Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 109 records · Page 6Linked to original sources

PubMed1997

X-ray studies of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP.

The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand, and consists of a curved, 8-stranded antiparallel beta-sheet, backed by three long alpha-helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. Additional protein-ligand interactions include salt bridges and hydrogen bonds, plus water-mediated hydrogen bonds. The observed mode of 5' m-RNA cap recognition is almost certainly conserved among all known eIF4Es.

Crystallography, X-Ray↗

PubMed1996

General RNA binding proteins render translation cap dependent.

Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start sites.

Animals↗

PubMed1996

Tissue distribution, genomic structure, and chromosome mapping of mouse and human eukaryotic initiation factor 4E-binding proteins 1 and 2.

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91. 5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans approximately 16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescence in situ hybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21-q22, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed1996

The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth.

Initiation in eukaryotes is the rate limiting step of translation. The binding of the mRNA to the 40S ribosomal subunit, which is mediated by the mRNA cap structure, is a key target for control of protein synthesis. The cap binding protein, eIF4E, is the most limiting of all initiation factors and its overexpression in NIH3T3 cells causes malignant transformation. 4E-binding protein 1 (BP1) and 4E-BP2 are small proteins that bind to eIF4E and inhibit translation. Here, 4E-BPs were expressed in cells transformed by eIF4E or by v-src to determine the effect of 4E-BPs on cell growth and tumorigenicity. We show that 4E-BPs cause a significant reversion of the transformed phenotype. Thus, we demonstrate that the eIF4E-binding proteins act as negative regulators of cell growth. We propose that 4E-BPs are members of a class of negative regulators of cell growth acting on the translation machinery of the cell.

3T3 Cells↗

PubMed1996

Double-stranded RNA-dependent protein kinase mediates c-Myc suppression induced by type I interferons.

The antiproliferative functions of interferons result from specific effects that these cytokines exert on several cell cycle-controlling genes. The possible coupling between the interferon-responsive genes that are directly transactivated by the interferon signaling and the genes that constitute the basic machinery of the cell cycle is not clear yet. We report in this work that interferon-induced double-stranded RNA-activated kinase (PKR) is one of the specific mediators of the antiproliferative effects of the cytokine. Transfections of M1 myeloid leukemia cells with two catalytically inactive mutant forms of PKR abrogated the ability of interferon to suppress c-Myc without interfering with the pRB/cyclin D responses. As a consequence, these genetically manipulated cells displayed a small but significant reduction in their growth sensitivity to interferons, a phenotype that characterizes a single pathway disruption. Transfection of the parental M1 cells with the functional wild-type human PKR restricted their proliferation in the absence of interferons. This PKR-mediated growth inhibition could be efficiently rescued by the ectopic expression of deregulated c-myc. Taken together these results prove the existence of direct or indirect links between PKR and c-Myc suppression, thereby placing this gene along one of the complementary growth suppressive pathways that are triggered by interferons.

Animals↗

PubMed1996

The kinase insert domain of interferon-induced protein kinase PKR is required for activity but not for interaction with the pseudosubstrate K3L.

Interferon-induced protein kinase (PKR) is a member of a family of kinases that regulate translation initiation through phosphorylation of eukaryotic initiation factor 2alpha. In addition to the conserved catalytic subdomains that are present in all serine/threonine kinases, the eukaryotic initiation factor 2alpha kinases possess an insert region between catalytic subdomains IV and V that has been termed the kinase insert domain. To investigate the importance of the kinase insert domain of PKR, several deletions and point mutations were introduced within this domain and analyzed for kinase activity both in vitro and in vivo. Here we show that deletion of the kinase insert sequence or mutation of serine 355, which lies within this region, abrogates kinase activity. In addition, the kinase insert domain of PKR and adjacent amino acids (LFIQME) in catalytic subdomain V are not required for binding of the pseudosubstrate inhibitor K3L from vaccinia virus. A portion of the catalytic domain of PKR between amino acids 366 and 415 confers K3L binding in vivo, suggesting a possible role for this region of PKR in substrate interaction.

Amino Acid Sequence↗

PubMed1996

Expression of a translationally regulated, dominant-negative CCAAT/enhancer-binding protein beta isoform and up-regulation of the eukaryotic translation initiation factor 2alpha are correlated with neoplastic transformation of mammary epithelial cells.

A translationally regulated, dominant-negative isoform of CCAAT/enhancer-binding protein beta is expressed in transplantable and primary mouse mammary tumors of different etiologies but is not expressed in preneoplastic mammary hyperplasias or in primary prostate, lung, lens, ovary or lymphoid tumors. The eukaryotic initiation factor 2alpha protein is also expressed at significantly higher levels (69.8 +/- 7.2%) in these mammary tumors compared with normal and hyperplastic tissues. Thus, misregulation of eukaryotic initiation factor 2alpha may promote the expression of a dominant-negative CCAAT/enhancer-binding protein beta isoform, which may inhibit terminal differentiation and facilitate uncontrolled proliferation of mammary epithelial cells.

Animals↗

PubMed1996

Alternatively spliced transcripts from the Drosophila eIF4E gene produce two different Cap-binding proteins.

Eukaryotic initiation factor 4E (eIF4E) is the subunit of eIF4F that binds to the cap structure at the 5' end of messenger RNA and is a critical component for the regulation of translation initiation. Using 7-methyl-GTP-Sepharose affinity chromatography, two distinct cap-binding proteins that migrate on SDS-polyacrylamide gel electrophoresis at approximately 35 kDa were purified from Drosophila adults. Peptide microsequence analysis indicated that these two proteins differ at their amino termini. Analysis of a set of cDNA clones encoding eIF4E led to the conclusion that the two different protein isoforms, which we term eIF4EI and eIF4EII, result from three alternatively spliced transcripts from a single eIF4E gene, which maps to region 67A8-B2 on polytene chromosomes. The three eIF4E transcripts also vary greatly in the lengths of their 5'-UTRs, suggesting the possibility of complex translational control of expression of the two eIF4E isoforms.

Alternative Splicing↗

PubMed1996

Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus.

Infection of cells with picornaviruses, such as poliovirus and encephalomyocarditis virus (EMCV), causes a shutoff of host protein synthesis. The molecular mechanism of the shutoff has been partly elucidated for poliovirus but not for EMCV. Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure to which the multisubunit translation initiation factor eIF4F binds to promote ribosome binding. Picornaviruses use a mechanism for the translation of their RNA that is independent of the cap structure. Poliovirus infection engenders the cleavage of the eIF4G (formerly p220) component of eIF4F and renders this complex inactive for cap-dependent translation. In contrast, EMCV infection does not result in eIF4G cleavage. Here, we report that both EMCV and poliovirus activate a translational repressor, 4E-BP1, that inhibits cap-dependent translation by binding to the cap-binding subunit eIF4E. Binding of eIF4E occurs only to the underphosphorylated form of 4E-BP1, and this interaction is highly regulated in cells. We show that 4E-BP1 becomes dephosphorylated upon infection with both EMCV and poliovirus. Dephosphorylation of 4E-BP1 temporally coincides with the shutoff of protein synthesis by EMCV but lags behind the shutoff and eIF4G cleavage in poliovirus-infected cells. Dephosphorylation of 4E-BP1 by specifically inhibiting cap-dependent translation may be the major cause of the shutoff phenomenon in EMCV-infected cells.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins.

Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser209) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.

3T3 Cells↗

PubMed1996

4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12-tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase.

3T3 Cells↗

PubMed1996

Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated leukemias.

The transcription factor IRF-1 (interferon regulatory factor 1) is an activator of type I interferon and interferon-inducible genes. IRF-1 manifests tumor suppressor activity. Its overexpression results in inhibition of cell growth, and deletions of the IRF-1 gene were demonstrated in a number of human leukemias and myelodysplasias. Although the mechanism by which IRF-1 affects cell growth is unknown, it is believed that IRF-1 activates a set of genes that negatively regulate cell growth. The double-stranded RNA-dependent protein kinase (PKR), which is an interferon-inducible gene, contains a promoter element for the binding of IRF-1 and exhibits antiproliferative properties. Consequently, we investigated the role of IRF-1 in PKR expression. Here, we show that in IRF-1-deficient embryonic fibroblasts, PKR expression is reduced relative to wild-type cells. This result predicts diminished expression of PKR as a potential consequence of deletion of the IRF-1 gene in human leukemias. We show that cells of the human leukemic U937 cell line contain a deletion of one IRF-1 gene and express low levels of PKR. We demonstrate that upregulation of IRF-1 expression in U937 cells by transfection is sufficient to induce PKR expression. We also found a marked reduction in the expression of PKR in blood samples from two patients with myelodysplasias, carrying a deletion of chromosome 5q, a locus to which IRF-1 was mapped. These results show that IRF-1 activates PKR expression and suggest that loss of one allele of the IRF-1 gene is sufficient to affect PKR expression. Therefore, PKR is a strong candidate for a mediator of the tumor suppressor activity of IRF-1.

Chromosomes, Human, Pair 5↗

PubMed1996

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

3T3 Cells↗

PubMed1996

Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at the G1 phase in mammalian cells and yeast. Here we show that rapamycin inhibits cap-dependent, but not cap-independent, translation in NIH 3T3 cells. Cap-dependent translation is also specifically reduced in extracts from rapamycin-treated cells, as determined by in vitro translation experiments. This inhibition is causally related to the dephosphorylation and consequent activation of 4E-BP1, a protein recently identified as a repressor of the cap-binding protein, eIF-4E, function. These effects of rapamycin are specific as FK506, a structural analogue of rapamycin, had no effect on either cap-dependent translation or 4E-BP1 phosphorylation. The rapamycin-FK506 binding protein complex is the effector of the inhibition of 4E-BP1 phosphorylation as excess of FK506 over rapamycin reversed the rapamycin-mediated inhibition of 4E-BP1 phosphorylation. Thus, inactivation of eIF-4E is, at least in part, responsible for inhibition of cap-dependent translation in rapamycin-treated cells. Furthermore, these results suggest that 4E-BP1 phosphorylation is mediated by the FRAP/TOR signalling pathway.

3T3 Cells↗

PubMed1996

The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase.

The 2A proteinases (2Apro) of certain picornaviruses induce the cleavage of the eIF4G subunit of the cap-binding protein complex, eIF4F. Several reports have demonstrated that 2Apro of rhinovirus and coxsackievirus B4 cleave eIF4G directly. However, it was suggested that in poliovirus infection, the 2Apro induces the activation of a cellular proteinase which in turn cleaves eIF4G. Furthermore, it is not clear whether eIF4G is cleaved as part of the eIF4F complex or as an individual polypeptide. To address these issues, recombinant eIF4G was purified from Sf9 insect cells and tested for cleavage by purified rhinovirus 2Apro. Here we report that eIF4G alone is a relatively poor substrate for cleavage by the rhinovirus 2Apro. However, an eIF4G-eIF4E complex is cleaved efficiently by the 2Apro, suggesting that eIF4F is a preferred substrate for cleavage by rhinovirus 2Apro. Furthermore, 2Apr drastically reduced the translation of a capped mRNA. An eIF4G-eIF4E complex, but not eIF4G alone, was required to restore translation.

Animals↗

PubMed1996

Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.

3T3 Cells↗

PubMed1996

Transdominant mutants of I kappa B alpha block Tat-tumor necrosis factor synergistic activation of human immunodeficiency virus type 1 gene expression and virus multiplication.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites for the NF-kappa B/Rel family of transcription factors which are required for the transcriptional activation of viral genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1. In the present study, we examined the effect of transdominant mutants of I kappa B alpha on the synergistic activation of the HIV-1 LTR by TNF-alpha and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I kappa B alpha inhibited Tat-TNF-alpha activation of HIV LTR in a dose-dependent manner. Transdominant forms of I kappa B alpha, mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or constitutive (S283A, T291A, T299A) phosphorylation of I kappa B alpha were tested for their capacity to block HIV-1 LTR transactivation. I kappa B alpha molecules mutated in the N-terminal sites were not degraded following inducer-mediated stimulation (t1/2, > 4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I kappa B alpha (S32A, S36A) transdominant mutant was at least five times as effective as wild-type I kappa B alpha in inhibiting synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1 gene expression by interfering with the NF-kappa B/Rel signaling pathway.

3T3 Cells↗

PubMed1996

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

Animals↗