Investigation of the eIF2alpha phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2alpha) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2alpha in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2alpha. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2alpha phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2alpha phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2alpha phosphorylation; futhermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eLF2alpha. Zinc (II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2alpha phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.     

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.     

Investigation of the eIF2α phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2α in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2α. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2α phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2α phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2α phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eIF2α. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2α phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.     

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.     

Phosphorylation of a reinitiation supporting protein,RISP, determines its function in translation reinitiation

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2β and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2β, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.     

Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3

The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3. Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6. We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes. FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits. An int6 deletion (int6Delta) mutant was viable but grew slowly in minimal medium. This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein. These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation. Interestingly, haploid int6Delta cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6Delta cells formed abnormal spores. We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S. pombe.     

Caliciviruses differ in their functional requirements for eIF4F components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.     

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.In eukaryotes, eukaryotic translation initiation factor 4G (eIF4G) plays a central role in the recruitment of ribosomes to the mRNA 5′ end and is therefore critical for the regulation of protein synthesis (14). Two homologues of eIF4G, eIF4GI and eIF4GII, have been cloned (15). Although they differ in various respects, both homologues clearly function in translation initiation. The most thoroughly studied of these is eIF4GI, which serves as a scaffolding protein for the assembly of eIF4F, a protein complex composed of eIF4E (the mRNA cap-binding factor) and eIF4A (an ATP-dependent RNA helicase). Thus, via its association with the mRNA cap-binding protein eIF4E and with another translation initiation factor (eIF3) which is bound to the 40S ribosomal subunit, eIF4GI creates a physical link between the mRNA cap structure and the ribosome, thus facilitating cap-dependent translation initiation (25). eIF4GI functions also in cap-independent, internal ribosome entry site (IRES)-mediated translation initiation. For instance, upon picornavirus infection, eIF4G is rapidly attacked by viral proteases. The resulting eIF4GI cleavage products serve to reprogram the cell''s translational machinery, as the N-terminal cleavage product inhibits cap-dependent translation of host cell mRNAs by sequestering eIF4E while the C-terminal cleavage product stimulates IRES-mediated translation of viral mRNAs (23). Similarly, apoptotic caspases cleave eIF4G into an N-terminal fragment that blocks cap-dependent translation and a C-terminal fragment that is utilized for IRES-mediated translation of mRNAs encoding proapoptotic proteins (22).The regulation of eIF4GI cleavage by viral proteases or apoptotic caspases has been extensively studied. Little is known, however, about the regulation of eIF4GI steady-state levels. Yet the eIF4GI amount that exists at a given moment results from the sum of the effects of de novo synthesis and ongoing degradation. Many cellular proteins are physiologically degraded by the proteasome. This has been shown to be true for eIF4GI, as the factor can be degraded by the proteasome in vitro (5) and in living cells (6). However, how eIF4GI targeting for or protection from destruction by the proteasome is regulated remains unknown.There are two major routes to degradation by the proteasome. In the more conventional route, polyubiquitinated proteins are targeted to the 26S proteasome. Alternatively, a few proteins can be degraded by the 20S proteasome (and sometimes by the 26S proteasome) in a ubiquitin-independent manner (16). Interestingly, it has been shown recently that a few of these proteins (1, 2, 13) can be protected from degradation by the 20S proteasome by binding to the NAD(P)H quinone-oxydoreductase 1 (NQO1). It has been proposed that NQO1 may interact with the 20S proteasome and may consequently block access of target proteins to the 20S degradation core. Because eIF4GI can be degraded in vitro by the 20S proteasome (5) and since it appears that proteasomes can degrade eIF4GI in living cells independently of ubiquitination (6), we asked whether NQO1 could protect eIF4GI from degradation by the proteasome.     

The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m⁷GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m⁷GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled.     

Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo

Translation initiation factor 1A (eIF1A) is predicted to bind in the decoding site of the 40S ribosome and has been implicated in recruitment of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and ribosomal scanning. We show that the unstructured C-terminus of eIF1A interacts with the C-terminus of eIF5B, a factor that stimulates 40S-60S subunit joining, and removal of this domain of eIF1A diminishes translation initiation in vivo. These findings support the idea that eIF1A-eIF5B association is instrumental in releasing eIF1A from the ribosome after subunit joining. A larger C-terminal truncation that removes a 3(10) helix in eIF1A deregulates GCN4 translation in a manner suppressed by overexpressing TC, implicating eIF1A in TC binding to 40S ribosomes in vivo. The unstructured N-terminus of eIF1A interacts with eIF2 and eIF3 and is required at low temperatures for a step following TC recruitment. We propose a modular organization for eIF1A wherein a core ribosome-binding domain is flanked by flexible segments that mediate interactions with other factors involved in recruitment of TC and release of eIF1A at subunit joining.     

Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)

Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e-subunit (p48) of mammalian initiation factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic initiation factor-3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full-length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation.     

Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits

The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.     

20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3

The molecular basis for coordinated regulation of protein synthesis and degradation is not understood. Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3. The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components. Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factors in vitro and in vivo, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. These studies demonstrate the importance of the endoproteolytic activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation.     

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate–bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway.     

Eucaryotic initiation factor 4B controls eIF3-mediated ribosomal entry of viral reinitiation factor

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.