scholarly journals Substrate recognition determinants of human eIF2α phosphatases

Open Biology ◽  
2021 ◽  
Vol 11 (12) ◽  
Author(s):  
George Hodgson ◽  
Antonina Andreeva ◽  
Anne Bertolotti
Keyword(s):  
Mammalian Cells ◽  
Substrate Binding ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Binding Motif ◽  
Binding Region ◽  
Catalytic Subunits ◽  
Eif2α Phosphorylation ◽  
Cellular Defence

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A 325–554 and R15B 340–639 . G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A 325–674 and R15B 340–713 , encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

scholarly journals Substrate recognition determinants of human eIF2α phosphatases

2021 ◽  
Author(s):  
George Hodgson ◽  
Antonina Andreeva ◽  
Anne Bertolotti
Keyword(s):  
Mammalian Cells ◽  
Biological Significance ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type ◽  
Binding Region ◽  
Antagonistic Action ◽  
Catalytic Subunits ◽  
Eif2α Phosphorylation ◽  
Cellular Defence

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.

scholarly journals Inhibition of a constitutive translation initiation factor 2α phosphatase, CReP, promotes survival of stressed cells

2003 ◽  
Vol 163 (4) ◽  
pp. 767-775 ◽  
Author(s):  
Céline Jousse ◽  
Seiichi Oyadomari ◽  
Isabel Novoa ◽  
Phoebe Lu ◽  
Yuhong Zhang ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Gene Encoding ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
Stressed Cells

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on serine 51 is effected by specific stress-activated protein kinases. eIF2α phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2α dephosphorylation, however, GADD34 mutant cells retain significant eIF2α-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2α. RNAi of this gene, which we named constitutive repressor of eIF2α phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2α-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2α dephosphorylation by targeting the CReP-protein–phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

scholarly journals The Cellular Protein P58IPK Regulates Influenza Virus mRNA Translation and Replication through a PKR-Mediated Mechanism

2006 ◽  
Vol 81 (5) ◽  
pp. 2221-2230 ◽  
Author(s):  
Alan G. Goodman ◽  
Jennifer A. Smith ◽  
Siddharth Balachandran ◽  
Olivia Perwitasari ◽  
Sean C. Proll ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mammalian Cells ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Virus Protein ◽  
Mrna Levels ◽  
Viral Mrna ◽  
Eif2α Phosphorylation ◽  
In Cells

ABSTRACT We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58IPK, the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. P58IPK also inhibits PERK, an eIF2α kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58IPK to interact with and inhibit multiple eIF2α kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58IPK−/− mice, we demonstrated that the absence of P58IPK led to an increase in eIF2α phosphorylation and decreased influenza virus mRNA translation. The absence of P58IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58IPK target, PKR, the trends were reversed—eIF2α phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2α phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2α. Taken together, our results support a model in which P58IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

scholarly journals Specific Isoforms of Translation Initiation Factor 4GI Show Differences in Translational Activity

2006 ◽  
Vol 26 (22) ◽  
pp. 8448-8460 ◽  
Author(s):  
Mark J. Coldwell ◽  
Simon J. Morley
Keyword(s):  
Translation Initiation ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Protein Isoforms ◽  
Initiation Factor ◽  
Functional Studies ◽  
Eif2α Phosphorylation ◽  
Translational Activity

ABSTRACT The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2α phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

scholarly journals The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Heather P Harding ◽  
Adriana Ordonez ◽  
Felicity Allen ◽  
Leopold Parts ◽  
Alison J Inglis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Ribosome Stalling ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

scholarly journals Eap1p, a Novel Eukaryotic Translation Initiation Factor 4E-Associated Protein in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (13) ◽  
pp. 4604-4613 ◽  
Author(s):  
Gregory P. Cosentino ◽  
Tobias Schmelzle ◽  
Ashkan Haghighat ◽  
Stephen B. Helliwell ◽  
Michael N. Hall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Tor Signaling

ABSTRACT Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m7G(5′)ppp(5′)N, where N is any nucleotide] present at the 5′ termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of theEAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.

scholarly journals The Leader Protein of Theiler's Virus Prevents the Activation of PKR

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Teresa Cesaro ◽  
Thomas Michiels
Keyword(s):  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Granule Formation ◽  
Double Stranded Rna ◽  
Eif2α Phosphorylation ◽  
Leader Protein ◽  
Infected Cells ◽  
L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

scholarly journals Unconventional Translation Initiation Factor EIF2A is required for Drosophila spermatogenesis

2021 ◽  
Author(s):  
David D Lowe ◽  
Denise Montell
Keyword(s):  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Male Sterile ◽  
Specific Expression ◽  
Transposon Insertion ◽  
Stress Related Genes ◽  
Male Specific ◽  
Multicellular Organisms

The eukaryotic initiation factor EIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related genes in mammalian cells. Its function in multicellular organisms has not been reported. Here, we identify and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. We identified that CG7414 undergoes sex-specific splicing that regulates its male-specific expression. We characterized a Mi{Mic} transposon insertion that disrupts the coding regions of all predicted isoforms and is a genetic null allele, and a PBac transposon insertion into an intron, which is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility. These results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entree into the elucidation of the role of EIF2A and unconventional translation in vivo.

scholarly journals GCN2 phosphorylation of eIF2α activates NF-κB in response to UV irradiation

2005 ◽  
Vol 385 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Hao-Yuan JIANG ◽  
Ronald C. WEK
Keyword(s):  
Uv Irradiation ◽  
Mammalian Cells ◽  
Transcriptional Control ◽  
Control Cell ◽  
Nuclear Factor Κb ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
Secondary Function

In response to UV irradiation, mammalian cells elicit a gene expression programme designed to repair damage and control cell proliferation and apoptosis. Important members of this stress response include the NF-κB (nuclear factor-κB) family. However, the mechanisms by which UV irradiation activates NF-κB are not well understood. In eukaryotes, a variety of environmental stresses are recognized and remediated by a family of protein kinases that phosphorylate the α subunit of eIF2 (eukaryotic initiation factor-2). In the present study we show that NF-κB in MEF (murine embryo fibroblast) cells is activated by UV-C and UV-B irradiation through a mechanism requiring eIF2α phosphorylation. The primary eIF2α kinase in response to UV is GCN2 (general control non-derepressible-2), with PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase) carrying out a secondary function. Our studies indicate that lowered protein synthesis accompanying eIF2α phosphorylation, combined with eIF2α kinase-independent turnover of IκBα (inhibitor of κBα), reduces the levels of IκBα in response to UV irradiation. Release of NF-κB from the inhibitory IκBα would facilitate NF-κB entry into the nucleus and targeted transcriptional control. We also find that loss of GCN2 in MEF cells significantly enhances apoptosis in response to UV exposure similar to that measured in cells deleted for the RelA/p65 subunit of NF-κB. These results demonstrate that GCN2 is central to recognition of UV stress, and that eIF2α phosphorylation provides resistance to apoptosis in response to this environmental insult.

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