eIF2α Phosphorylation Tips the Balance to Apoptosis during Osmotic Stress

Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2α, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2α phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2α phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2α promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2α phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2α phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2α phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2α phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.     

GCN2-like eIF2α kinase manages the amino acid starvation response in Toxoplasma gondii

The apicomplexan protozoan Toxoplasma gondii is a significant human and veterinary pathogen. As an obligate intracellular parasite, Toxoplasma depends on nutrients provided by the host cell and needs to adapt to limitations in available resources. In mammalian cells, translational regulation via GCN2 phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) is a key mechanism for adapting to nutrient stress. Toxoplasma encodes two GCN2-like protein kinases, TgIF2K-C and TgIF2K-D. We previously showed that TgIF2K-D phosphorylates T. gondii eIF2α (TgIF2α) upon egress from the host cell, which enables the parasite to overcome exposure to the extracellular environment. However, the function of TgIF2K-C remained unresolved. To determine the functions of TgIF2K-C in the parasite, we cloned the cDNA encoding TgIF2K-C and generated knockout parasites of this TgIF2α kinase to study its function during the lytic cycle. The TgIF2K-C knockout did not exhibit a fitness defect compared with parental parasites. However, upon infection of human fibroblasts that were subsequently cultured in glutamine-free medium, the intracellular TgIF2K-C knockout parasites were impeded for induced phosphorylation of TgIF2α and showed a 50% reduction in the number of plaques formed compared with parental parasites. Furthermore, we found that this growth defect in glutamine-free media was phenocopied in parasites expressing only a non-phosphorylatable TgIF2α (TgIF2α-S71A), but not in a TgIF2K-D knockout. These studies suggest that Toxoplasma GCN2-like kinases TgIF2K-C and TgIF2K-D evolved to have distinct roles in adapting to changes in the parasite’s environment.     

Mechanical stimulation suppresses phosphorylation of eIF2α and PERK-mediated responses to stress to the endoplasmic reticulum

Cellular perturbations such as stress to the endoplasmic reticulum induce an integrated stress response, which activates phosphorylation of eIF2α and leads to alleviation of cellular injury or apoptosis. This study investigated the role of mechanical stimulation in the regulation of eIF2α and cell death. Mechanical stimulation was applied to mouse ulnae, MC3T3 cells, and mesenchymal stem cells. The results demonstrate that mechanical stimulation reduces phosphorylation of eIF2α through inactivation of Perk. Furthermore, flow pre-treatment reduces thapsigargin-induced cell mortality through suppression of phosphorylation of Perk. However, H₂O₂-driven cell mortality, which is not mediated by Perk, is not suppressed by mechanical stimulation. Taken together, our observations suggest a pro-survival role of mechanical stimulation in Perk-mediated stress responses.     

Deletion of the eIF2α Kinase GCN2 Fails to Rescue the Memory Decline Associated with Alzheimer’s Disease

Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer’s disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2^−/− and GCN2^+/− deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2^−/− and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.     

Development of transgenic mice expressing a conditionally active form of the eIF2α kinase PKR

Phosphorylation of the alpha (α) subunit of the eukaryotic initiation factor 2 (eIF2) at serine 51 is an important mechanism of translational control in response to various forms of environmental stress. In metazoans, eIF2α phosphorylation is mediated by four kinases each of which becomes activated by distinct stimuli. Previous work established that expression of a chimera protein comprising of the bacteria Gyrase B N-terminal (GyrB) domain fused to the kinase domain (KD) of the eIF2α kinase PKR is capable of inducing eIF2α phosphorylation in cultured cells after treatment with the antibiotic coumermycin. Herein, we report the development of transgenic mice expressing the fusion protein GyrB.PKR ubiquitously. Treatment of mice with coumermycin induces eIF2α phosphorylation in vivo as demonstrated by immunoblotting and immunoshistochemistry of mouse tissues. The GyrB.PKR transgene represents a useful model system to investigate the biological effects of the conditional induction of eIF2α phosphorylation in vivo in the absence of parallel signaling pathways that are elicited in response to stress.     

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Investigation of the eIF2α phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2α in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2α. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2α phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2α phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2α phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eIF2α. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2α phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.     

The non-steroidal anti-inflammatory drug indomethacin activates the eIF2α kinase PKR, causing a translational block in human colorectal cancer cells

The NSAID (non-steroidal anti-inflammatory drug) indomethacin, a cyclo-oxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against CRC (colorectal cancer) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. In the present study we show that indomethacin selectively activates the dsRNA (double-stranded RNA)-dependent protein kinase PKR in a cyclo-oxygenase-independent manner, causing rapid phosphorylation of eIF2α (the α-subunit of eukaryotic translation initiation factor 2) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of the eIF2α kinases PERK (PKR-like endoplasmic reticulum-resident kinase), GCN2 (general control non-derepressible-2) and HRI (haem-regulated inhibitor kinase), and induced eIF2α phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in vitro, an effect reversed by the PKR inhibitor 2-aminopurine, suggests a direct effect of the drug in kinase activation. The results of the present study identify PKR as a novel target of indomethacin, suggesting new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.     

Brain-specific disruption of the eIF2α kinase PERK decreases ATF4 expression and impairs behavioral flexibility

Translational control depends on phosphorylation of eIF2α by PKR-like ER kinase (PERK). To examine the role of PERK in cognitive function, we selectively disrupted PERK expression in the adult mouse forebrain. In the prefrontal cortex (PFC) of PERK-deficient mice, eIF2α phosphorylation and ATF4 expression were diminished and were associated with enhanced behavioral perseveration, decreased prepulse inhibition, reduced fear extinction, and impaired behavioral flexibility. Treatment with the glycine transporter inhibitor SSR504734 normalized eIF2α phosphorylation, ATF4 expression, and behavioral flexibility in PERK-deficient mice. Moreover, the expression levels of PERK and ATF4 were reduced in the frontal cortex of human patients with schizophrenia. Together, our findings reveal that PERK plays a critical role in information processing and cognitive function and that modulation of eIF2α phosphorylation and ATF4 expression may represent an effective strategy for treating behavioral inflexibility associated with several neurological disorders such as schizophrenia.     

Phosphorylation of a heme-regulated eukaryotic initiation factor 2α kinase enhances the interaction with heat-shock protein 90 and substantially upregulates kinase activity

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.     

Phosphorylation of serine 323 of ASB2α is pivotal for the targeting of filamin A to degradation

ASB proteins are the specificity subunits of cullin5-RING E3 ubiquitin ligases (CRL5) that play roles in ubiquitin-mediated protein degradation. However, how their activity is regulated remains poorly understood. Here, we unravel a novel mechanism of regulation of a CRL5 through phosphorylation of its specificity subunit ASB2α. Indeed, using mass spectrometry, we showed for the first time that ASB2α is phosphorylated and that phosphorylation of serine-323 (Ser-323) of ASB2α is crucial for the targeting of the actin-binding protein filamin A (FLNa) to degradation. Mutation of ASB2α Ser-323 to Ala had no effect on intrinsic E3 ubiquitin ligase activity of ASB2α but abolished the ability of ASB2α to induce degradation of FLNa. In contrast, the ASB2α Ser-323 to Asp phosphomimetic mutant induced acute degradation of FLNa. Moreover, inhibition of the extracellular signal-regulated kinases 1 and 2 (Erk1/2) activity reduced ASB2α-mediated FLNa degradation. We further showed that the subcellular localization of ASB2α to actin-rich structures is dependent on ASB2α Ser-323 phosphorylation and propose that the interaction with FLNa depends on the electrostatic potential redistribution induced by the Ser-323 phosphate group. Taken together, these data unravel an important mechanism by which ASB2α-mediated FLNa degradation can be regulated.