The eIF4E RNA regulon promotes the Akt signaling pathway

Eukaryotic initiation factor 4E (eIF4E) promotes cellular proliferation and can rescue cells from apoptotic stimuli such as serum starvation. However, the mechanisms underlying apoptotic rescue are not well understood. In this study, we demonstrate that eIF4E overexpression leads to enhanced survival signaling through Akt and that eIF4E requires Akt1 to rescue serum-deprived fibroblasts. Furthermore, a mutant form of eIF4E (W73A), which is messenger RNA (mRNA) export competent but does not promote translation, rescues cells as readily as wild-type eIF4E. We show that eIF4E mediates Akt activation via up-regulation of Nijmegen breakage syndrome 1 (NBS1), a phosphoinositide-3 kinase-Akt pathway upstream activator. Additionally, eIF4E coordinately up-regulates the expression of downstream effectors of the Akt pathway, thereby amplifying Akt signaling effects. A negative regulator of eIF4E, the promyelocytic leukemia protein (PML), suppresses Akt activation and apoptotic rescue. These PML activities likely arise, at least in part, through its inhibition of eIF4E-mediated NBS1 mRNA export. In summary, eIF4E coordinately regulates gene expression to potentiate Akt activation, an activity required for apoptotic rescue.     

Translation initiation factor 4E blocks endoplasmic reticulum-mediated apoptosis

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA cap-binding protein required for translation of cellular mRNAs utilizing the 5' cap structure. The rate-limiting factor for mRNA recruitment to ribosomes, eIF4E is a major target for regulation of translation by growth factors, hormones, and other extracellular stimuli. When overexpressed, eIF4E exerts profound effects on cell growth and survival, leading to suppression of oncogene-dependent apoptosis, causing malignant transformation and conferring tumors with multiple drug resistance. We found previously that overexpressed eIF4E interdicts the apoptotic pathway induced by growth factor withdrawal and cytotoxic drugs by selectively activating the expression of Bcl-X(L), thus preventing mitochondrial release of cytochrome c. In this study, we examined the impact of ectopic eIF4E expression on apoptosis mediated by the endoplasmic reticulum (ER). Here we show that eIF4E rescued cells from the ER stressors brefeldin A, tunicamycin, thapsigargin, and the Ca(2+) ionophore A23187. In addition, we found that cells rescued from Ca(2+) ionophore-triggered apoptosis did not release calcium from their ER nor did they translocate caspase-12 from the ER to the cytoplasm. These data lend strong support to the concept that eIF4E functions as a pleiotropic regulator of cell viability and that integration of critical organelle-mediated checkpoints for apoptosis can be controlled by the cap-dependent translation apparatus.     

Conducting the initiation of protein synthesis: the role of eIF4G

The eukaryotic initiation factor eIF4G is a large modular protein which serves as a docking site for initiation factors and proteins involved in RNA translation. Together with eIF4E and eIF4A, eIF4G constitutes the eIF4F complex which is a key component in promoting ribosome binding to the mRNA. Thus, the central role of eIF4G in initiation makes it a valid target for events aimed at modulating translation. Such events occur during viral infection by picornaviruses and lentiviruses and result in the hijack of the translational machinery through cleavage of eIF4G. Proteolysis of eIF4G is also mediated by caspases during the onset of apoptosis causing inhibition of protein synthesis. We will review the role of eIF4G and protein partners as well as the cellular and viral events that modulate eIF4G activity in the initiation of translation.     

Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.     

Sumoylation of eIF4E activates mRNA translation

Eukaryotic translation initiation factor 4E (eIF4E) is the cap‐binding protein that binds the 5′ cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap‐dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin‐related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E‐dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.     

Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.     

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.     

Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.     

Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg

Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus's ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary 'arms race' between Capsicum eIF4E and potyviral VPg.     

Development of biochemical assays for the identification of eIF4E-specific inhibitors

Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5' cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.     

Ribavirin targets eIF4E dependent Akt survival signaling

The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m⁷G) cap moiety on the 5′-end of mRNAs. We demonstrate that a physical mimic of the m⁷G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m⁷G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m⁷G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.     

Targeting the eIF4F translation initiation complex for cancer therapy

Abstract In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.     

Regulation of Host Translational Machinery by African Swine Fever Virus

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host''s defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.     

Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G.

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.     

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.