Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.     

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.     

Nerve growth factor specifically stimulates translation of eukaryotic elongation factor 1A-1 (eEF1A-1) mRNA by recruitment to polyribosomes in PC12 cells

During postnatal brain development the level of peptide elongation factor-1A (eEF1A-1) expression declines and that of the highly homologous isoform, eEF1A-2, increases in neurons. eEF1A-1 is implicated in cytoskeletal interactions, tumorigenesis, differentiation, and the absence of eEF1A-2 is implicated in neurodegeneration in the mouse mutant, wasted. The translation of eEF1A-1 mRNA is up-regulated via mitogenic stimulation. However, it is not known if eEF1A-1 mRNA translation is regulated by neurotrophins or if its synthesis is differentially regulated than that of the neuronal isoform, eEF1A-2. Regulated translation of these factors by neurotrophins, particularly by the Trk class of neurotrophin receptors, would implicate them in differentiation, survival, and neuronal plasticity. In this study, we investigated the effect of nerve growth factor (NGF) stimulation on the synthesis of eEF1A-1 and eEF1A-2. We found that NGF stimulation causes a preferential synthesis of eEF1A-1 over eEF1A-2 in PC12 cells. We analyzed the co-sedimentation of eEF1A-1 mRNA with polyribosome fractions in sucrose gradients, and found that NGF stimulation enriched the presence of eEF1A-1 mRNA in polyribosomes, indicating that the translation of eEF1A-1 mRNA is regulated by NGF. Inhibitors of phosphatidylinositol 3-kinase (LY 294002), mammalian target of rapamycin (rapamycin), and the NGF receptor, TrkA (K-252a), but not of mitogen-activated protein kinase (PD 98059), prevented the recruitment of eEF1A-1 mRNA to polyribosomes. The mobilization of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive in both proliferating and differentiated PC12 cells, indicating the importance of this pathway during differentiation. Our data shows that after growth factor withdrawal, an NGF-signaling pathway stimulates eEF1A-1 mRNA translation in proliferating and differentiated PC12 cells. Therefore, eEF1A-1 mRNA is a specific translational target of TrkA signaling.     

Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis.

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1alpha, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1alpha to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1alpha in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1alpha, whereas cells transfected with antisense eEF1A-1/EF-1alpha are protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1alpha and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.     

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control nonderepressible 2 (Gcn2p) protein kinase. Gcn2p causes down-regulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation.     

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

eEF1A: Thinking Outside the Ribosome

Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.     

eIF5A interacts functionally with eEF2

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.     

Improper organization of the actin cytoskeleton affects protein synthesis at initiation

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.     

衰老细胞中热休克转录因子1的异常调节和定位

为评估人热休克转录因子1（HSF1）在衰老细胞中呈现年龄依赖功能失调机制, 通过凝胶电泳迁移率改变实验(EMSA)和RNA酶保护实验等了解低总体倍增水平（PDLs）的年轻和高PDLs的衰老IMR90双倍体人肺纤维母细胞的HSF1 DNA 结合活性、HSF1蛋白质及其编码转录子mRNA水平和亚细胞分布.使用H2O2诱导年轻IMR90细胞成为“应激诱导早熟性老化(SIPS)” 细胞,并与复制性衰老细胞比较HSF1 DNA 结合活性、HSF1亚细胞分布和细胞内过氧化物含量.在不同年龄的IMR90细胞中,无论体内或体外,HSF1激活能力与细胞年龄呈反相关,但细胞内HSF1蛋白质与其mRNA水平并无改变.HSF1的亚细胞定位分析显示,HSF1主要存在于年轻细胞胞质中,热刺激促使三体形成和核转移;而在衰老细胞中,37℃时HSF1大部分存在于细胞核内,热刺激后形成三体,与DNA结合能力明显比年轻细胞弱;用H2O2诱导的应激成熟前老化细胞内,HSF1功能和亚细胞分布都与复制性衰老细胞相似.结果显示,细胞年龄与HSF1的激活和定位相关,而与HSF1含量无关,这些变化可能是通过氧化修饰所致.     

A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling. The ability of eEF1A1 and eEF1A2 to interact with SH2 and SH3 domains of different signalling molecules in vitro was compared. Indeed, contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and C-terminal part of Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and phospholipase C-gamma1. Interestingly, the interaction of both isoforms with Shp2 in vivo was found using stable cell lines expressing eEF1A1-His or eEF1A2-His. The formation of a complex between endogenous eEF1A and Shp2 was also shown. Importantly, a higher level of tyrosine phosphorylation of eEF1A2 as compared to eEF1A1 was demonstrated in several independent experiments and its importance for interaction of eEF1A2 with Shp2 in vitro was revealed. Thus, despite the fact that both isoforms of eEF1A could be involved in the phosphotyrosine-mediated processes, eEF1A2 apparently has greater potential to participate in such signalling pathways. Since tyrosine kinases/phosphatases play a prominent role in human cancerogenesis, our observations may gave a basis for recently found oncogenicity of the eEF1A2 isoform.     

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.     

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.     

Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Synthesized peptides corresponded to the selected peptide fragments were conjugated to bovine serum albumin (BSA) and used for immunizations of mice. Antibodies, produced against the eEF1A2 fragment 330–343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.     

Enhancement of translation elongation in neurons by brain-derived neurotrophic factor: implications for mammalian target of rapamycin signaling

The effects and signaling mechanisms of brain-derived neurotrophic factor (BDNF) on translation elongation were investigated in cortical neurons. BDNF increased the elongation rate approximately twofold, as determined by measuring the ribosomal transit time. BDNF-accelerated elongation was inhibited by rapamycin, implicating the mammalian target of rapamycin (mTOR). To explore the mechanisms underlying these effects, we examined the protein phosphorylation cascades that lead to the activation of translation elongation in neurons. BDNF increased eukaryote elongation factor 1A (eEF1A) phosphorylation and decreased eEF2 phosphorylation. Whereas eEF2 phosphorylation levels altered by BDNF were inhibited by rapamycin, eEF1A phosphorylation was not affected by rapamycin or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. BDNF induced phosphorylation of eEF2 kinase (Ser366), as well as decreased its kinase activity. All these events were inhibited by rapamycin. Furthermore, mTOR siRNA, which reduced mTOR levels up to 50%, inhibited the BDNF-induced enhancement in elongation rate and decrease in eEF2 phosphorylation. These results strongly suggest that BDNF enhances translation elongation through the activation of the mTOR-eEF2 pathway.     

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity

The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.     

Tetrahymena eukaryotic translation elongation factor 1A (eEF1A) bundles filamentous actin through dimer formation

Eukaryotic translation elongation factor 1A (eEF1A) is known to be a multifunctional protein. In Tetrahymena, eEF1A is localized to the division furrow and has the character to bundle filamentous actin (F-actin). eEF1A binds F-actin and the ratio of eEF1A and actin is approximately 1:1 (Kurasawa et al., 1996). In this study, we revealed that eEF1A itself exists as monomer and dimer, using gel filtration column chromatography. Next, eEF1A monomer and eEF1A dimer were separated using gel filtration column, and their interaction with F-actin was examined with cosedimentation assay and electron microscopy. In the absence of Ca2+/calmodulin (CaM), eEF1A dimer bundled F-actin and coprecipitated with F-actin at low-speed centrifugation, but eEF1A monomer did not. In the presence of Ca2+/CaM, eEF1A monomer increased, while dimer decreased. To examine that Ca2+/CaM alters eEF1A dimer into monomer and inhibits bundle formation of F-actin, Ca2+/CaM was added to F-actin bundles formed by eEF1A dimer. Ca2+/CaM separated eEF1A dimer to monomer, loosened F-actin bundles and then dispersed actin filaments. Simultaneously, Ca2+/CaM/ eEF1A monomer complexes were dissociated from actin filaments. Therefore, Ca2+/CaM reversibly regulates the F-actin bundling activity of eEF1A.