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.     

The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells

Serum stimulation of cultured Xenopus kidney cells results in enhanced phosphorylation of the translational initiation factor (eIF) 4E and promotes a 2.8-fold increase in the binding of the adapter protein eIF4G to eIF4E, to form the functional initiation factor complex eIF4F. Here we demonstrate the serum-stimulated co-isolation of the poly(A)-binding protein (PABP) with the eIF4F complex. This apparent interaction of PABP with eIF4F suggests that a mechanism shown to be important in the control of translation in the yeast Saccharomyces cerevisiae also operates in vertebrate cells. We also present evidence that the signaling pathways modulating eIF4E phosphorylation and function in Xenopus kidney cells differ from those in several mammalian cell types studied previously. Experiments with the immunosuppressant rapamycin suggest that the mTOR signaling pathway is involved in serum-promoted eIF4E phosphorylation and association with eIF4G. Moreover, we could find little evidence for regulation of eIF4E function via interaction with the specific binding proteins 4E-BP1 or 4E-BP2 in these cells. Although rapamycin abrogated serum-enhanced rates of protein synthesis and the interaction of eIF4G with eIF4E, it did not prevent the increase in association of eIF4G with PABP. This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E.     

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

Clinical outcome in stage I to III breast carcinoma and eIF4E overexpression

OBJECTIVE: The objective of this study is to determine if high eukaryotic initiation factor 4E (eIF4E) overexpression (sevenfold elevation or more over benign breast tissue) is associated with a worse clinical outcome. SUMMARY BACKGROUND DATA: Dysregulation of cellular functions by selective overexpression of specific proteins can lead to malignant transformation. The overexpression of eIF4E preferentially increases translation of mRNAs with long, G-C rich 5'-untranslated regions. Selective gene products, such as tumor neoangiogenic factors, ornithine decarboxylase, and cyclin D1, are upregulated. METHODS: One hundred fourteen breast specimens were analyzed and eIF4E overexpression was quantified by Western blot analysis. Quantification for eIF4E protein level was accomplished using a rabbit anti-eIF4E antibody and colorimetric development of Western blots using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The blots were scanned and analyzed by densitometry. Treatment, pathologic, and clinical outcome data variables were analyzed. Statistical analysis was performed to determine if eIF4E overexpression is associated with breast cancer clinical outcome. RESULTS: In the 55 benign specimens, the mean eIF4E expression was 1.1+/-0.4 fold (mean +/- standard deviation). All 59 malignant breast carcinoma specimens were noted to have eIF4E overexpression (range, 1.9-fold to 30.6-fold), with a mean overexpression of 10.8+/-6.3-fold. The mean level of eIF4E expression in malignant specimens was higher than benign specimens (p < 0.05, unpaired t test). The degree of eIF4E overexpression appears to be independent of T and N stage. In the 21 patients with eIF4E overexpression of less than sevenfold, there was one cancer recurrence but no cancer-related deaths. In the 38 patients with high eIF4E overexpression (sevenfold or more), 14 patients had breast cancer recurrences (p = 0.03, log rank test), of whom 11 have died from the disease (p = 0.04, log rank test). The average follow-up interval in this study was 40 months. CONCLUSIONS: Patients with stage I to III breast cancer and high eIF4E overexpression had a higher rate of cancer recurrence and a higher rate of cancer-related death when compared to similar-stage breast cancer patients with low eIF4E overexpression. Therefore, eIF4E protein overexpression may be of prognostic value in stage I to III breast carcinoma.     

Overexpression of eukaryotic initiation factor 4E (eIF4E) in breast carcinoma

BACKGROUND: Eukaryotic initiation factor 4E (eIF-4E) is a 25-kilodalton phosphoprotein that binds specifically to mRNA as the initial step for mRNA translation. An elevated level of eIF4E has been associated with the up-regulation of various protooncogene products. Transfection of cell lines by viral vectors with eIF4E overexpression has resulted in malignant transformation. The objective in this study was twofold: to examine benign and malignant breast specimens for eIF4E expression, and to determine whether eIF4E overexpression may have prognostic potential. METHODS: Western blot analysis was performed on benign and malignant breast specimens using anti-eIF4E rabbit antiserum. Quantification was accomplished by developing blots with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate and densitometry. Confirmation of eIF4E overexpression at the cellular level was performed using immunohistologic staining in situ. RESULTS: The authors examined 112 breast specimens for eIF4E protein expression. Of the 52 benign breast specimens examined, none showed eIF4E overexpression. All 12 ductal carcinoma in situ specimens were found to overexpress eIF4E in the intermediate range (mean elevation: 2.5-fold). Of the 48 breast carcinoma specimens examined, all had eIF4E elevation at levels of 3-30-fold (mean: 10.5 +/- 0.9-fold). Charts from 39 patients with Stage I, II, and III breast carcinoma were reviewed. In ten patients with eIF4E overexpression of < sevenfold, there was no recurrence or death from breast carcinoma. In the 29 breast carcinoma patients with > or = 7-fold eIF4E overexpression, 9 patients had breast carcinoma recurrences and 5 had died from disease at last follow-up. The median follow-up in this study was 34.5 months. CONCLUSIONS: Overexpression of eIF4E was observed in malignant breast specimens but not in normal or benign breast tissues. In patients with breast carcinoma, the group with high eIF4E overexpression (> or = 7-fold) experienced a worse clinical outcome (higher recurrences and death) compared with the group with low eIF4E overexpression (< 7-fold).     

Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation

In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the messenger RNA results from recognition of the 5' cap structure (m7GpppX) of the mRNA by the cap-binding complex eIF4F. eIF4F is itself a three-subunit complex comprising the cap-binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF4G, which interacts with both eIF4A and eIF4E and enhances cap binding by eIF4E. The mRNA 3' polyadenylate tail and the associated poly(A)-binding protein (PABP) also regulate translational initiation, probably by interacting with the 5' end of the mRNA. In yeast and plants, PABP interacts with eIF4G but no such interaction has been reported in mammalian cells. Here, we describe a new human PABP-interacting protein, PAIP-I, whose sequence is similar to the central portion of eIF4G and which interacts with eIF4A. Overexpression of PAIP-1 in COS-7 cells stimulates translation, perhaps by providing a physical link between the mRNA termini.     

Transcription factor E2F controls the reversible gamma delta T cell growth arrest mediated through WC1

IL-2-stimulated expansion of T cells requires continued and sequential passage of the dividing cells through a major cell cycle check point in the G1 phase. We have previously shown that a gamma delta T cell-specific surface receptor, WC1, induces G0/G1 growth arrest, reversible with Con A, in proliferating IL-2-dependent gamma delta T cells. We now show that this reversible WC1-induced cell cycle arrest is correlated with induction of the cyclin kinase inhibitor p27kip1 and an associated down-regulation in cyclins A, D2, and D3 expression, along with dephosphorylation of pocket proteins p107, p130, and pRb. Together with diminished pocket protein phosphorylation, p107 expression levels are significantly down-regulated in response to WC1 stimulation. This coordinated sequence of signaling events is focused on E2F regulation so that, downstream of the pocket proteins, WC1 stimulation results in a diminished DNA binding activity for free E2F as a consequence of reduced E2F1 expression, whereas E2F4 expression is unaffected. Consistent with this interpretation, overexpression of E2F1 overcomes the growth-arresting effects induced by WC1 stimulation. Finally, in accordance with our previous observations at both the cellular and molecular level, subsequent mitogen stimulation can reverse all the above changes induced by WC1. These results, focused on E2F regulation, therefore provide a first insight into the effects of both positive (mitogen) and negative (anti-WC1) stimuli on cell cycle control in IL-2-dependent gamma delta T cells.     

Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G

Crk, which belongs to the adaptor family of proteins composed of Src homology 2 (SH2) and SH3 domains, has a putative role in signaling. However, the downstream events of Crk signaling remain unclear. In this study, we found that Jun kinase (JNK) is moderately activated by v-Crk in both NIH 3T3 cells and chicken embryo fibroblasts. Transient expression of v-Crk, c-Crk-I, or c-Crk-II activated JNK1 in human embryo kidney cells, 293T. Coexpression of a guanine nucleotide exchange protein C3G, which specifically binds to Crk's SH3 domain, further enhanced the JNK activity as well as growth rate and anchorage-independent growth of v-Crk NIH 3T3 cells. Furthermore, overexpression of a dominant-negative form of C3G lacking the guanine nucleotide exchange domain abolished both the JNK activity and the colony forming potential of v-Crk NIH 3T3 cells. The requirement for JNK activation in v-Crk induced transformation was demonstrated by the suppression of colony forming activity of v-Crk NIH 3T3 cells when a dominant-negative form of JNK kinase, Sek1/MKK4 is expressed in these cells. These data strongly suggest the existence of a novel signaling cascade involving an adaptor protein v-Crk, which transmits signals through C3G toward JNK activation.     

A novel functional human eukaryotic translation initiation factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.     

Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence

Phosphoinositide-specific phospholipase Cgamma (PLCgamma) is a key regulatory enzyme that binds to the phosphoryl-tyrosine residues in the cytoplasmic domain of certain activated receptors and catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] forming IP3 and diacylglycerol (DAG) in response to several mitogenic factors. Previously, we determined that microinjected PLCgamma induces DNA synthesis in G0-arrested NIH 3T3 cells, suggesting the possibility that PLCgamma may have an oncogenic potential. In this report, we demonstrate that overexpression of PLCgamma in NIH 3T3 cells results in altered growth properties and cellular transformation. The PLCgamma/3T3 transfectants do not require serum growth factors to proliferate, display anchorage-independent growth in soft agar and induce tumors when transplanted into nude mice. These findings suggest that overexpression of PLCgamma facilitates the transformation of NIH 3T3 cells. Furthermore, PLCgamma expression and activity have been shown to be elevated in many human tumors. Thus, PLCgamma signaling may contribute to the promotion and/or progression of human cancers.     

Vaccinia virus protein synthesis has a low requirement for the intact translation initiation factor eIF4F, the cap-binding complex, within infected cells

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both beta-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (beta-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5' noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5' and 3' termini.     

Differential regulation of translation and eIF4E phosphorylation during human thymocyte maturation

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+ CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+ CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.     

Impaired glucose-stimulated insulin release in islets from adult rats malnourished during foetal-neonatal life

Initiation factor (eIF) 4G plays a key role in the regulation of translation, acting as a bridge between eIF4E and eIF3, to allow an mRNA molecule to associate with the 40S ribosomal subunit. In this study, we show that activation of the Fas/CD95 receptor complex in Jurkat cells induces the degradation of eIF4G, the inhibition of total protein synthesis and cell death. These responses were prevented by the caspase inhibitors, zVAD.FMK and zDEVD.FMK. We also show that, in contrast to Saccharomyces cerevisiae, although rapamycin caused a modest inhibition of protein synthesis it did not induce apoptosis or the cleavage of eIF4G. Studies with the specific inhibitor, SB203580, have shown that signalling through the p38 MAP kinase pathway is not required for either the Fas/CD95-induced cleavage of eIF4G or cell death. These data suggest that the cleavage of eIF4G and the inhibition of translation play an integral role in Fas/CD95-induced cell death in Jurkat cells.     

A link between cyclin A expression and adhesion-dependent cell cycle progression

Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.     

Modulation of translation initiation in rat skeletal muscle and liver in response to food intake

Protein synthesis is altered in both skeletal muscle and liver in response to nutritional status with food deprivation being associated with an inhibition of mRNA translation. In the present study, the effect of food-intake on the initiation of mRNA translation was examined in rats fasted for 18-h and then refed a complete diet. Fasting and refeeding caused alterations in translation initiation in both skeletal muscle and liver that were not associated with any detectable changes in the activity of eIF2B or in the phosphorylation state of eIF2 alpha. Instead, alterations in initiation were associated with changes in the phosphorylation state of eIF4E and/or the association of eIF4E with eIF4G as well as the eIF4E binding protein, 4E-BP1. In muscle from fasted rats, the amount of eIF4E present in an inactive complex with 4E-BP1 was increased 5-fold compared to freely fed control animals. One hour after refeeding a complete diet, the amount of 4E-BP1 bound to eIF4E was reduced to freely fed control values. Reduced association of the two proteins was the result of increased phosphorylation of 4E-BP1. Refeeding a complete diet also stimulated the binding of eIF4E to eIF4G to form the active eIF4F complex. In liver, the amount of eIF4E associated with eIF4G, but not the amount of eIF4E associated with 4E-BP1, was altered by fasting and refeeding. Furthermore, in liver, but not in skeletal muscle, fasting and refeeding resulted in modulation of the phosphorylation state of eIF4E. Overall, the results suggest that protein synthesis may be differentially regulated in muscle and liver in response to fasting and refeeding. In muscle, protein synthesis is regulated through modulation of the binding of eIF4E to eIF4G and in liver through modulation of both phosphorylation of eIF4E as well as binding of eIF4E to eIF4G.