins using tandem affinity purification tagging. We find Sty1, but not Mkp1, to bind to the translation elongation factor eukaryotic elongation factor 2 (eEF2) and the translation initiation factor eukaryotic initiation factor 3a (eIF3a). The Sty1-eIF3a interaction is weakened under oxidative or hyperosmotic stress, whereas the Sty1-eEF2 interaction is stable. Nitrogen deprivation causes a transient strengthening of both the Sty1-eEF2 and the Sty1-Mkp1 interactions, overlapping with the time of maximal Sty1 activation. Analysis of polysome profiles from cells under oxidative stress, or after hyperosmotic shock or nitrogen deprivation, shows that translation in sty1 mutant cells recovers considerably less efficiently than that in the wild type. Cells lacking the Sty1-regulated transcription factor Atf1 are deficient in maintaining and recovering translational activity after hyperosmotic shock but not during oxidative stress or nitrogen starvation. In cells lacking Sty1, eIF3a levels are decreased, and phosphorylation of eIF3a is reduced. Taken together, our data point to a central role in translational adaptation for the stress-activated MAPK pathway in fission yeast similar to that in other investigated eukaryotes, with the exception that fission yeast MAPK-activated protein kinases seem not to be directly involved in this process.

scholarly journals Importance of eIF2α Phosphorylation and Stress Granule Assembly in Alphavirus Translation Regulation

2005 ◽  
Vol 16 (8) ◽  
pp. 3753-3763 ◽  
Author(s):  
Gerald M. McInerney ◽  
Nancy L. Kedersha ◽  
Randal J. Kaufman ◽  
Paul Anderson ◽  
Peter Liljeström
Keyword(s):  
Semliki Forest Virus ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
Translation Regulation ◽  
Viral Gene ◽  
Enhancer Element ◽  
Translation Inhibition ◽  
Eif2α Phosphorylation

Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2α subunit (eIF2α). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2α does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2α phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2α and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.

scholarly journals Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4

2020 ◽  
Vol 4 (2) ◽  
pp. e202000865
Author(s):  
Andreia Mendes ◽  
Julien P Gigan ◽  
Christian Rodriguez Rodrigues ◽  
Sébastien A Choteau ◽  
Doriane Sanseau ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Stress Response ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Translation Arrest ◽  
Eif2α Phosphorylation ◽  
Signaling Axis ◽  
Stressed Cells ◽  
Cytoskeleton Dynamics

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by the subtilase cytotoxin (SubAB). PERK inactivation, increases globally protein synthesis levels and regulates IFN-β expression, while impairing LPS-stimulated DC migration. Although the loss of PERK activity does not impact DC development, the cross talk existing between actin cytoskeleton dynamics; PERK and eIF2α phosphorylation is likely important to adapt DC homeostasis to the variations imposed by the immune contexts.

Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

2013 ◽  
Vol 451 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Janete Chung ◽  
Antonio A. Rocha ◽  
Renata R. Tonelli ◽  
Beatriz A. Castilho ◽  
Sergio Schenkman
Keyword(s):  
Protein Synthesis ◽  
Stationary Phase ◽  
Phosphorylation Site ◽  
Elongation Factor ◽  
Growth Conditions ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Wild Type ◽  
Translational Arrest ◽  
Aspartate Residue

The protein known as eIF5A (eukaryotic initiation factor 5A) has an elusive role in translation. It has a unique and essential hypusine modification at a conserved lysine residue in most eukaryotes. In addition, this protein is modified by phosphorylation with unknown functions. In the present study we show that a phosphorylated state of eIF5A predominates in exponentially growing Trypanosoma cruzi cells, and extensive dephosphorylation occurs in cells in stationary phase. Phosphorylation occurs mainly at Ser2, as shown in yeast eIF5A. In addition, a novel phosphorylation site was identified at Tyr21. In exponential cells, T. cruzi eIF5A is partially associated with polysomes, compatible with a proposed function as an elongation factor, and becomes relatively enriched in polysomal fractions in stationary phase. Overexpression of the wild-type eIF5A, or eIF5A with Ser2 replaced by an aspartate residue, but not by alanine, increases the rate of cell proliferation and protein synthesis. However, the presence of an aspartate residue instead of Ser2 is toxic for cells reaching the stationary phase, which show a less-pronounced protein synthesis arrest and a decreased amount of eIF5A in dense fractions of sucrose gradients. We conclude that eIF5A phosphorylation and dephosphorylation cycles regulate translation according to the growth conditions.

scholarly journals eIF2α kinases GCN2 and PERK modulate transcription and translation of distinct sets of mRNAs in mouse liver

2009 ◽  
Vol 38 (3) ◽  
pp. 328-341 ◽  
Author(s):  
An N. Dang Do ◽  
Scot R. Kimball ◽  
Douglas R. Cavener ◽  
Leonard S. Jefferson
Keyword(s):  
Mouse Liver ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Cellular Stress Response ◽  
Initiation Factor ◽  
Future Research ◽  
Internal Ribosomal Entry Sites ◽  
Α Subunit ◽  
Eif2α Phosphorylation ◽  
Initiation Factor 2

In eukaryotes, selective derepression of mRNA translation through altered utilization of upstream open reading frames (uORF) or internal ribosomal entry sites (IRES) regulatory motifs following exposure to stress is regulated at the initiation stage through the increased phosphorylation of eukaryotic initiation factor 2 on its α-subunit (eIF2α). While there is only one known eIF2α kinase in yeast, general control nonderepressible 2 (GCN2), mammals have evolved to express at least four: GCN2, heme-regulated inhibitor kinase (HRI), double-stranded RNA-activated protein kinase (PKR), and PKR-like endoplasmic reticulum-resident kinase (PERK). So far, the main known distinction among these four kinases is their activation in response to different acute stressors. In the present study, we used the in situ perfused mouse liver model and hybridization array analyses to assess the general translational response to stress regulated by two of these kinases, GCN2 and PERK, and to differentiate between the downstream effects of activating GCN2 versus PERK. The resulting data showed that at least 2.5% of mouse liver mRNAs are subject to derepressed translation following stress. In addition, the data demonstrated that eIF2α kinases GCN2 and PERK differentially regulate mRNA transcription and translation, which in the latter case suggests that increased eIF2α phosphorylation is not sufficient for derepression of translation. These findings open an avenue for more focused future research toward groups of mRNAs that code for the early cellular stress response proteins.

scholarly journals PKR-Mediated Stress Response Enhances Dengue and Zika Virus Replication

2021 ◽  
Author(s):  
Taissa Ricciardi-Jorge ◽  
Edroaldo Lummertz da Rocha ◽  
Edgar Gonzalez-Kozlova ◽  
Gabriela Flavia Rodrigues-Luiz ◽  
Brian J Ferguson ◽  
...  
Keyword(s):  
Stress Response ◽  
Zika Virus ◽  
Mrna Translation ◽  
Denv Infection ◽  
Host Cells ◽  
Type I ◽  
Reporter System ◽  
Translation Arrest ◽  
Eif2α Phosphorylation ◽  
Translational Arrest

ABSTRACTThe mechanisms by which Flaviviruses use non-canonical translation to support their replication in host cells are largely unknown. Here we investigated how the integrated stress response (ISR), which promotes translational arrest by eIF2α phosphorylation (p–eIF2α), regulates Flavivirus replication. During Dengue virus (DENV) and Zika virus (ZIKV) infection, eIF2α activation peaked at 24 hours post infection and was dependent on PKR but not type I interferon. The ISR is activated downstream of p-eIF2α during infection with either virus, but translation arrest only occurred following DENV infection. Despite this difference, both DENV and ZIKV replication was impaired in cells lacking PKR, independent of IFN-I/NF-kB signaling or cell viability. By using a ZIKV 5’UTR reporter system as a model, we found that this region of the genome is sufficient to promote an enhancement of viral mRNA translation in the presence of an active ISR. Together we provide evidence that Flaviviruses escape ISR translational arrest and co-opt this response to increase viral replication.

scholarly journals Translation Initiation Control by Heme-Regulated Eukaryotic Initiation Factor 2α Kinase in Erythroid Cells under Cytoplasmic Stresses

2001 ◽  
Vol 21 (23) ◽  
pp. 7971-7980 ◽  
Author(s):  
Linrong Lu ◽  
An-Ping Han ◽  
Jane-Jane Chen
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Stress Responses ◽  
Fetal Liver ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Erythroid Cells ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
In Erythroid Cells

ABSTRACT Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2α (eIF2α) by eIF2α kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2α kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2α kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2α phosphorylation upon arsenite treatment. HRI is also the major eIF2α kinase responsible for the increased eIF2α phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.

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