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.     

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.     

4E-BP1, a multifactor regulated multifunctional protein

Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) is a member of a family of translation repressor proteins, and a well-known substrate of mechanistic target of rapamycin (mTOR) signaling pathway. Phosphorylation of 4E-BP1 causes its release from eIF4E to allow cap-dependent translation to proceed. Recently, 4E-BP1 was shown to be phosphorylated by other kinases besides mTOR, and overexpression of 4E-BP1 was found in different human carcinomas. In this review, we summarize the novel findings on mTOR independent 4E-BP1 phosphorylation in carcinomas. The implications of overexpression and possible multi-function of 4E-BP1 are also discussed.     

Protein Phosphatase PPM1G Regulates Protein Translation and Cell Growth by Dephosphorylating 4E Binding Protein 1 (4E-BP1)

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.     

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E,and the mRNA 5′ cap

Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.     

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

Suppression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation

In this study, we document that the overall rate of protein synthesis decreases during in vitro maturation (IVM) of pig oocytes despite enhanced formation of the 5' cap structure eIF4F. Within somatic/interphase cells, formation of the eIF4F protein complex correlates very well with overall rates of protein translation, and the formation of this complex is controlled primarily by the availability of the 5' cap binding protein eIF4E. We show that the eIF4E inhibitory protein, 4E-BP1, becomes phosphorylated during IVM, which results in gradual release of eIF4E from 4E-BP1, as documented by immunoprecipitation analyses. Isoelectric focusing and Western blotting experiments show conclusively that eIF4E becomes gradually phosphorylated with a maximum at metaphase II (M II). The activity of eIF4E and its ability to bind mRNA also increases during oocyte maturation as documented in experiments with m7-methyl GTP-Sepharose, which mimics the cap structure of mRNA. Complementary analysis of flow-through fraction for 4E-BP1, and eIF4G proteins additionally provides evidence for enhanced formation of cap-binding protein complex eIF4F. Altogether, our results bring new insights to the regulation of translation initiation during meiotic division, and more specifically clarify that 4E-BP1 hyper-phosphorylation is not the cause of the observed suppression of overall translation rates.     

4E-BP1、4E-BP1-4A 重组慢病毒载体的构建及对胃癌细胞 HGC27生长的影响

目的:构建4E-BP1及其 T37A、T46A、S65A、T70A 突变体4E-BP1-4A 基表达的重组慢病毒载体,研究其对胃癌 HGC27细胞生长的影响.方法:PCR 扩增4E-BP1基及其突变体4E-BP1-4A 基并克隆到 pCDH 载体,构建成 pCDH-4E-BP1、pCDH-4E-BP1-4A,将其与包装载体共转染293T 细胞,包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A重组慢病毒载体,将此慢病毒感染胃癌 HGC27细胞,Western 印迹鉴定病毒载体介导的4E-BP1、4E-BP1-4A 蛋白的表达,MTT、克隆形成和软琼脂方法研究过量表达4E-BP1、4E-BP1-4A 对胃癌 HGC27细胞生长的影响.结果:包装成 Lenti-4E-BP1及 Lenti-4E-BP1-4A 重组慢病毒载体,并将此慢病毒载体感染胃癌 HGC27细胞;MTT、克隆形成、软琼脂实验表明过量表达4E-BP1可抑制胃癌 HGC27细胞的生长,过量表达4E-BP1-4A 时抑制效果更明显.结论:构建了4E-BP1、4E-BP1-4A 的重组慢病毒表达载体,在胃癌 HGC27细胞中过量表达4E-BP1可抑制细胞生长,过量表达4E-BP1-4A 的抑制效果更明显.     

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.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.     

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.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.     

Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Reduction of serum FABP4 level by sitagliptin,a DPP-4 inhibitor,in patients with type 2 diabetes mellitus

Fatty acid binding protein 4 (FABP4), also known as adipocyte FABP or aP2, is secreted from adipocytes in association with lipolysis as a novel adipokine, and elevated serum FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the modulation of serum FABP4 level by therapeutic drugs. Sitagliptin (50 mg/day), a dipeptidyl peptidase 4 (DPP-4) inhibitor that increases glucagon-like peptide 1 (GLP-1), was administered to patients with type 2 diabetes (n = 24) for 12 weeks. Treatment with sitagliptin decreased serum FABP4 concentration by 19.7% (17.8 ± 1.8 vs. 14.3 ± 1.5 ng/ml, P < 0.001) and hemoglobin A1c without significant changes in adiposity or lipid variables. In 3T3-L1 adipocytes, sitagliptin or exendin-4, a GLP-1 receptor agonist, had no effect on short-term (2 h) secretion of FABP4. However, gene expression and long-term (24 h) secretion of FABP4 were significantly reduced by sitagliptin, which was not mimicked by exendin-4. Treatment with recombinant DPP-4 increased gene expression and long-term secretion of FABP4, and the effects were cancelled by sitagliptin. Furthermore, knockdown of DPP-4 in 3T3-L1 adipocytes decreased gene expression and long-term secretion of FABP4. In conclusion, sitagliptin decreases serum FABP4 level, at least in part, via reduction in the expression and consecutive secretion of FABP4 in adipocytes by direct inhibition of DPP-4.     

Structural scaffold for eIF4E binding selectivity of 4E‐BP isoforms: crystal structure of eIF4E binding region of 4E‐BP2 and its comparison with that of 4E‐BP1

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E‐binding protein 2 (4E‐BP2) than of 4E‐BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E‐BP2 in complex with m⁷GTP‐bound human eIF4E has been determined by X‐ray diffraction analysis and compared with that of 4E‐BP1. The crystal structure revealed that the Pro47‐Ser65 moiety of 4E‐BP2 adopts a L ‐shaped conformation involving extended and α‐helical structures and extends over the N‐terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E‐BP1. Although the pattern of the overall interaction of 4E‐BP2 with eIF4E was similar to that of 4E‐BP1, a notable difference was observed for the 60–63 sequence in relation to the conformation and binding selectivity of the 4E‐BP isoform, i.e. Met‐Glu‐Cys‐Arg for 4E‐BP1 and Leu‐Asp‐Arg‐Arg for 4E‐BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E‐BP2 over 4E‐BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59‐Leu60 sequence of 4E‐BP2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.