Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP

The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.     

Initiation factors eIF4: from sea urchin embryonic development to chronic lymphocytic leukemia

mRNA translation is now recognized as a important regulatory step for gene expression in different physiological and pathophysiological processes including cell proliferation and apoptosis. B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes and defective apoptosis. The mRNA cap-binding protein eIF4E (eukaryotic Initiation Factor 4E) and its repressor 4E-BP (eIF4E Binding protein) are crucial translational regulators that have been involved in survival and apoptosis processes of cells. We have shown that the release of eIF4E from its translational repressor 4E-BP is an important event for the first mitotic division triggered by fertilization and that the degradation of 4E-BP is a new means to regulate 4E-BP function that has to be analyzed in other physiological and physiopathological processes. In this chapter, we describe recent advances illustrating the importance of eIF4E and 4E-BP in cancer processes, suggesting that these actors can be targeted for potential therapy against cancer in general and LLC in particular.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

Rapamycin-insensitive regulation of 4e-BP1 in regenerating rat liver

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1).

BACKGROUND INFORMATION: The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. RESULTS: We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. CONCLUSIONS: The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.     

内蒙古白绒山羊4E-BP1基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊4E-BP1(真核细胞翻译起始因子4E结合蛋白1)基因并进行生物信息学及表达模式分析。根据已报道物种4E-BP1基因cDNA序列,用primer premier5软件设计引物,通过RT-PCR从绒山羊胎儿成纤维细胞总RNA中扩增出4E-BP1基因编码区cDNA序列,对目的片段进行测序及表达模式分析。克隆到的内蒙古白绒山羊4E-BP1基因cDNA全长357 bp,包含了完整的的ORF,编码118个氨基酸残基。核酸序列与牛、马、人、大鼠及小鼠的同源性分别为98%、90%、90%、88%和87%。4E-BP1基因在绒山羊脑、心脏、睾丸及胰腺组织中均有表达。     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

4E binding protein 1 expression is inversely correlated to the progression of gastrointestinal cancers

Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Overexpression of the initiation factor eIF4E occurs in a variety of human tumours. The aim of this study was to determine the level of expression and the phosphorylation state of eIF4E and 4E-binding protein 1 (4E-BP1) in gastrointestinal cancer, and to ascertain whether or not these factors can be used as diagnostic or prognostic markers within this type of cancer. The eIF4E levels were significantly higher in tumours compared with normal tissue (51. 5+/-4.4 vs 30.9+/-2.5 arbitrary units (A.U.)/mg of protein, p<0.001). However, phosphorylated eIF4E did not change in stomach cancers and decreased in colorectal cancers (67.1+/-1.2 vs 60.8+/-2.8%, p<0.05). 4E-BP1 expression increased in most of the gastrointestinal cancers studied. In addition, an inverse correlation between 4E-BP1 elevation and N and M stages was found, showing significant higher elevation of 4E-BP1 in Node-negative patients (11.21+/-5.74 vs 4. 03+/-2.36 n-fold, p<0.05) as well as in patients without distant metastasis (8.41+/-3.29 vs 0.97+/-0.35 n-fold, p<0.05). These results suggest that 4E-BP1 could function as a tumour suppressor. Moreover, the data show a significant dephosphorylation of 4E-BP1 in gastrointestinal tumours that correlated with an increase in the association of 4E-BP1 and eIF4E indicating a lower availability to eIF4E to recruit to the ribosomes. Our results support a possible role of 4E-BP1 as a prognostic factor in gastrointestinal carcinoma.     

Localisation and regulation of the eIF4E-binding protein 4E-BP3

The cap-binding protein eIF4E-binding protein 3 (4E-BP3) was identified some years ago, but its properties have not been investigated in detail. In this report, we investigated the regulation and localisation of 4E-BP3. We show that 4E-BP3 is present in the nucleus as well as in the cytoplasm in primary T cells, HEK293 cells and HeLa cells. 4E-BP3 was associated with eIF4E in both cell compartments. Furthermore, 4E-BP3/eIF4E association in the cytoplasm was regulated by serum or interleukin-2 starvation in the different cell types. Rapamycin did not affect the association of eIF4E with 4E-BP3 in the cytoplasm or in the nucleus.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1

Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) represses translation initiation by binding to eukaryotic initiation factor 4E (eIF4E). 4E-BP1 also binds to the eIF4E homologous protein (4EHP). We show that eIF4E-binding mutants of 4E-BP1 (Y54A and L59A) fail to form heterodimeric complexes with wild-type 4EHP. In addition, the W95A mutant of 4EHP, similar to a homologous mutation in eIF4E, inhibits its binding to wild-type 4E-BP1. Interestingly, 4EHP over-expression instigates a negative feedback loop that inhibits upstream signaling to 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) whereas the 4E-BP1-binding-deficient mutant of 4EHP(W95A) was unable to trigger this feedback loop. Thus, the interaction of 4EHP with 4E-BP1 is necessary for this observed impaired signaling to 4E-BP1 and S6K1.     

Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes

Incubation of hepatocytes under hypoxia increases binding of translation initiation factor eIF-4E to its inhibitory regulator 4E-BP1, and this correlates with dephosphorylation of 4E-BP1. Rapamycin induced the same effect in aerobic cells but no additive effect was observed when hypoxic cells were treated with rapamycin. This enhanced association of 4E-BP1 with eIF-4E might be mediated by mTOR. Nevertheless, only hypoxia produces a rapid inhibition of protein synthesis. Although hypoxia might be signalling via the rapamycin-sensitive pathway by changing eIF-4E availability, such a pathway is unlikely to be responsible for the depression in overall protein synthesis under hypoxia.     

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 ∼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 fluorescencein situhybridization 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.     

The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson''s disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.