The interferon-induced double-stranded RNA-activated protein kinase PKR will phosphorylate serine, threonine, or tyrosine at residue 51 in eukaryotic initiation factor 2alpha.

The family of eukaryotic initiation factor 2alpha (eIF2alpha) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.     

Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.     

The alpha subunit of eukaryotic initiation factor 2B (eIF2B) is required for eIF2-mediated translational suppression of vesicular stomatitis virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

Functional complementation by wheat eIF2alpha in the yeast GCN2-mediated pathway

Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway.     

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.     

A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.     

Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum

Exposure to ultraviolet light can cause inflammation, premature skin aging, and cancer. UV irradiation alters the expression of multiple genes that encode functions to repair DNA damage, arrest cell growth, and induce apoptosis. In addition, UV irradiation inhibits protein synthesis, although the mechanism is not known. In this report, we show that UV irradiation induces phosphorylation of eukaryotic translation initiation factor 2 on the alpha-subunit (eIF2alpha) and inhibits protein synthesis in a dosage- and time-dependent manner. The UV-induced phosphorylation of eIF2alpha was prevented by the overexpression of a non-phosphorylatable mutant of eIF2alpha (S51A). PERK is an eIF2alpha protein kinase localized to the endoplasmic reticulum that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. Expression of trans-dominant-negative mutants of PERK also prevented eIF2alpha phosphorylation upon UV treatment and protected from the associated translation attenuation. The luminal domain of dominant-negative mutant PERK formed heterodimers with endogenous PERK to inhibit the PERK signaling pathway. In contrast, eIF2alpha phosphorylation was not inhibited by overexpression of a trans-dominant-negative mutant kinase, PKR, supporting the theory that UV-induced eIF2alpha phosphorylation is specifically mediated by PERK. These results support a novel mechanism by which UV irradiation regulates translation via an endoplasmic reticulum-stress signaling pathway.     

Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis

As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2alpha)atSer(51) is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2alpha stress kinases. We used gene targeting to replace the wild-type Ser(51) allele with an Ala in the eIF2alpha gene to test the hypothesis that translational control through eIF2alpha phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2alpha mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-alpha, and serum deprivation. TNFalpha treatment induced eIF2alpha phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2alpha kinase PKR. In addition, expression of a phospho-mimetic Ser(51) to Asp mutant eIF2alpha-activated caspase 3, indicating that eIF2alpha phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2alpha phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2alpha kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2alpha. We propose that eIF2alpha phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.     

The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA-activated protein kinase R

The translation initiation factor 2 alpha (eIF2alpha)-kinase, dsRNA-activated protein kinase (PKR), constitutes one of the major antiviral proteins activated by viral infection of vertebrates. PKR is activated by viral double-stranded RNA and subsequently phosphorylates the alpha-subunit of translation initiation factor eIF2. This results in overall down regulation of protein synthesis in the cell and inhibition of viral replication. Fish appear to have a PKR-like protein that has Z-DNA binding domains instead of dsRNA binding domains in the regulatory domain, and has thus been termed Z-DNA binding protein kinase (PKZ). We present the cloning of the Atlantic salmon PKZ cDNA and show its upregulation by interferon in Atlantic salmon TO cells and poly inosinic poly cytodylic acid in head kidney. We also demonstrate that recombinant Atlantic salmon PKZ, expressed in Escherichia coli, phosphorylates eIF2alphain vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2alpha. PKZ activity, as measured by phosphorylation of eIF2alpha, was increased after addition of Z-DNA, but not by dsRNA. In addition, we show that wild-type Atlantic salmon PKZ, but not the kinase defective variant K217R, has a direct inhibitory effect on protein synthesis after transient expression in Chinook salmon embryo cells. Overall, the results support a role for PKZ, like PKR, in host defense against virus infection.     

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.     

Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells.

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.     

Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2alpha

Eukaryotic translation initiation factor 2alpha (eIF2alpha) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2alpha at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2alpha by x-ray diffraction at 1.9 A resolution. This structure contains two major domains. The N terminus is a beta-barrel with five antiparallel beta-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting beta3 and beta4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction.