Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.     

Phosphorylation of only serine-51 in protein synthesis initiation factor-2 is associated with inhibition of peptide-chain initiation in reticulocyte lysates

We have examined the phosphorylation of the alpha-subunit of initiation factor-2 (eIF-2 alpha) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2 alpha, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2 alpha in isoelectric focusing/immunoblotting analyses of lysates treated as described above.     

Zinc inhibits protein synthesis in neurons. Potential role of phosphorylation of translation initiation factor-2alpha.

In the central nervous system, Zn(2+) is concentrated in the cerebral cortex and hippocampus and has been found to be toxic to neurons. In this study, we show that exposure of cultured cortical neurons from mouse to increasing concentrations of Zn(2+) (10-300 microM) induces a progressive decrease in global protein synthesis. The potency of Zn(2+) was increased by about 2 orders of magnitude in the presence of Na(+)-pyrithione, a Zn(2+) ionophore. The basal rate of protein synthesis was restored 3 h after Zn(2+) removal. Zn(2+) induced a sustained increase in phosphorylation of the alpha subunit of the translation eukaryotic initiation factor-2 (eIF-2alpha), whereas it triggered a transient increase in phosphorylation of eukaryotic elongation factor-2 (eEF-2). Protein synthesis was still depressed 60 min after the onset of Zn(2+) exposure while the state of eEF-2 phosphorylation had already returned to its basal level. Moreover, Zn(2+) was less effective than glutamate to increase eEF-2 phosphorylation, whereas it induced a more profound inhibition of protein synthesis. These results suggest that Zn(2+)-induced inhibition of protein synthesis mainly correlates with the increase in eIF-2alpha phosphorylation. Supporting further that Zn(2+) acts at the initiation step of protein synthesis, it strongly decreased the amount of polyribosomes.     

Phosphorylation of maize eukaryotic translation initiation factor on Ser2 by catalytic subunit CK2

Alignment of eukaryotic translation initiation factor 5A (eIF5A) sequences has shown, for plants, in contrast to most other eukaryotes, the presence of N-terminal serine residue (Ser2) which could be phosphorylated by CK2. Using point directed mutagenesis, we demonstrate here that in recombinant maize ZmeIF5Awt Ser2 is exclusively phosphorylated by catalytic subunit of CK2 (CK2α), whereas its mutated variant Ser2Ala is not phosphorylated. To shed light on the physiological significance of this Ser2 phosphorylation, transient expression of fluorescence-labeled proteins was performed in maize protoplast. Wild-type ZmeIF5A was distributed evenly between nucleus and cytoplasm, but the replacement of Ser2 by aspartic acid, which mimics the phosphorylated serine, influences its intracellular localization. We postulate that phosphorylation of Ser2 in maize eIF5A, and most probably in other plant cells, plays a role in specific regulation of nuclear export of eIF5A-bound mRNAs.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) is a major mechanism regulating protein synthesis in rabbit reticulocytes. To determine whether phosphorylation of eIF-2 alpha is a likely regulatory mechanism in the Ehrlich cell, we have measured the percent of cellular eIF-2 alpha which is phosphorylated in cells exposed to heat shock, 2-deoxyglucose, or amino acid deprivation, conditions which rapidly decrease the concentration of 40 S initiation complexes and inhibit protein synthesis. eIF-2 alpha and eIf-2 alpha (P) were separated by isoelectric focusing and were detected by immunoblotting with a monoclonal antibody we developed for this purpose. Under the above three inhibitory conditions, phosphorylation of eIF-2 alpha increased rapidly, and this increase correlated in time with the rapid inhibition of protein synthesis. In heat-shocked cells which were returned to 37 degrees C, both phosphorylation and protein synthesis remained unchanged for 10 min and then returned toward control values slowly and in parallel. The close temporal correspondence between changes in protein synthesis and phosphorylation supports an important regulatory role for phosphorylation in protein synthesis. An increase of 25-35 percentage points, to 50-60% phosphorylation from control levels of 20-30% phosphorylation, correlated with an 80-100% inhibition of protein synthesis. This steep curve of inhibition is consistent with a mechanism in which eIF-2 alpha (P) saturates and inhibits the guanine-nucleotide exchange factor.     

Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates.

The data presented here show that serine-51 of the alpha-subunit of eukaryotic initiation factor eIF-2 is the only residue phosphorylated by the eIF-2 alpha-specific kinases HCR (haem-controlled repressor) and dsI (double-stranded RNA-activated inhibitor) in vitro. This confirms our earlier finding that serine-48 is not labelled by either kinase. Methodology appropriate for the examination of phosphorylation sites in eIF-2 alpha in whole cells and their extracts has been developed, and used to study the site(s) in eIF-2 alpha labelled in reticulocyte lysates. Only serine-51 became phosphorylated under conditions of haem-deficiency or in the presence of double-stranded RNA. No evidence for a second phosphorylation site on the alpha-subunit was obtained with the lysates and conditions used here.     

Alpha subunit of eukaryotic translational initiation factor-2 is a heat-shock protein

The use of ultra high resolution giant two-dimensional gel electrophoresis has expanded the number of recognizable heat-shock proteins to 68 inductions in rat thymic lymphocytes, many of which are among the less abundant cellular proteins (Maytin, E. V., Colbert, R. A., and Young, D. A. (1985) J. Biol. Chem. 260, 2384-2392). Previous studies also show that cells receiving a prior heat shock recover more rapidly from the inhibition of protein synthesis induced by a second heat shock. In this report we use a monoclonal antibody to identify the alpha subunit of eukaryotic initiation factor-2 (eIF-2 alpha) as a heat-shock protein. Its relative rate of synthesis increases approximately 40% in the 2nd h and 5-fold in the 4th h of a continuous heat shock and is stimulated more dramatically, 15-fold, in the 3rd h of recovery from a 1-h heat shock. These results suggest that the induction of eIF-2 alpha in the heat-shock response may be important for restoring the cell's ability to initiate protein synthesis. In addition to identifying a function for one of the heat-shock proteins, our findings draw attention to the likelihood that other low-abundance heat-shock proteins may play critical roles in the heat-shock response.     

ICP34.5 protein of herpes simplex virus facilitates the initiation of protein translation by bridging eukaryotic initiation factor 2alpha (eIF2alpha) and protein phosphatase 1

The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.     

Regulation of antioxidant metabolism by translation initiation factor 2alpha

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.     

Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.     

Protein synthesis inhibition by flavonoids: roles of eukaryotic initiation factor 2alpha kinases

Flavonoids such as genistein and quercetin suppress tumor cell growth in vitro and in vivo. Many metabolic enzymes, including protein kinases, are known to be inhibited by flavonoids, yet the molecular targets and biochemical mechanisms of the tumor growth suppression remain unclear. Here, we find that flavonoids inhibit protein synthesis in both mouse and human leukemia cells. This inhibition is associated with phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), a key regulatory mechanism of protein translation. Three mammalian eIF2alpha kinases have been identified: the interferon-inducible double-stranded RNA-dependent kinase (PKR), the heme-regulated inhibitor (HRI), and the very recently discovered PERK/PEK. We find that all of these eIF2alpha kinases can be activated by quercetin and genistein, indicating redundant roles of the eIF2alpha kinases. Thus, activation of eIF2alpha kinases appears to be a mechanism by which flavonoids can inhibit the growth of tumor and leukemia cells.     

Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral infection.     

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.     

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

Kinetic modelling of the effect of alpha subunit phosphorylation on the activity of the protein synthesis initiation factor eIF-2

The ability of eIF-2.GDP in which the alpha subunit of eIF-2 is phosphorylated (eIF-2(alpha P).GDP) to act as a competitive inhibitor of eIF-2B-catalysed exchange of eIF-2-bound GDP has been investigated by modelling data provided by Rowlands et al. (J. Biol. Chem. 263, 5526-5533:1988). Some revision of previously determined dissociation and rate constants proved to be necessary. Under the conditions employed it was not possible to demonstrate significant inhibition of GDP exchange by eIF-2 (alpha P).GDP without substantial increase in its affinity for eIF-2B over that of eIF-2.GDP. Classic double reciprocal plots for competitive inhibition were found only when [eIF-2B] was low in relation to [eIF-2 (alpha P).GDP]. Relatively high cellular [eIF-2B] lessens the inhibitory effect of eIF-2(alpha P).GDP and suggests the possibility of other potential controls of initiation.