Perk is essential for translational regulation and cell survival during the unfolded protein response

Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress.     

Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation

The parasitic protozoan Leishmania is the etiological agent of human leishmaniasis worldwide. It undergoes cellular differentiation from the sandfly promastigote form into amastigotes within mammalian macrophages, a process that is essential for its intracellular survival. Here, we characterized the Leishmania infantum PERK eIF2alpha kinase homologue and addressed its role in the parasite's cytodifferentiation. We show that Leishmania PERK is an endoplasmic reticulum (ER) transmembrane protein that largely colocalizes with the ER BiP chaperone. The Leishmania PERK catalytic kinase domain undergoes autohyperphosphorylation and phosphorylates the translation initiation factor 2-alpha subunit (eIF2alpha) in vitro at threonine 166. We also report that PERK is post-translationally regulated specifically in the intracellular stage of the parasite or under ER stress, most likely through extensive autohyperphosphorylation. We have generated a PERK dominant negative mutant overexpressing a truncated PERK protein lacking the N-terminal luminal domain and showed that this mutant is impaired in eIF2alpha phosphorylation in response to ER stress or during amastigote differentiation. Most importantly, we showed that lack of eIF2alpha phosphorylation markedly delays the Leishmania differentiation process towards amastigote forms both in parasites grown axenically or within macrophages. These data highlight the importance of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation in the intracellular development of Leishmania.     

Role of the endoplasmic reticulum unfolded protein response in glomerular epithelial cell injury

C5b-9-induced glomerular epithelial cell (GEC) injury in vivo (in passive Heymann nephritis) and in culture is associated with damage to the endoplasmic reticulum (ER) and increased expression of ER stress proteins. Induction of ER stress proteins is enhanced via cytosolic phospholipase A(2) (cPLA(2)) and limits complement-dependent cytotoxicity. The present study addresses another aspect of the ER unfolded protein response, i.e. activation of protein kinase R-like ER kinase (PERK or pancreatic ER kinase), which phosphorylates eukaryotic translation initiation factor 2-alpha (eIF2alpha), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2alpha was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2alpha and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2alpha was enhanced by overexpression of cPLA(2). Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2alpha phosphorylation and reduced protein synthesis. Complement and ischemia-reperfusion induced phosphorylation of PERK (which correlates with activation), and fibroblasts from PERK knock-out mice were more susceptible to complement- and ischemia-reperfusion-mediated cytotoxicity, as compared with wild type fibroblasts. The GEC protein, nephrin, plays a key role in maintaining glomerular permselectivity. In contrast to a general reduction in protein synthesis, translation regulated by the 5'-end of mouse nephrin mRNA during ER stress was paradoxically maintained, probably due to the presence of short open reading frames in this mRNA segment. Thus, phosphorylation of eIF2alpha and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury.     

Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinase PERK

The hepatitis C virus envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded RNA-activated protein kinase PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). We previously reported that E2 modulates global translation through inhibition of the interferon-induced antiviral protein PKR through its PKR-eIF2alpha phosphorylation site homology domain (PePHD). Here we show that the PKR-like ER-resident kinase (PERK) binds to and is also inhibited by E2. At low expression levels, E2 induced ER stress, but at high expression levels, and in vitro, E2 inhibited PERK kinase activity. Mammalian cells that stably express E2 were refractory to the translation-inhibitory effects of ER stress inducers, and E2 relieved general translation inhibition induced by PERK. The PePHD of E2 was required for the rescue of translation that was inhibited by activated PERK, similar to our previous findings with PKR. Here we report the inhibition of a second eIF2alpha kinase by E2, and these results are consistent with a pseudosubstrate mechanism of inhibition of eIF2alpha kinases. These findings may also explain how the virus promotes persistent infection by overcoming the cellular ER stress response.     

Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK

Regulated phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2alpha phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction.     

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.     

Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.     

Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state.     

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha 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 P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha 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 eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.     

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.     

Nck-1 antagonizes the endoplasmic reticulum stress-induced inhibition of translation

Eukaryotic cells have developed specific mechanisms to overcome environmental stress. Here we show that the Src homology 2/3 (SH2/SH3) domain-containing protein Nck-1 prevents the unfolded protein response normally induced by pharmacological endoplasmic reticulum (ER) stress agents. Overexpression of Nck-1 enhances protein translation, whereas it abrogates eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and inhibition of translation in response to tunicamycin or thapsigargin treatment. Nck-1 overexpression also attenuates induction of the ER chaperone, the immunoglobulin heavy chain-binding protein (BiP), and impairs cell survival in response to thapsigargin. We provided evidence that in these conditions, the effects of Nck on the unfolded protein response (UPR) involve its second SH3 domain and a calyculin A-sensitive phosphatase activity. In addition, we demonstrated that protein translation is reduced in mouse embryonic fibroblasts lacking both Nck isoforms and is enhanced in similar cells expressing high levels of Nck-1. In these various mouse embryonic fibroblasts, we also provided evidence that Nck modulates the activation of the ER resident eIF2alpha kinase PERK and consequently the phosphorylation of eIF2alpha on Ser-51 in response to stress. Our study establishes that Nck is required for optimal protein translation and demonstrates that, in addition to its adaptor function in mediating signaling from the plasma membrane, Nck also mediates signaling from the ER membrane compartment.     

Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR

Phosphorylation of the alpha (alpha) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2alpha kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2alpha kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK(-/-) mice are more susceptible to VSV-mediated apoptosis than PERK(+/+) MEFs. The higher replication capacity of VSV in PERK(-/-) MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2alpha phosphorylation. We also show that VSV-infected PERK(-/-) MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2alpha kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.