Inactivation of eukaryotic initiation factor 2B in vitro by heat shock

Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 °C or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2α phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.

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Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

Molecular and Cellular Biology ◽

10.1128/mcb.02254-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2384-2397 ◽

Cited By ~ 40

Author(s):

Jeanne M. Fringer ◽

Michael G. Acker ◽

Christie A. Fekete ◽

Jon R. Lorsch ◽

Thomas E. Dever

Keyword(s):

Translation Initiation ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation ◽

Cell Extracts ◽

Eukaryotic Translation Initiation ◽

Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

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Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B

Biochemical Journal ◽

10.1042/bj2940625 ◽

1993 ◽

Vol 294 (3) ◽

pp. 625-629 ◽

Cited By ~ 253

Author(s):

G I Welsh ◽

C G Proud

Keyword(s):

Kinase Activity ◽

Glycogen Synthase ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Glycogen Synthase Kinase ◽

Initiation Factor ◽

Glycogen Synthase Kinase 3 ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Cell Extracts

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

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A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5328 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5328-5334 ◽

Cited By ~ 118

Author(s):

N Méthot ◽

M S Song ◽

N Sonenberg

Keyword(s):

Aspartic Acid ◽

Ribosomal Subunit ◽

Direct Interaction ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Ribosome Binding ◽

Initiation Factors ◽

Self Association

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

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Mutation at the acidic residue-rich domain of eukaryotic initiation factor 2 (eIF2α)-associated glycoprotein p67 increases the protection of eIF2α phosphorylation during heat shock

Archives of Biochemistry and Biophysics ◽

10.1016/s0003-9861(03)00092-4 ◽

2003 ◽

Vol 413 (1) ◽

pp. 116-122 ◽

Cited By ~ 6

Author(s):

Bansidhar Datta ◽

Rekha Datta

Keyword(s):

Heat Shock ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Rich Domain ◽

Eif2α Phosphorylation ◽

Eukaryotic Initiation Factor 2 ◽

Initiation Factor 2

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.23.7971-7980.2001 ◽

2001 ◽

Vol 21 (23) ◽

pp. 7971-7980 ◽

Cited By ~ 206

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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Evidence that the dephosphorylation of Ser535 in the ∊-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin

Biochemical Journal ◽

10.1042/bj20020677 ◽

2002 ◽

Vol 367 (2) ◽

pp. 475-481 ◽

Cited By ~ 34

Author(s):

Xuemin WANG ◽

Maarten JANMAAT ◽

Anne BEUGNET ◽

Fiona E.M. PAULIN ◽

Christopher G. PROUD

Keyword(s):

Protein Synthesis ◽

Glycogen Synthase ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Ovary Cells

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (∊) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser535. GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser535in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser535in vivo and to explore the role of phosphorylation of Ser535 in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser535, which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

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Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

Cell Death and Disease ◽

10.1038/s41419-020-03324-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ai-Ling Tian ◽

Qi Wu ◽

Peng Liu ◽

Liwei Zhao ◽

Isabelle Martins ◽

...

Keyword(s):

Stress Response ◽

Immune Responses ◽

Initiation Factor ◽

Integrated Stress Response ◽

Serine Residue ◽

Eif2α Phosphorylation ◽

Lysosomotropic Agents ◽

Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1918459117 ◽

2020 ◽

Vol 117 (20) ◽

pp. 10935-10945 ◽

Cited By ~ 1

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.

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