A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.     

The small molecule ‘1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate’ and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha

By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

Cytoprotective roles of ERK and Akt in endoplasmic reticulum stress triggered by subtilase cytotoxin

Subtilase cytotoxin (SubAB) is the prototype of a distinct AB₅ toxin family produced by Shiga toxigenic Escherichia coli. Recent reports disclosed pro-apoptotic pathways triggered by SubAB, whereas its anti-apoptotic signals have not been elucidated. In the present study, we investigated pro-survival signaling elicited by SubAB, especially focusing on extracellular signal-regulated kinase (ERK) and Akt. We found that SubAB activated ERK and Akt, and inhibition of individual kinases enhanced SubAB-triggered apoptosis. SubAB induced endoplasmic reticulum (ER) stress, and other ER stress inducers mimicked the stimulatory effects of SubAB on ERK and Akt. Attenuation of ER stress reduced SubAB-induced phosphorylation of these kinases, suggesting involvement of the unfolded protein response (UPR). SubAB induced activation of protein kinase-like ER kinase (PERK) and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and phosphorylation of eIF2α by salubrinal caused activation of ERK and Akt, leading to cell survival. Dominant-negative inhibition of PERK enhanced SubAB-induced apoptosis and reduced phosphorylation of ERK and Akt. Furthermore, the anti-apoptotic effect of eIF2α was significantly reversed by inhibition of ERK and Akt. These results suggest cytoprotective roles of ERK and Akt in SubAB-triggered, ER stress-mediated apoptosis.     

Protein synthesis attenuation by phosphorylation of eIF2α is required for the differentiation of Trypanosoma cruzi into infective forms

Chagas' disease is a potentially life-threatening illness caused by the unicellular protozoan parasite Trypanosoma cruzi. It is transmitted to humans by triatomine bugs where T. cruzi multiplies and differentiates in the digestive tract. The differentiation of proliferative and non-infective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) can be correlated to nutrient exhaustion in the gut of the insect vector. In vitro, metacyclic-trypomastigotes can be obtained when epimastigotes are submitted to nutritional stress suggesting that metacyclogenesis is triggered by nutrient starvation. The molecular mechanism underlying such event is not understood. Here, we investigated the role of one of the key signaling responses elicited by nutritional stress in all other eukaryotes, the inhibition of translation initiation by the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), during the in vitro differentiation of T. cruzi. Monospecific antibodies that recognize the phosphorylated Tc-eIF2α form were generated and used to demonstrate that parasites subjected to nutritional stress show increased levels of Tc-eIF2α phosphorylation. This was accompanied by a drastic inhibition of global translation initiation, as determined by polysomal profiles. A strain of T. cruzi overexpressing a mutant Tc-eIF2α, incapable of being phosphorylated, showed a block on translation initiation, indicating that such a nutritional stress in trypanosomatids induces the conserved translation inhibition response. In addition, Tc-eIF2α phosphorylation is critical for parasite differentiation since the overexpression of the mutant eIF2α in epimastigotes abolished metacyclogenesis. This work defines the role of eIF2α phosphorylation as a key step in T. cruzi differentiation.     

Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human

PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2α’s N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2α, we expressed human eIF2α’s with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2α by all mammalian kinases, thus defining amino acid residues in eIF2α (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2α by GCN2. We propose that mammalian eIF2α kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2α in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms.     

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca²⁺sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺([Ca²⁺]_i) due to plasmalemmal Ca²⁺influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca²⁺stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺]_iper se causes slowing of peptide chain elongation is lacking. L-type Ca²⁺channel activity of GH₃pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺]_ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺]_ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca²⁺channel antagonist, nisoldipine, lowered [Ca²⁺]_iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺]_ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca²⁺contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺]_ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca²⁺]_ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.     

Large G3BP-induced granules trigger eIF2α phosphorylation

Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2α. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2α (eIF2α) kinases, we identified protein kinase R as the principal kinase that mediates eIF2α phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2α phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.     

A GCN2-like eukaryotic initiation factor 2 kinase increases the viability of extracellular Toxoplasma gondii parasites

Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2α (eIF2α) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma parasites and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.     

Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2

Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress.     

6-Shogaol induces apoptosis in human hepatocellular carcinoma cells and exhibits anti-tumor activity in vivo through endoplasmic reticulum stress

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.     

Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.