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Two human eukaryotic initiation factor 4E (eIF4E) genes were isolated and characterized from placental and chromosome 4-specific genomic libraries. One of the genes (EIF4E1) contained six introns, but the other gene (EIF4E2) was intronless, flanked by Alu sequences and 14-base pair (bp) direct repeats, and terminated by a short poly(A) stretch, all characteristics of retrotransposons. Numerous additional intronless eIF4E pseudogenes were found, but unlike EIF4E2, all contained premature in-frame stop codons. The entire EIF4E1 gene spanned >50 kilobase pairs. The coding regions of these two genes differed in four nucleotide residues, resulting in two amino acid differences in the predicted proteins. The promoter of EIF4E1 has been characterized previously. The putative promoter of EIF4E2 contained no TATA box but did contain a transcription initiator region (Inr) and numerous other sequence motifs characteristic of regulated promoters. EIF4E2 contained only two of the three polyadenylation signals present in EIF4E1. Evidence for transcription of both genes was obtained from primer extension, S1 mapping, ribonuclease protection, and reverse transcriptase-polymerase chain reaction experiments. Transcription was found to initiate 19 bp upstream of the translational initiation codon in the case of EIF4E1 and 80 bp in the case of EIF4E2. The two genes were differentially expressed in four human cell lines, Wish, Chang, K562, and HeLa.

Eukaryotic protein synthesis initiation factor (eIF) 4 gamma, also known as p220, is a component of the protein complex eIF-4, which is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5'-terminal secondary structure and recruitment of mRNA to the ribosome. Peptide sequence data from rabbit reticulocyte eIF-4 gamma was used to synthesize oligonucleotide probes and polymerase chain reaction primers. These were used to screen lambda-cDNA libraries from rabbit and human brain, yielding a partial rabbit and a complete human cDNA sequence of 5.1 kilobases. Northern blot and primer extension analysis indicated that the cDNA sequence was complete. To confirm that the cDNA represented that of eIF-4 gamma, three peptides were synthesized based on cDNA sequences and used to produce anti-peptide antibodies. The antibodies specifically recognized intact eIF-4 gamma and its cleavage products following poliovirus infection. The eIF-4 gamma mRNA contains AUG codons at nucleotides 6, 67, 90, 165, and 369, but only the last is followed by a long open reading frame. The eIF-4 gamma polypeptide is 154 kDa (1396 amino acid residues) and contains sequence motifs of potential interest: a sequence (AGLGPR) that is similar to the substrate recognition sequence of protease 2A from rhinovirus serotype 14, five PEST regions with scores greater than 10, which are characteristic of rapidly degraded proteins, stretches of polyglutamic acid, and numerous potential phosphorylation sites.

We investigated the possibility that insulin could stimulate translation of ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that expresses large numbers of human insulin receptors (HIR 3.5 cells). Within 3 h after exposure to 70 nM insulin, ODC enzyme activity increased approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but not in normal fibroblasts. Pretreatment of cells with cycloheximide completely inhibited insulin-stimulated ODC expression; actinomycin D partially inhibited this effect. To determine the influence of the 5' untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC expression, plasmids were constructed which contained sequences from the 5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the coding region of the human growth hormone gene. These constructions were then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold change in growth hormone accumulation in the medium of cells transiently expressing plasmids containing the entire 5'UTR of ODC mRNA or just the 5'-most 115 bases, a G/C-rich conserved sequence predicted to form a stem-loop structure and shown previously to be responsible for constitutive inhibition of translation. There was a direct correlation between the extent of insulin stimulation and the predicted secondary structure of the added 5'UTR sequences. To determine whether this effect might be due to insulin activation of initiation factors responsible for melting mRNA secondary structure, we examined the effect of insulin on the phosphorylation states of two such factors, eucaryotic initiation factors eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both initiation factors; this stimulation was evident at 15 min and maximal by 60 min. These results suggest a potential general mechanism by which insulin could preferentially stimulate translation of mRNAs whose 5'UTRs exhibit significant secondary structure by activating initiation factors involved in melting such secondary structures.

A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Ribosomal protein L32 mRNA moved from messenger ribonucleoprotein particles into polysomes following serum activation of quiescent Swiss 3T3 cells. This redistribution of the mRNA into a translationally active state began by 1 h and was complete by 3 h after activation. In contrast, actin mRNA showed no translational control, being found predominantly in polysomes in both quiescent and activated cultures. The phosphorylation state of eukaryotic initiation factor (eIF) 4E, which binds mRNA caps, was examined in parallel. eIF-4E phosphorylation was elevated by 1 h following serum activation and reached a peak by 3-5 h. Treatment of resting cells with phorbol ester also simultaneously stimulated eIF-4E phosphorylation and the movement of L32 mRNA into polysomes. These results are consistent with a model in which mitogen-induced phosphorylation increases the pool of active eIF-4E molecules, which in turn cause the recruitment of translationally controlled mRNAs to actively synthesizing ribosomes.

Site-directed mutagenesis was used to replace the serine residue at the primary phosphorylation site of human eukaryotic initiation factor (eIF) 4E with an alanine residue. The mutated cDNA was transcribed in vitro, and the transcript was used to direct protein synthesis in a reticulocyte lysate system. The variant protein (eIF-4EAla) was retained on a 7-methylguanosine 5'-triphosphate (m7GTP)-Sepharose affinity column and was specifically eluted by m7GTP. Examination of eIF-4EAla by isoelectric focusing revealed two species which had the same pI values as the phosphorylated and nonphosphorylated forms of unaltered eIF-4E (here designated eIF-4ESer). However, conversion of unphosphorylated eIF-4EAla to the putative phosphorylated eIF-4EAla in the reticulocyte lysate system was slower than the corresponding conversion of eIF-4ESer. The possibility that the more acidic form of eIF-4EAla was due to NH2-terminal acetylation was ruled out by an experiment in which the acetyl-CoA pool of the reticulocyte lysate system was depleted with oxaloacetate and citrate synthase. The more acidic form of eIF-4EAla was, however, eliminated by treatment with calf intestine alkaline phosphatase, suggesting that it results from a second-site phosphorylation. When translation reaction mixtures were resolved on sucrose density gradients, the 35S-labeled eIF-4ESer was found on the 48 S initiation complex in the presence of guanylyl imidodiphosphate, as reported earlier (Hiremath, L.S., Hiremath, S.T., Rychlik, W., Joshi, S., Domier, L.L., and Rhoads, R.E. (1989) J. Biol. Chem. 264, 1132-1138). eIF-4EAla, by contrast, was not found on the 48 S complex, suggesting that phosphorylation of eIF-4E is necessary for it to carry out its role of transferring mRNA to the 48 S complex. Supporting this interpretation was the finding that eIF-4ESer isolated from 48 S initiation complexes consisted predominantly of the phosphorylated form.

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.

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.