Protein Synthesis Initiation Factor Research Articles

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF‐2B

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.

Angiotensin II induces phosphorylation of eukaryotic protein synthesis initiation factor 4E in vascular smooth muscle cells.

Angiotensin II has been shown to induce hypertrophy of cultured vascular smooth muscle cells (VSMC). To understand the mechanisms of induction of the hypertrophy, we studied its effect on the phosphorylation state of eIF-4E, a rate-limiting eukaryotic protein synthesis initiation factor whose activity has been shown to be regulated by phosphorylation. Angiotensin II induced a 2-3-fold increase in the phosphorylation of eIF-4E in VSMC. The stimulation of phosphorylation was apparent at 20 min and persisted for at least 12 h. Phosphoamino acid analysis revealed that serine is the major residue of eIF-4E phosphorylated by angiotensin II. Staurosporine and calphostin C, two potent inhibitors of the serine/threonine protein kinase, protein kinase C, significantly attenuated the angiotensin II-induced eIF-4E phosphorylation. Staurosporine and calphostin C also blunted the angiotensin II-stimulated protein synthesis. Together, these observations indicate that angiotensin II induces phosphorylation of eIF-4E in a protein kinase C-dependent manner and suggest that this pathway may play an important role in the mechanism by which angiotensin II causes hypertrophy of VSMC.

Mechanism of differential regulation of IL-2 in murine Th1 and Th2 T cell subsets. 1. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E.

Abstract Regulation of IL-2 gene expression in response to receptor-mediated stimuli is known to be mediated primarily by the IL-2 transcriptional enhancer and multiple transcription factors. However, the mechanism that controls the differential expression of the IL-2 gene in both human and murine CD4+ Th cell subsets (Th1-IL-2+ and Th2-IL-2-) is not clearly understood. Differential IL-2 gene expression was assessed in murine Th1 and Th2 subsets by analyzing the expression of a Escherichia coli lacZ reporter gene under control of the human IL-2 enhancer (IL2ZH) transfected in both T cell subsets. Stimulation of transfected T cells with the mitogen Con A, anti-CD3 Ab, or PMA plus ionomycin activated the IL2ZH construct in Th1 but not Th2 cells. However, IL2ZH was activated in stimulated Th2 cells that were co-transfected with a vector that overexpressed the eukaryotic initiation factor 4E (eIF-4E). It has been shown that eIF-4E is rate limiting for protein synthesis and its overexpression leads to increased rates of protein synthesis. Hence, eIF-4E overexpression could have overcome a deficiency in transcriptionally active levels of IL-2 regulatory factors in Th2 cells leading to IL-2 enhancer activation. This possibility was supported by demonstrating that transcriptionally active levels of the critical IL-2 transcription factor, nuclear factor of activated T cells (NF-AT), occurred only in Th2 cells overexpressing eIF-4E but not in normal Th2 cells, thus indicating that the inability of Th2 cells to express IL-2 was associated with inadequate levels of at least one transcription factor, NF-AT. Moreover, these results were confirmed by the observation that eIF-4E overexpression augmented NF-AT binding activity in Th2 cells. These data suggest that concentrations of inducible transcription factors are a major component of the regulatory mechanisms dictating IL-2 expression and may be under translational control in Th1/Th2 T cell subsets.

In vitro synthesis of human protein synthesis initiation factor 4 gamma and its localization on 43 and 48 S initiation complexes.

The rate of protein synthesis is controlled in a large number of physiological situations at the stage of 48 S initiation complex formation, a phase that involves the recruitment of mRNA to the 40 S ribosomal subunit. This process is mediated by the eukaryotic initiation factor-4 (eIF-4) group of translation initiation factors consisting of eIF-4E, eIF-4A, eIF-4B, and eIF-4 gamma. In order to develop a new tool to study this process, we have produced radiolabeled eIF-4 gamma by in vitro transcription and translation. Despite the fact that eIF-4 gamma is predicted from the cDNA sequence to be 154 kDa, the major synthetic product migrated on SDS-polyacrylamide gel electrophoresis at 205 kDa. Although this is similar to the migration of the fastest polypeptide of authentic eIF-4 gamma (approximately 206 kDa), no products were found to co-migrate with the slowest forms of authentic eIF-4 gamma (210-220 kDa), suggesting that these forms derive from extensive modification of the initial polypeptide. The in vitro product also formed a complex with eIF-4E, as judged by its ability to bind to m7GTP-Sepharose. Sucrose gradient sedimentation studies demonstrated that eIF-4 gamma was present on both 43 and 48 S initiation complexes but not 80 S complexes. This supports a model in which free eIF-4E binds to mRNA followed by binding of the eIF-4E.mRNA complex to a 43 S initiation complex already containing eIF-4 gamma.

Is hypusine essential for eukaryotic cell proliferation?

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)-L-lysine] is a most remarkable amino acid, occurring in all eukaryotic cells, yet occupying only a single position in one protein, eukaryotic protein synthesis initiation factor 5A (eIF-5A). The unusual structure of hypusine, its derivation from the polyamine spermidine, and its increased formation in response to growth stimulation, as well as its limited occurrence in the highly conserved amino acid sequence of eIF-5A, have aroused keen interest in the biological significance of its existence and in its relationship to eIF-5A function.

Decreasing the level of translation initiation factor 4E with antisense RNA causes reversal of ras-mediated transformation and tumorigenesis of cloned rat embryo fibroblasts.

Transformation of cloned rat embryo fibroblasts (CREF) with the T24-ras oncogene results in loss of contact inhibition, growth in soft agar and tumor formation in nude mice. Previously we showed that in such cells (CREF T24), the phosphorylation rate of protein synthesis initiation factor 4E (eIF-4E) is increased, correlating with an increase in the general rate of protein synthesis. In the present study, we have expressed antisense RNA complementary to eIF-4E mRNA in CREF T24 cells using a stably integrated vector. Cells expressing antisense RNA (CREF T24/AS) contained 30-50% of the normal level of eIF-4E and exhibited many of the properties of untransformed cells. CREF T24 had a spindle-shaped, refractile appearance, whereas CREF T24/AS grew in ordered, parallel patterns and exhibited contact inhibition similar to untransformed CREF. The rates of growth and protein synthesis in CREF T24/AS were decreased compared to CREF T24 but were not as low as in CREF. The efficiency of growth in soft agar was 11-fold lower for CREF T24/AS compared with CREF T24. The latency period for tumor formation in nude mice was increased from 8 days for CREF T24 to 17-27 days for CREF T24/AS and various clonal lines derived from them. Cell lines established from these CREF T24/AS-derived tumors were shown to have partially regained the eIF-4E levels characteristic of CREF T24. These results demonstrate that many of the phenotypic alterations associated with ras-induced malignant transformation can be reversed by a moderate reduction of the translational initiation capacity and therefore may be mediated through a translational mechanism.

Characterization of wheat germ protein synthesis initiation factor eIF-4C and comparison of eIF-4C from wheat germ and rabbit reticulocytes.

Eukaryotic protein synthesis initiation factor (eIF)-4C was purified from wheat germ and the molecular weight was calculated to be approximately 19,000 by SDS-polyacrylamide gel electrophoresis. A similar molecular weight was determined by gel filtration chromatography indicating that wheat germ eIF-4C is functional as a single polypeptide chain. An efficient in vitro translation system dependent upon the addition of eIF-4C was developed. This system was used to determine the concentrations of eIF-4C required for the half-maximal rate of translation of satellite tobacco necrosis virus RNA, alfalfa mosaic virus RNA 4, and barley alpha-amylase mRNA. No significant differences in the concentrations of eIF-4C required for the translation of these mRNAs were observed, although differences were noted for eIF-4A and eIF-4F. This finding suggests that eIF-4C is not involved in the binding of mRNA to 40 S ribosomal subunits. In heterologous assays, rabbit reticulocyte eIF-4C was as active as wheat germ eIF-4C in the wheat germ eIF-4C-dependent system. In addition, wheat germ eIF-4C substituted for rabbit reticulocyte eIF-4C in in vitro assay systems from rabbit reticulocytes. These results indicate that eIF-4C from wheat and rabbit contain conserved functional domains.

Heat-shocked neuronal PC12 cells reveal Alzheimer's disease--associated alterations in amyloid precursor protein and tau.

The Alzheimer's disease (AD) brain contains many abnormal protein modifications. These include the abnormal processing of amyloid precursor protein (APP) to form the amyloidogenic beta/A4 peptide and the abnormal phosphorylation of tau to form A68, the major constituent of the neurofibrillary tangle. In addition, many of the biochemical alterations found in the AD brain are also found in heat-shocked or stressed cells. We used heat-shocked neuronal PC12 cells to investigate the effects of stress on APP and tau. We found that by simply exposing neuronal PC12 cells to an elevated temperature (45 degrees C) for 30 minutes, they exhibited several features characteristic of the heat shock response. These included a 45% reduction in total protein synthesis, the induction of heat shock protein (hsp) 72, and increased phosphorylation of the protein synthesis initiation factor eIF-2 alpha. The heat-shocked cells also exhibited alterations in the metabolism and phosphorylation of APP. Under heat shock conditions, we found two additional APP-like polypeptides not present in controls and a significant decrease in the phosphorylation state of APP. We also found that an A68-like protein is formed in neuronal PC12 cells when subjected to elevated temperature. This A68-like protein was formed with heat shock even in the absence of protein synthesis, suggesting that its production occurred post-translationally. The tau/A68 polypeptides were identified as phosphoproteins, and the phosphorylation of tau to form A68 was reversed with recovery of the cells from heat shock. Immunoprecipitation of lysates from heat shocked cells with antibodies to hsp72/73 resulted in co-precipitation of tau, but not A68 with hsp72 indicating a stable complex formation between these two proteins. These results suggest that heat shock proteins may play either a protective or promoting role in the formation of A68 and/or the amyloidogenic C-terminal fragment of APP.

Characterization of the ATP-dependent binding of wheat germ protein synthesis initiation factors eIF-(iso)4F and eIF-4A to mRNA.

The ATP-dependent binding of wheat germ protein synthesis initiation factors eIF-(iso)4F and eIF-4A to an oligoribonucleotide has been investigated by direct fluorescence titration techniques. In addition, the effect of ATP on the interaction between another cap-binding initiation factor, eIF-4F, and eIF-4A was studied using the same methods. Comparison of the equilibrium association constants (K(eq)) indicate that 1) hydrolyzable ATP affects the affinity of eIF-(iso)4F for eIF-4A, regardless of whether or not mRNA was previously bound to the eIF-(iso)4F; in contrast, ATP had no effect on the eIF-(iso)4F/oligoribonucleotide interaction; 2) in the presence of ATP, the binding of the binary eIF-(iso)4F.eIF-4A complex to the oligoribonucleotide is of similar affinity as the binding of the oligoribonucleotide to the eIF-(iso)4F alone; the stoichiometry of this ternary eIF-(iso)4F.eIF-4A.mRNA complex was found to be 1:1:1; and 3) a similar ATP effect is observed for the eIF-4F/eIF-4A interaction as for the eIF-(iso)4F.eIF-4A complex.

Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A

A cDNA encoding wheat translation initiation factor 4A (eIF-4A) was isolated from a wheat cDNA library and sequenced. The deduced wheat eIF-4A amino acid sequence from the cDNA is compared to eIF-4A from Arabidopsis thaliana, tobacco, mouse and Saccharomyces cerevisiae. Putative RNA helicase motifs, and putative ATP-binding and hydrolysis sites are identified.

Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus.

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4 gamma in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH2- and COOH-terminal fragments of eIF-4 gamma were separated by reverse phase HPLC and identified with specific antibodies, and the NH2-terminal sequence of the COOH-terminal fragment was determined by automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR decreases GPPRGGPG494 in rabbit eIF-4 gamma, corresponding to 478GRTTLSTR decreases GPPRGGPG493 in human eIF-4 gamma.

Use of Nonreducing SDS-PAGE for Monitoring Renaturation of Recombinant Protein Synthesis Initiation Factor, eIF-4α

The purification of biologically active human protein synthesis initiation factor 4α, eIF-4α, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines. Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond. A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations. The production of biologically active eIF-4α only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea. This requires a minimum dithiothreitol concentration of 50 mM. Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents. Once renatured, as described, eIF-4α can be purified by affinity chromatography on m7GTP-Sepharose. Approximately 20 μg of biologically active eIF-4α per milliliter of bacterial culture can be obtained. The affinity-purified eIF-4α has activity equivalent to that reported for purified native human eIF-4α, as measured by its activity in a rabbit reticulocyte translation system. The method described is applicable to the purification of other cysteine-contaming polypeptides that accumulate to high levels in inclusion bodies.

Interferon-induced dsRNA-activated protein kinase (PKR)*: antiproliferative, antiviral and antitumoral functions

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a serine/threonine kinase induced in mammalian cells by interferon. It is responsible for the phosphorylation of the protein synthesis initiation factor eIF-2, thus mediating inhibition of protein synthesis. Studies on the expression of the human protein kinase in the yeast Saccharomyces cerevisae and in murine NIH 3T3 cells, provided evidence for the antiproliferative, antiviral and antitumoral functions of this dsRNA activated protein kinase.

The role of the β-subunit of initiation factor eIF-2 in initiation complex formation

The functional properties of preparations of protein synthesis initiation factor eIF-2 which lack the β-subunit (as confirmed immunologically) were compared with those of the heterotrimeric factor. The former can bind guanine nucleotides but not initiator tRNA, and also exhibits a substantially reduced rate of initiation factor eIF-2B-mediated GDP/GTP-exchange.

Altered expression and phosphorylation of amyloid precursor protein in heat shocked neuronal PC12 cells

The pathology of the Alzheimer's disease (AD) brain, including amyloid plaques, neurofibrillary tangles and neuronal degeneration, indicates that neurons affected by AD exist under conditions of stress. In fact, the brains of AD patients undergo many changes classically associated with the heat shock response, which is one form of a stress response. These changes include reduced protein synthesis, disrupted cytoskeleton, increased number of proteins associated with ubiquitin, and the induction of heat shock proteins. To investigate the response of neurons to stress, we examined neuronal PC12 cells incubated at either 37°C (control cells) or 45°C (heat-shocked cells). After a 30 min exposure at 45°C, the heat shocked cells exhibited several features characteristic of the classical heat shock response including a 45% reduction in total protein synthesis, the induction of heat shock protein 72, and an increased phosphorylation of the protein synthesis initiation factor eIF-2α. We used this cellular model system to study the neuronal response to stress specifically focusing on protein synthesis elongation factor 2 (EF-2) and the Alzheimer's amyloid precursor protein (APP), the precursor form of β-amyloid peptide. Hyperphosphorylation of EF-2 has been observed in the neocortex and hippocampus of AD brain 18. However, in our system, we find no hyperphosphorylation of EF-2 in response to heat shock. Heat-shocked neuronal PC12 cells exhibited two additional APP-like polypeptides not present in controls. We also found a significant decrease in the phosphorylation state of APP in response to heat shock. Protein kinase C phosphorylated APP in the neuronal PC12 cells, as evidenced by the response to PMA, a phorbol ester which stimulated the phosphorylation of APP and staurosporine which reduced its phosphorylation. Therefore, heat-shocked neuronal PC12 cells undergo several biochemical alterations which are also associated with the AD brain. We have used heat-shocked neuronal PC12 cells as a cell culture model system to investigate changes in several proteins (EF-2, APP, and tau) known to be altered in the AD brain.

Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase

Deletion of the vaccinia virus K3L gene, a homologue of the alpha subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E. (1991) Virology 183, 419-422). Purified recombinant K3L gene product, pK3r, has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2 alpha (eIF-2 alpha)-specific protein kinase (PKR) in in vitro reactions. Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2 alpha. In the cell-free translation system, pK3r exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2 alpha. In addition, pK3r reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the alpha subunit of purified eIF-2. At 400 mM NaCl, in vitro translated [35S]methionine-radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR. This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2 alpha as the domain recognized by the eIF-2 alpha-specific protein kinases. In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3. Recombinant pK3 also prevents activation of the heme-sensitive eIF-2 alpha-specific protein kinase, eIF-2 alpha-PKh, in both cell-free translation systems as well as in partially purified preparations. This suggests some similarity between the eIF-2 alpha binding domains of the two eIF-2 alpha specific protein kinases.

Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase.

Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2. We have analyzed the transient expression of mutant and wild-type kinase molecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene products. The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40-fold higher levels. Protein stability measurements and primer-extension analysis of human kinase-specific mRNA levels provided evidence that kinase expression was regulated at the level of mRNA translation. Further, cotransfection experiments revealed that the domain II catalytically inactive mutant could stimulate reporter gene protein synthesis in a transdominant manner. We also examined the expression of mutants with deletions in the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expressed at levels 70-fold higher. Both the inactive domain II P68 mutant and the deletion mutant lacking aa 91-243 were less inhibitory to growth in yeast due to the reduced ability to phosphorylate initiation factor 2 alpha in vivo. In conclusion we have demonstrated that the P68 kinase can regulate mRNA translation primarily of its own mRNA and to a lesser extent of a heterologous mRNA and that this regulation is notably affected by mutations in either the catalytic or N-terminal regulatory domains.

The functional genes for protein synthesis initiation factor 4AI and 4AII map to mouse Chromosomes 11 and 16

The functional genes for protein synthesis initiation factor 4AI and 4AII map to mouse Chromosomes 11 and 16

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli Over-expression of infBB.su in E. coli and purification of α- and β-forms of IF2 B.su

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, α and β, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti- E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infB B.su. In this work we have cloned infB B.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2α and -β were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2α and -β, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2β is Met 94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infB B.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.

Amino acid sequence of the human protein synthesis initiation factor eIF-4 gamma

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.