Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

Eukaryotic Initiation Factors (eIF) 2�� and 4E Expression, Localization, and Phosphorylation in Brain Tumors

Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2α. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2α levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2α in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well. There are significant differences in the phosphorylated eIF4E levels between the tumors studied, being the highest in meningiomas and the lowest in the oligodendroglial tumors. Relative to subcellular localization, eIF4E is frequently found in the nucleus of the oligodendroglial tumors and rarely in the same compartment of the meningiomas, whereas eIF2α showed an inverse pattern. Finally, cyclin D1 levels directly correlate with the phosphorylation status of both factors. The different expression, phosphorylation, or/and subcellular distribution of eIF2α and eIF4E within the brain types of tumors studied could indicate that different pathways are activated for promoting cell cycle proliferation, for instance, leading to increased cyclin D1 expression. (J Histochem Cytochem 57:503–512, 2009)     

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.     

Respiratory Syncytial Virus Limits α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation to Maintain Translation and Viral Replication

The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, M. M., Powers, L. S., Yarovinsky, T. O., Look, D. C., and Hunninghake, G. W. (2006) J. Immunol. 176, 1733–1740). It is activated by autophosphorylation, likely triggered by a double-stranded RNA intermediate during replication of the virus. In most instances, ph-PKR targets the α subunit of eukaryotic translation initiation factor 2 (eIF2α) protein via phosphorylation, leading to an inhibition of translation of cellular and viral protein. However, we found that although ph-PKR increases in RSV infection, significant eIF2α phosphorylation is not observed, and inhibition of protein translation does not occur. RSV infection attenuates eIF2α phosphorylation by favoring phosphatase rather than kinase activity. Although PKR is activated, RSV sequesters PKR away from eIF2α by binding of the kinase to the RSV N protein. This occurs in conjunction with an increase in the association of the phosphatase, PP2A, with eIF2α following PKR activation. The result is limited phosphorylation of eIF2α and continued translation of cellular and viral proteins.     

Expression and Purification of the α-Subunit of Eukaryotic Initiation Factor eIF2: Use as a Kinase Substrate

The α-subunit of eukaryotic initiation factor eIF2 (eIF2α) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulatedin vivoby phosphorylation of eIF2α at Ser⁵¹. In the present study, rat eIF2α was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2α kinase activity and was rapidly phosphorylated by the eIF2α kinases HCR and PKR. A variant of eIF2α in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2α was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2α has been developed. Use of the wildtype and variant forms of eIF2α to measure eIF2α kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.     

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex.     

Modulation of Dcytb (Cybrd 1) expression and function by iron,dehydroascorbate and Hif-2α in cultured cells

Background

Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines.

Methods

Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and ⁵⁹Fe uptake assays.

Results

Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake.

Conclusions

Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α.

General significance

Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.     

Eukaryotic Initiation Factor 2α - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells'' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.     

Cloning of hHRI, Human Heme-regulated Eukaryotic Initiation Factor 2α Kinase: Down-regulated in Epithelial Ovarian Cancers

Protein synthesis is regulated in response to environmental stimuli by covalent modification, phosphorylating the components of the translational machinery. Phosphorylation of the subunit of eIF-2 is one of the bestcharacterized mechanisms for down-regulating protein synthesis in higher eukaryotes in response to various stress conditions. One of mammalian eIF-2 kinases is a hemeregulated inhibitor kinase (HRI), which is activated by heme deficiency and plays an important role in translational control. In this work, we have analyzed the differentially expressed genes between epithelial ovarian cancer and normal ovary. We have screened a total of 1,408 genes isolated from a human dermal papilla cell cDNA library by cDNA array hybridization. Among many differentially expressed genes, eIF2 kinase, a heme regulated inhibitor was down-regulated in ovarian epithelium cancer. The down-regulation of hHRI was also confirmed in other ovarian cancer tissues by Northern blot hybridization. The hHRI gene is 2,887 bp in length and the amino acid sequence deduced from the cDNA clone encodes a protein of 630 amino acids with molecular mass of 73 kDa. It contains all 12 catalytic domains of the protein kinases with consensus sequences of the proteinserine/threonine kinases. The expression pattern of hHRI mRNA showed approximately 3.0 kb bands which were expressed ubiquitously in all human tissues examined, which indicates that eIF-2 kinase could play an important role in the translational regulation of nonerythroid tissues.     

A Subunit of Eukaryotic Translation Initiation Factor 2α-Phosphatase (CreP/PPP1R15B) Regulates Membrane Traffic

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.     

β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (P_i release) by the eIF2·GTP·Met-tRNA_i ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNA_i to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui⁻ phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17–21) known to impede eIF1 dissociation in vitro. The eIF1 Sui⁻ mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd⁻ phenotype is likewise suppressed by eIF1 overexpression or the 17–21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.     

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.