Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export‐competent RNP

The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap‐dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E‐sensitivity element (4E‐SE), and its overexpression alters the nuclear export of several eIF4E‐sensitive mRNAs. LRPPRC‐mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E‐binding site and it coincides with the subcellular re‐distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E‐ and eIF4E‐sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.     

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.     

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G

Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.     

Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E

The initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

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.     

Serotonin increases phosphorylation of synaptic 4EBP through TOR, but eukaryotic initiation factor 4E levels do not limit somatic cap-dependent translation in aplysia neurons

The target of rapamycin (TOR) plays an important role in memory formation in Aplysia californica. Here, we characterize one of the downstream targets of TOR, the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP) from Aplysia. Aplysia 4EBP contains the four critical phosphorylation sites regulated by TOR as well as an N-terminal RAIP motif and a C-terminal TOS site. Aplysia 4EBP was hypophosphorylated in synaptosomes, and serotonin addition caused a rapamycin-sensitive increase in 4EBP phosphorylation both in synaptosomes and in isolated neurites. Aplysia 4EBP was regulated in a fashion similar to that of mammalian 4EBPs, binding to eIF4E when dephosphorylated and releasing eIF4E after phosphorylation. Overexpression of 4EBP in the soma of Aplysia neurons caused a specific decrease in cap-dependent translation that was rescued by concomitant overexpression of eIF4E. However, eIF4E overexpression by itself did not increase cap-dependent translation, suggesting that increasing levels of free eIF4E by phosphorylating 4EBP is not important in regulating cap-dependent translation in the cell soma. Total levels of eIF4E were also regulated by 4EBP, suggesting that 4EBP can also act as an eIF4E chaperone. These studies demonstrate the conserved nature of 4EBP regulation and its role in cap-dependent translation and suggest differential roles of 4EBP phosphorylation in the soma and synapse.     

Phosphorylation of the mRNA cap-binding protein eIF4E and cancer

Dysregulated protein synthesis is frequently involved in oncogenesis and cancer progression. Translation initiation is thought to be the rate-limiting step in protein synthesis, and the mRNA 5′ cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is a pivotal factor that initiates translation. The activities of eIF4E are regulated at multiple levels, one of which is through its phosphorylation at Serine 209 by the mitogen-activated protein kinase-interacting kinases (MNKs, including MNK1 and MNK2). Benefiting from novel mouse genetic tools and pharmacological MNK inhibitors, our understanding of a role for eIF4E phosphorylation in tumor biology and cancer therapy has greatly evolved in recent years. Importantly, recent studies have found that the level of eIF4E phosphorylation is frequently upregulated in a wide variety of human cancer types, and phosphorylation of eIF4E drives a number of important processes in cancer biology, including cell transformation, proliferation, apoptosis, metastasis and angiogenesis. The MNK-eIF4E axis is being assessed as a therapeutic target either alone or in combination with other therapies in different cancer models. As novel MNK inhibitors are being developed, experimental studies bring new hope to cure human cancers that are not responsive to traditional therapies. Herein we review recent progress on our understanding of a mechanistic role for phosphorylation of eIF4E in cancer biology and therapy.     

Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor

Mammalian hibernation involves cessation of energetically costly processes typical of homeostatic regulation including protein synthesis. To further elucidate the mechanisms employed in depressing translation, we surveyed key eukaryotic initiation factors [eIF2, eIF4B, eIF4E, eIF4GI and -II, and 4E-binding protein-1 (4E-BP1), -2, and -3] for their availability and phosphorylation status in the livers of golden-mantled ground squirrels (Spermophilus lateralis) across the hibernation cycle. Western blot analyses indicated only one significant locus for regulation of translational initiation in ground squirrel liver: control of eIF4E. We found seasonal variation in a potent regulator of eIF4E activity, 4E-BP1. Summer squirrels lack 4E-BP1 and apparently control eIF4E activity through direct phosphorylation. In winter, eIF4E is regulated through binding with 4E-BP1. During the euthermic periods that separate bouts of torpor (interbout arousal), 4E-BP1 is hyperphosphorylated to promote initiation. However, during torpor, 4E-BP1 is hypophosphorylated and cap-dependent initiation of translation is restricted. The regulation of cap-dependent initiation of translation may allow for the differential expression of proteins directed toward enhancing survivorship.     

Regulation of eukaryotic initiation factor 4E phosphorylation in the nervous system of Aplysia californica

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.     

Ubiquitination and proteasome-dependent degradation of human eukaryotic translation initiation factor 4E

Translation initiation factor 4E (eIF4E) is a cytoplasmic cap-binding protein that is required for cap-dependent translation initiation. Here, we have shown that eIF4E is ubiquitinated primarily at Lys-159 and incubation of cells with a proteasome inhibitor leads to increased eIF4E levels, suggesting the proteasome-dependent proteolysis of ubiquitinated eIF4E. Ubiquitinated eIF4E retained its cap binding ability, whereas eIF4E phosphorylation and eIF4G binding were reduced by ubiquitination. The W73A mutant of eIF4E exhibited enhanced ubiquitination/degradation, and 4E-BP overexpression protected eIF4E from ubiquitination/degradation. Because heat shock or the expression of the carboxyl terminus of heat shock cognate protein 70-interacting protein (Chip) dramatically increased eIF4E ubiquitination, Chip may be at least one ubiquitin E3 ligase responsible for eIF4E ubiquitination.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.     

Translational homeostasis via the mRNA cap-binding protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.