Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myoblasts

The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the alpha-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B epsilon-subunit, because deprivation of either amino acid caused a decrease in eIF2Bepsilon kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E.4E-BP1 complex to the active eIF4E.eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine- and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not rate-controlling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.     

The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells

Serum stimulation of cultured Xenopus kidney cells results in enhanced phosphorylation of the translational initiation factor (eIF) 4E and promotes a 2.8-fold increase in the binding of the adapter protein eIF4G to eIF4E, to form the functional initiation factor complex eIF4F. Here we demonstrate the serum-stimulated co-isolation of the poly(A)-binding protein (PABP) with the eIF4F complex. This apparent interaction of PABP with eIF4F suggests that a mechanism shown to be important in the control of translation in the yeast Saccharomyces cerevisiae also operates in vertebrate cells. We also present evidence that the signaling pathways modulating eIF4E phosphorylation and function in Xenopus kidney cells differ from those in several mammalian cell types studied previously. Experiments with the immunosuppressant rapamycin suggest that the mTOR signaling pathway is involved in serum-promoted eIF4E phosphorylation and association with eIF4G. Moreover, we could find little evidence for regulation of eIF4E function via interaction with the specific binding proteins 4E-BP1 or 4E-BP2 in these cells. Although rapamycin abrogated serum-enhanced rates of protein synthesis and the interaction of eIF4G with eIF4E, it did not prevent the increase in association of eIF4G with PABP. This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways

Initiation factor eIF4E binds to the 5'-cap of eukaryotic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through its own phosphorylation and through inhibitory binding proteins (4E-BPs), which modulate its availability for initiation complex assembly. eIF4E phosphorylation is enhanced by phorbol esters. We show, using specific inhibitors, that this involves both the p38 mitogen-activated protein (MAP) kinase and Erk signaling pathways. Cell stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin-1beta also cause increased phosphorylation of eIF4E, which is abolished by the specific p38 MAP kinase inhibitor, SB203580. These changes in eIF4E phosphorylation parallel the activity of the eIF4E kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase and increase Mnk1 activity, do not increase phosphorylation of eIF4E. The latter stresses increase the binding of eIF4E to 4E-BP1, and we show that this blocks the phosphorylation of eIF4E by Mnk1 in vitro, which may explain the absence of an increase in eIF4E phosphorylation under these conditions.     

Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein

eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.     

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.     

Differential regulation of translation and eIF4E phosphorylation during human thymocyte maturation

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+ CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+ CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.     

Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation

In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the messenger RNA results from recognition of the 5' cap structure (m7GpppX) of the mRNA by the cap-binding complex eIF4F. eIF4F is itself a three-subunit complex comprising the cap-binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF4G, which interacts with both eIF4A and eIF4E and enhances cap binding by eIF4E. The mRNA 3' polyadenylate tail and the associated poly(A)-binding protein (PABP) also regulate translational initiation, probably by interacting with the 5' end of the mRNA. In yeast and plants, PABP interacts with eIF4G but no such interaction has been reported in mammalian cells. Here, we describe a new human PABP-interacting protein, PAIP-I, whose sequence is similar to the central portion of eIF4G and which interacts with eIF4A. Overexpression of PAIP-1 in COS-7 cells stimulates translation, perhaps by providing a physical link between the mRNA termini.     

The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth

Initiation in eukaryotes is the rate limiting step of translation. The binding of the mRNA to the 40S ribosomal subunit, which is mediated by the mRNA cap structure, is a key target for control of protein synthesis. The cap binding protein, eIF4E, is the most limiting of all initiation factors and its overexpression in NIH3T3 cells causes malignant transformation. 4E-binding protein 1 (BP1) and 4E-BP2 are small proteins that bind to eIF4E and inhibit translation. Here, 4E-BPs were expressed in cells transformed by eIF4E or by v-src to determine the effect of 4E-BPs on cell growth and tumorigenicity. We show that 4E-BPs cause a significant reversion of the transformed phenotype. Thus, we demonstrate that the eIF4E-binding proteins act as negative regulators of cell growth. We propose that 4E-BPs are members of a class of negative regulators of cell growth acting on the translation machinery of the cell.     

The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ?1H?-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ?1H?-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.     

Angiotensin II stimulates phosphorylation of the translational repressor 4E-binding protein 1 by a mitogen-activated protein kinase-independent mechanism

To investigate the molecular basis of the hypertrophic action of angiotensin II (AII) in vascular smooth muscle cells (SMC), we have examined the ability of the hormone to regulate the function of the translational repressor 4E-binding protein 1 (4E-BP1). Addition of AII to quiescent aortic SMC potently increased the phosphorylation of 4E-BP1 as revealed by a decreased electrophoretic mobility and an increased phosphate content of the protein. The stimulation of 4E-BP1 phosphorylation was maximal at 15 min and persisted up to 120 min. Results from affinity chromatography on m7GTP-agarose demonstrated that AII-induced phosphorylation of 4E-BP1 promotes its dissociation from eIF4E in target cells. Further characterization of 4E-BP1 phosphorylation by phosphoamino acid analysis and phosphopeptide mapping revealed that 4E-BP1 is phosphorylated on eight distinct peptides containing serine and threonine residues in AII-treated cells. The combination of results obtained from kinetics experiments, phosphopeptide analysis of in vitro and in vivo phosphorylated 4E-BP1, and pharmacological studies with the MAP kinase kinase inhibitor PD 98059 provided strong evidence that the MAP kinases ERK1/ERK2 are not involved in the regulation of 4E-BP1 phosphorylation in aortic SMC. Together, our results demonstrate that AII treatment of vascular SMC leads to hyperphosphorylation of the translational regulator 4E-BP1 and to its dissociation from eIF4E by a MAP kinase-independent mechanism.     

Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA

A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5'-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the approximately 450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES.eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.     

The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae

Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5' cap-dependent pathway. It interacts with eIF4E (the mRNA 5' cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.     

Malignant transformation by overproduction of translation initiation factor eIF4G

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.     

A novel functional human eukaryotic translation initiation factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.     

mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control

The mu-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other mu-opioid agonists have been described. We report that mu-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the mu-opioid receptor cDNA, we show that the specific agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon mu-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the mu-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.     

Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F

Most eukaryotic mRNAs contain a 5'cap structure and a 3'poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3' end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C-terminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5' and the 3' ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.