Angiotensin IV enhances phosphorylation of 4EBP1 by multiple signaling events in lung endothelial cells

Angiotensin IV (Ang IV)-stimulated cell proliferation is regulated through activation of multiple signaling modules in lung endothelial cells (EC). Because eukaryotic intitiation factor 4E (eIF4E) binding protein 1 (4EBP1) plays a critical role in the RNA translation and the regulation of cell growth, we examined whether Ang IV modulates expression and/or phosphorylation of eIF4E and 4EBP1 as well as the role of multiple signaling events associated with 4EBP1 phosphorylation in EC. Ang IV stimulation increased phosphorylation but not expression of eIF4E and 4EBP1 proteins. Ang IV stimulation selectively phosphorylated Thr46 > Thr70 > Ser65 but not Thr37 residues in 4EBP1. Pretreatment of cells with PD-98059 and rapamycin, inhibitors of mitogen-activated protein kinase (ERK1/2) and mammalian target for rapamycin (mTOR), respectively, partially blocked Ang IV-mediated phosphorylation of 4EBP1. In contrast, overexpression of p70 ribosomal S6 kinase (p70S6K) and protein kinase B (Akt) enhanced phosphorylation of 4EBP1 and eIF4E binding affinity to the cap region of mRNA. These results support critical roles of multiple signaling and phosphorylation of 4EBP1 by Ang IV in translation process and protein synthesis.     

Arachidonic acid activation of translation initiation signaling in vascular smooth muscle cells

To understand the role of arachidonic acid (AA) in regulating vascular smooth muscle cell (VSMC) growth, its effects on phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E were studied. Arachidonic acid stimulated phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E in a time-dependent manner in VSMC. Arachidonic acid stimulation of phosphorylation of the above signaling molecules is specific, as these events were not affected by other unsaturated or saturated fatty acids. Metabolic conversion of AA via the LOX/MOX and/or COX pathways, to some extent, was required for its effects on the phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E. In addition, AA increased PI3K activity in a time-dependent manner in VSMC. LY294002, an inhibitor of PI3K, completely blocked AA-induced phosphorylation of Akt, S6K1, ribosomal protein S6, 4EBP1, and eIF4E, suggesting a role for PI3K in these effects. Consistent with its effects on translation initiation signaling events, AA induced global protein synthesis in VSMC and this response was dependent, to some extent, on its metabolism via the LOX/MOX and/or COX pathways, and mediated by the PI3K/Akt/mTOR pathway. Thus, the above observations provide the first biochemical evidence for the role of AA in the activation of translation initiation signaling in VSMC.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

Phosphorylation of eukaryotic translation initiation factor 4B (EIF4B) by open reading frame 45/p90 ribosomal S6 kinase (ORF45/RSK) signaling axis facilitates protein translation during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication

Open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinase (RSK), which is crucial for KSHV lytic replication, but the exact functional roles remain to be determined. To characterize the biological consequence of persistent RSK activation by ORF45, we screened known cellular substrates of RSK. We found that ORF45 induced phosphorylation of eukaryotic translation initiation factor 4B (eIF4B), increased its assembly into translation initiation complex, and subsequently facilitated protein translation. The ORF45/RSK-mediated eIF4B phosphorylation was distinguishable from that caused by the canonical AKT/mammalian target of rapamycin/ribosomal S6 kinase and MEK/ERK/RSK pathways because it was resistant to both rapamycin (an mammalian target of rapamycin inhibitor) and U1026 (an MEK inhibitor). The rapamycin and U1026 doubly insensitive eIF4B phosphorylation was induced during KSHV reactivation but was abolished if either ORF45 or RSK1/2 were ablated by siRNA, a pattern that is correlated with reduced lytic gene expression as we observed previously. Ectopic expression of eIF4B but not its phosphorylation-deficient mutant form increased KSHV lytic gene expression and production of progeny viruses. Together, these results indicated that ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication.     

Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.     

IGF-I activates the eIF4F system in cardiac muscle in vivo

IGF-I acutely stimulates protein synthesis in cardiac muscle through acceleration of mRNA translation. In the present study, we examined the regulatory signaling pathways and translation protein factors that potentially contribute to the myocardial responsiveness of protein synthesis to IGF-I in vivo. IGF-I was injected IV into rats and 20 min later the hearts were excised and homogenized for assay of regulatory proteins. IGF-I increased assembly of the translationally active eukaryotic initiation factor (eIF)4GeIF4E complex. The increased assembly of eIF4GeIF4E was associated with an enhanced eIF4G phosphorylation and increased availability of eIF4E. Increased availability of eIF4E occurred as a consequence of diminished abundance of the inactive 4E-BP1eIF4E complex following IGF-I. The assembly of the 4E-BP1eIF4E complex appeared to be decreased through an IGF-I-induced phosphorylation of 4E-BP1. IGF-I also caused an increase in the phosphorylation of S6K1. Activation of the potential upstream regulators of 4E-BP1 and S6K1 phosphorylation via PKB and mTOR was also observed. In contrast, there was no effect of IGF-I on phosphorylation of elongation factor (eFE)2. The results suggest the major impact of IGF-I in cardiac muscle occurred via stimulation of translation initiation rather than elongation. Furthermore, the results are consistent with a role for assembly of active eIF4GeIF4E complex and activation of S6K1 in mediating the stimulation of mRNA translation initiation by IGF-I through a PKB/mTOR signaling pathway.     

Helicobacter pylori promotes eukaryotic protein translation by activating phosphatidylinositol 3 kinase/mTOR

The innate immune response elicited by Helicobacter pylori in the human gastric mucosa involves a range of cellular signalling pathways, including those implicated in metabolism regulation. In this study, we analysed H. pylori-induced PI3K/Akt/mTOR signalling, which regulates glycolysis and protein synthesis and associates thereby with cellular energy- and nutrients-consuming processes such as growth and proliferation. The immunohistochemical analysis demonstrated that Akt kinase phosphorylation is abundant in gastric biopsies obtained from gastritis, gastric adenoma and adenocarcinoma patients. Infection with H. pylori led to the phosphorylation of Akt effectors mTOR and S6 in a type 4 secretion system (T4SS)-independent manner in AGS cells. We observed that the activation of these molecules was dependent on PI3K and the Src family tyrosine kinases. Furthermore, H. pylori induced the phosphorylation of 4E-BP1 and eIF4E and suppressed the phosphorylation of eEF2, which are important regulators of protein synthesis. Inhibition of PI3K and Akt kinase prevented the phosphorylation of 4E-BP1, suggesting that PI3K signalling is involved in the regulation of translation initiation during H. pylori infection. Metabolic labelling showed that infected cells had higher rates of [³⁵S]methionine/cysteine incorporation, and this effect could be prevented using LY294002, an PI3K inhibitor. Thus, H. pylori activates PI3K/Akt signalling, mTOR, eIFs and protein translation, which might impact H. pylori-related gastric pathophysiology.     

Hepatitis C virus NS5A binds to the mRNA cap-binding eukaryotic translation initiation 4F (eIF4F) complex and up-regulates host translation initiation machinery through eIF4E-binding protein 1 inactivation

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.     

Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases

The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5' untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum-responsive eIF4B phosphorylation site, Ser422, located in an RNA-binding region required for eIF4A helicase-promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide-3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin-resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.     

敲除eIF4B基因对小鼠胚胎肝脏细胞凋亡的影响北大核心CSCD

真核翻译起始因子4B(eukaryotic translation initiation factor 4B,eIF4B)在mRNA翻译起始、细胞存活和增殖过程中发挥着关键作用,然而其在生物体内的生物学功能仍存在许多疑问。本研究利用eIF4B基因敲除小鼠模型,结合苏木精和伊红(hematoxylin-eosin,HE)染色、流式细胞术、Western blotting和免疫组化等一系列实验技术手段,探究了eIF4B在胚胎发育过程中的功能及作用机理。结果显示,eIF4B敲除小鼠胚胎的肝脏呈现严重病理损伤,主要表现为胚肝细胞的凋亡和坏死,而小鼠胚胎的肺脏、脑、胃和胰腺则发育正常。进一步研究发现,在eIF4B敲除小鼠的胚胎肝脏中活化型caspase 3(cleaved-caspase 3)的表达水平显著升高。此外,eIF4B敲除小鼠的胚胎成纤维细胞和胚胎肝脏中mTOR通路下游信号分子p70S6K的表达和磷酸化水平以及4EBP1的磷酸化水平显著升高。综上所述,eIF4B敲除导致cleaved-caspase 3表达增加和mTOR信号通路过度活化,促进胚肝细胞的凋亡,致使小鼠胚肝发育异常,最终引发小鼠胚胎死亡。本研究揭示了eIF4B在胚胎发育过程中的重要作用,为深入了解eIF4B在生物体内的生物学功能提供了新的科学依据。     

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.     

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.     

ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.     

A citrus polymethoxyflavonoid, nobiletin, is a novel MEK inhibitor that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells

The activation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) is well known to be associated with tumor invasion and metastasis. We previously reported that a polymethoxyflavonoid, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), derived from Citrus depressa (Hayata), inhibits the phosphorylation of MEK and thereby suppresses matrix metalloproteinase (MMP) expression in a tumor-metastasis stimulator, 12-O-tetradecanoyl phorbol 13-acetate (TPA)-stimulated human fibrosarcoma HT-1080 cells [Mol. Cancer Ther. 3 (2004) 839-847]. In the present study, we investigated whether or not nobiletin might directly influence MEK activity to exhibit the antitumor metastatic activity in vitro. MEK kinase assay using myelin basic protein (MBP) revealed that TPA-augmented MEK activity in HT-1080 cells and that the augmented MEK activity was diminished by nobiletin treatment. In addition, the decrease in MEK activity caused by nobiletin was found to inhibit the phosphorylation of extracellular regulated kinases (ERK), a downstream signaling factor for MEK. Furthermore, when an immunoprecipitated active MEK was incubated with nobiletin under cell-free conditions, nobiletin was found to inhibit the MEK-mediated MBP phosphorylation. In contrast, other citrus polymethoxyflavonoids such as 3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone (natsudaidain) and 3,5,6,7,8,3′,4′-heptamethoxyflavone, did not directly inhibit MEK activity. Moreover, natsudaidain and 3,5,6,7,8,3′,4′-heptamethoxyflavone exhibited no or less inhibitory effect than nobiletin on the proMMP-9/progelatinase B production in HT-1080 cells. Therefore, these results provide novel evidence that nobiletin directly inhibits MEK activity and decreases the sequential phosphorylation of ERK, exhibiting the antitumor metastatic activity by suppressing MMP expression in HT-1080 cells.     

Phosphorylation of Eukaryotic Translation Initiation Factor 4E and Eukaryotic Translation Initiation Factor 4E-binding Protein (4EBP) and Their Upstream Signaling Components Undergo Diurnal Oscillation in the Mouse Hippocampus: IMPLICATIONS FOR MEMORY PERSISTENCE*

Translation of mRNA plays a critical role in consolidation of long-term memory. Here, we report that markers of initiation of mRNA translation are activated during training for contextual memory and that they undergo diurnal oscillation in the mouse hippocampus with maximal activity observed during the daytime (zeitgeber time 4–8 h). Phosphorylation and activation of eukaryotic translation initiation factor 4E (eIF4E), eIF4E-binding protein 1 (4EBP1), ribosomal protein S6, and eIF4F cap-complex formation, all of which are markers for translation initiation, were higher in the hippocampus during the daytime compared with night. The circadian oscillation in markers of mRNA translation was lost in memory-deficient transgenic mice lacking calmodulin-stimulated adenylyl cyclases. Moreover, disruption of the circadian rhythm blocked diurnal oscillations in eIF4E, 4EBP1, rpS6, Akt, and ERK1/2 phosphorylation and impaired memory consolidation. Furthermore, repeated inhibition of translation in the hippocampus 48 h after contextual training with the protein synthesis inhibitor anisomycin impaired memory persistence. We conclude that repeated activation of markers of translation initiation in hippocampus during the circadian cycle might be critical for memory persistence.