Phosphorylation of eukaryotic translation initiation factor 4E is increased in Src-transformed cell lines.

Eukaryotic initiation factor 4F (eIF-4F) is a three-subunit complex that binds the 5' cap structure (m7GpppX, where X is any nucleotide) of eukaryotic mRNAs. This factor facilitates ribosome binding by unwinding the secondary structure in the mRNA 5' noncoding region. The limiting component of the 4F complex is believed to be the 24-kDa cap-binding phosphoprotein, eIF-4E. In this report, we describe the phosphorylation of eIF-4E in response to expression of the tyrosine kinase oncoproteins pp60v-src and pp60c-src527F. The results suggest that eIF-4E functions as a downstream target of the phosphorylation cascade induced by tyrosine-specific protein kinases as well as by effectors of the mitogenic response.     

Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth

Eukaryotic translation initiation factor 4E (eIF4E) binds to the cap structure at the 5' end of mRNAs and is a critical target for the control of protein synthesis. eIF4E is phosphorylated in many systems in response to extracellular stimuli, but biochemical evidence to date has been equivocal as to the biological significance of this modification. Here we use a genetic approach to this problem. We show that, in Drosophila melanogaster, homozygous eIF4E mutants arrest growth during larval development. In Drosophila eIF4EI, Ser251 corresponds to Ser209 of mammalian eIF4E, which is phosphorylated in response to extracellular signals. We find that, in vivo, eIF4EI Ser251 mutants cannot incorporate labeled phosphate. Furthermore, transgenic Drosophila organisms expressing eIF4E(Ser251Ala) in an eIF4E mutant background have reduced viability. Escapers develop more slowly than control siblings and are smaller. These genetic data provide evidence that eIF4E phosphorylation is biologically significant and is essential for normal growth and development.     

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.     

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells

Translation of the small G protein RhoA in neurons is regulated by the eukaryotic translation initiation factor eIF4E. Here we show that this translation factor also regulates RhoA expression and activity in breast cancer cells. The introduction of eIF4E into breast tumor cells increased RhoA protein levels, while expression of an eIF4E siRNA reduced RhoA expression. Previous studies indicate that the axon repulsion factor Semaphorin3A (Sema3A) stimulates the eIF4E-dependent translation of RhoA in neurons, and breast tumor cells support autocrine Sema3A signaling. Accordingly, we next examined if autocrine Sema3A signaling drives eIF4E-dependent RhoA translation in breast cancer cells. The incubation of breast tumor cells with recombinant Sema3A rapidly increased eIF4E activity, RhoA protein levels, and RhoA activity. This Sema3A activity was blocked in tumor cells expressing an shRNA-specific for the Sema3A receptor, Neuropilin-1 (NP-1), as well as in cells incubated with an eIF4E inhibitor. Importantly, RhoA protein levels were reduced in Sema3A shRNA-expressing compared to control shRNA-expressing breast tumor cells, demonstrating that autocrine Sema3A increases RhoA expression in breast cancer. Considering that Sema3A suppresses axon extension by stimulating RhoA translation, we next examined if the Sema3A/RhoA axis impacts breast tumor cell migration. The incubation of control breast tumor cells, but not RhoA shRNA-expressing cells, with rSema3A significantly reduced their migration. Collectively, these studies indicate that Sema3A impedes breast tumor cell migration in part by stimulating RhoA. These findings identify common signaling pathways that regulate the navigation of neurons and breast cancer cells, thus suggesting novel targets for suppressing breast tumor cell migration.     

Phosphorylation site of eukaryotic initiation factor 4E

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.     

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.     

Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E

Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.     

Translational regulation of the mRNA encoding the eukaryotic translation initiation factor 4E in Xenopus

We have cloned the cDNA for Xenopus eukaryotic translation initiation factor 4E (eIF4E). Here we show that translation of a luciferase mRNA that contains the 5' untranslated region derived from Xenopus eIF4E is active in fertilized eggs, but is repressed in oocytes. The results suggest that the expression of Xenopus eIF4E is regulated at the translation level.     

Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.     

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.     

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes compete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.     

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.     

Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.