Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20

The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast. This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15. In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids.     

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.     

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.     

The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.     

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

A 24 000-dalton protein [yeast eukaryotic initiation factor 4E (eIF-4E)] was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography. The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures. Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.     

Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C

Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.     

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.     

Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G.

Although the cap structure and the poly(A) tail are on opposite ends of the mRNA molecule, previous work has suggested that they interact to enhance translation and inhibit mRNA degradation. Here we present biochemical data that show that the proteins bound to the mRNA cap (eIF-4F) and poly(A) tail (Pab1p) are physically associated in extracts from the yeast Saccharomyces cerevisiae. Specifically, we find that Pab1p co-purifies and co-immunoprecipitates with the eIF-4G subunit of eIF-4F. The Pab1p binding site on the recombinant yeast eIF-4G protein Tif4632p was mapped to a 114-amino-acid region just proximal to its eIF-4E binding site. Pab1p only bound to this region when complexed to poly(A). These data support the model that the Pablp-poly(A) tail complex on mRNA can interact with the cap structure via eIF-4G.     

Variations in cap-binding complexes from uninfected and poliovirus-infected HeLa cells

Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.     

Identification and characterization of cap-binding proteins from yeast

Photochemical cross-linking of Saccharomyces cerevisiae ribosomal salt wash preparations to cap-labeled mRNA reveals, in addition to the previously characterized 24-kDa cap-binding protein (eIF-4E), the presence of two novel cap-binding proteins (CBPs) of apparent molecular masses of 96 and 150 kDa. Cross-linking of the 96-kDa CBP was found to occur spontaneously without UV light induction. Based on the ATP/Mg2+ requirements, the three CBPs can be subdivided into two classes: 1) ATP/Mg2+ independent (24- and 150 kDa) and 2) Mg2+ dependent (96 kDa). The co-purification of the 24- and 150-kDa CBPs through several different chromatographic steps is consistent with the existence of a yeast CBP complex, possibly analogous to mammalian eIF-4F.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.     

Maturation hormone induced an increase in the translational activity of starfish oocytes coincident with the phosphorylation of the mRNA cap binding protein,eIF-4E,and the activation of several kinases

The stimulation of translation in starfish oocytes by the maturation hormone, 1-methyladenine (1-MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF-4F, and the guanine nucleotide exchange factor for eIF-2, eIF-2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish, Pisaster orchraceus. Increased phosphorylation of eIF-4E, the cap binding subunit of the eIF-4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF-4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen-activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF-4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF-4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12-myristate 13-acetate, an activator of protein kinase C, for 30 min prior to the addition of 1-MA resulted in the inhibition of 1-MA-induced phosphorylation of eIF-4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF-4E, after very early events of maturation. Another possibility is that eIF-4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1-MA. In conclusion, our results suggest a role for the phosphorylation of eIF-4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth in mammalian cells. © 1993 Wiley-Liss, Inc.     

Ras transformation of cloned rat embryo fibroblasts results in increased rates of protein synthesis and phosphorylation of eukaryotic initiation factor 4E.

Eukaryotic initiation factor 4E (eIF-4E) is a 25-kDa phosphoprotein that binds to the 7-methylguanosine cap of mRNA and acts, along with other eIF-4 polypeptides, to unwind mRNA secondary structure at the 5' terminus. Recent studies have indicated that eIF-4E acts as a protooncogene, but only in its phosphorylated state. In order to determine the role of eIF-4E in oncogenesis, we examined its regulation and expression in cloned rat embryo fibroblasts transformed with the Harvey ras (Ha-ras) oncogene. The expression of Ha-ras increased the rate of protein synthesis but did not increase the levels of eIF-4E mRNA or protein. However, a dramatic increase (7-fold) in phosphate incorporation into eIF-4E was observed. The percentage of eIF-4E in the phosphorylated state was the same in transfected and control cells, indicating that both phosphorylation and dephosphorylation of eIF-4E were increased. Phosphopeptide mapping of eIF-4E from transformed cells indicated a single site of phosphorylation at Ser-53, which is the same as that identified previously in eIF-4E from reticulocytes and HeLa cells. These results indicate that p21ras is part of the signal transduction pathway leading to phosphorylation of eIF-4E. These findings also provide a potential mechanism for cell transformation by p21ras which involves the preferential stimulation of translation of certain mRNAs.     

Translation initiation factors induce DNA synthesis and transform NIH 3T3 cells

Several polypeptide factors that are essential for the initiation of protein synthesis bind to eukaryotic mRNAs and facilitate the formation of ribosome initiation complexes. Purified mRNA-binding translation initiation factors were microinjected into quiescent NIH 3T3 cells to study the possible growth-promoting role of these factors in living cells. We report that recombinant eIF-4E and rabbit reticulocyte eIF-4F induce a dose-dependent increase of DNA synthesis and morphologically transform NIH 3T3 cells. These results suggest that polypeptides involved in activating the rate-limiting step of protein synthesis (initiation complex formation) can be mitogenic and oncogenic when overexpressed in a cell by direct injection. Thus, eIF-4E and eIF-4F represent a class of proto-oncogenic proteins that is cytoplasmic, is involved in protein synthesis initiation, and is distinct from the proto-oncogenes that have been identified previously.     

Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.     

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.