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1.
Raptor-rictor axis in TGFbeta-induced protein synthesis   总被引:1,自引:0,他引:1  
Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.  相似文献   

2.
Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.  相似文献   

3.
Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.  相似文献   

4.
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.  相似文献   

5.
Recently, we identified an N‐terminally truncated form of the mRNA cap binding protein eIF4E in the porcine luminal epithelium during implantation. EIF4E truncation is accompanied by degradation of the eIF4E‐repressor protein 4E‐BP1. In this study, we investigated whether or not the other members of the eIF4E‐repressor family, namely 4E‐BP2 and 4E‐BP3, were also modified during early pregnancy. We did not detect 4E‐BP3 in the uterine tissue; however, 4E‐BP2 emerged in one or two stable fragments on pregnancy day 15. 4E‐BP2 truncation most likely occurs at the N‐terminus, and this calcium‐stimulated processing depends on progesterone and estradiol. The activities targeting eIF4E, 4E‐BP1, and 4E‐BP2 were found in different fractions after anionic exchange chromatography, indicating the action of different proteases. Detailed protein interaction studies with immobilized anti‐eIF4E and m7GTP‐Sepharose showed a differential binding of the 4E‐BP2 isoforms to the eIF4E variants and to the cap structure. In general, truncation of eIF4E reduces the inhibitory impact of 4E‐BP2, whereas truncation of 4E‐BP2 restores repression by binding the prototype eIF4E. In this context, we suggest long‐term translational repression by the truncated 4E‐BP2 is affected by the loss of the RAIP motif located at the N‐terminus, which is indispensable for phosphorylation and deactivation of the molecule. In conclusion, we propose a tightly balanced regulation of the truncation of the cap‐binding complex component eIF4F and degradation of 4E‐BP1 and/or truncation of 4E‐BP2 that together ensures correct translational control during the dynamic process of conceptus implantation. Mol. Reprod. Dev. 79: 767–776, 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

6.
Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap‐binding protein eIF4E, which is released from its repressor 4E‐BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E‐BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E‐BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E‐BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E‐BP and eIF4G. Mol. Reprod. Dev. 77: 83–91, 2010. © 2009 Wiley‐Liss, Inc.  相似文献   

7.
Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.  相似文献   

8.
9.
Eukaryotic translation initiation factor 4E (eIF4E) is the cap‐binding protein that binds the 5′ cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap‐dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin‐related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E‐dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.  相似文献   

10.
Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.  相似文献   

11.
Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha- or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor.  相似文献   

12.
13.
System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function. amino acid transport; hepatocellular carcinoma; apoptosis; protein synthesis  相似文献   

14.
Signaling mediated by the cellular kinase mammalian target of rapamycin (mTOR) activates cap-dependent translation under normal (nonstressed) conditions. However, translation is inhibited by cellular stress responses or rapamycin treatment, which inhibit mTOR kinase activity. We show that during human cytomegalovirus (HCMV) infection, viral protein synthesis and virus production proceed relatively normally when mTOR kinase activity is inhibited due to hypoxic stress or rapamycin treatment. Using rapamycin inhibition of mTOR, we show that HCMV infection induces phosphorylation of two mTOR effectors, eucaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) and eIF4G. The virally induced phosphorylation of eIF4G is both mTOR and phosphatidylinositol 3-kinase (PI3K) independent, whereas the phosphorylation of 4E-BP is mTOR independent, but PI3K dependent. HCMV infection does not induce mTOR-independent phosphorylation of a third mTOR effector, p70S6 kinase (p70S6K). We show that the HCMV-induced phosphorylation of eIF4G and 4E-BP correlates with the association of eIF4E, the cap binding protein, with eIF4G in the eIF4F translation initiation complex. Thus, HCMV induces mechanisms to maintain the integrity of the eIF4F complex even when mTOR signaling is inhibited.  相似文献   

15.
Shiga toxins (Stxs) cause irreversible damage to eukaryotic ribosomes, yet cellular intoxication of intestinal epithelial cells (IECs) results in increased synthesis of selected proteins, notably cytokines. How mRNA translation is maintained in this circumstance is unclear. This study was designed to assess whether Stx-induced alterations in host signal transduction machinery permit translation despite protein synthesis inhibition. A key step of translation is recruitment of initiation machinery to the 5' mRNA cap. This event occurs in part via interaction of the 5' cap with the cap binding protein, eIF4E, whose activity is positively regulated by phosphorylation and negatively regulated by binding to the translational repressor 4E-BP1. Following Stx treatment of IECs, eIF4E phosphorylation was detected by Western blotting using phospho-specific antibodies. Treatment with the p38 inhibitor, SB202190, or either of the ERK1/2 inhibitors, PD98059 and U0126, partially blocked Stx1-induced eIF4E phosphorylation. The Mnk1 inhibitor, CGP57380, blocked both basal and Stx-induced eIF4E phosphorylation. Interestingly, pretreatment with CGP57380 did not alter basal protein synthesis, but diminished the ability of cells to maintain translation following Stx1 challenge. Stx1 also induced hyperphosphorylation of 4E-BP1 and phosphorylation of S6Kinase; both effects were blocked by rapamycin. These data are novel observations showing that Stxs regulate multiple signal transduction pathways controlling translation in host cells, and support a role for eIF4E phosphorylation in maintaining host cell translation despite ribosomal intoxication.  相似文献   

16.
An important function of growth hormone (GH) is to promote cell and tissue growth, and a key component of these effects is the stimulation of protein synthesis. In this study, we demonstrate that, in H4IIE hepatoma cells, GH acutely activated protein synthesis through signaling via the mammalian target of rapamycin (mTOR) and specifically through the rapamycin-sensitive mTOR complex 1 (mTORC1). GH treatment enhanced the phosphorylation of two targets of mTOR signaling, 4E-BP1 and ribosomal protein S6. Phosphorylation of S6 and 4E-BP1 was maximal at 30-45 min and 10-20 min after GH stimulation, respectively. Both proteins modulate components of the translational machinery. The GH-induced phosphorylation of 4E-BP1 led to its dissociation from eIF4E and increased binding of eIF4E to eIF4G to form (active) eIF4F complexes. The ability of GH to stimulate the phosphorylation of S6 and 4E-BP1 was blocked by rapamycin. GH also led to the dephosphorylation of a third translational component linked to mTORC1, the elongation factor eEF2. Its regulation followed complex biphasic kinetics, both phases of which required mTOR signaling. GH rapidly activated both the MAP kinase (ERK) and PI 3-kinase pathways. Signaling through PI 3-kinase alone was, however, sufficient to activate the downstream mTORC1 pathway. Consistent with this, GH increased the phosphorylation of TSC2, an upstream regulator of mTORC1, at sites that are targets for Akt/PKB. Finally, the activation of overall protein synthesis by GH in H4IIE cells was essentially completely inhibited by wortmannin or rapamycin. These results demonstrate for the first time that mTORC1 plays a major role in the rapid activation of protein synthesis by GH.  相似文献   

17.
Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.  相似文献   

18.
The mRNA cap‐binding oncoprotein “eIF4E” is phosphorylated at residue S209 by Mnk kinases, and is closely associated with tumor development and progression. Despite being well‐established, mechanistic details at the molecular level of mRNA recognition by eIF4E due to phosphorylation have not been clearly elucidated. We investigated this through molecular modeling and simulations of the S209 phosphorylated derivative of eIF4E and explored the associated implication on the binding of the different variants of mRNA‐cap analogs. A key feature that emerges as a result of eIF4E phosphorylation is a salt‐bridge network between the phosphorylated S209 (pS209) and a specific pair of lysine residues (K159 and K162) within the cap‐binding interface on eIF4E. This interaction linkage stabilizes the otherwise dynamic C‐terminal region of the protein, resulting in the attenuation of the overall plasticity and accessibility of the binding pocket. The pS209‐K159 salt‐bridge also results in an energetically less favorable environment for the bound mRNA‐cap primarily due to electrostatic repulsion between the negative potentials from the phosphates in the cap and those appearing as a result of phosphorylation of S209. These observations collectively imply that the binding of the mRNA‐cap will be adversely affected in the phosphorylated derivative of eIF4E. We propose a mechanistic model highlighting the role of eIF4E phosphorylation as a regulatory tool in modulating eIF4E: mRNA‐cap recognition and its potential impact on translation initiation.  相似文献   

19.
Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.  相似文献   

20.
Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient protein synthesis in cap-dependent translation. The protein specifically binds the cap structure at the mRNA 5' terminus and facilitates the assembly of the mRNA with other initiation factors and the 40S ribosomal subunit. Phosphorylation of eIF4E is implicated in the regulation of the initiation step of translation. However, the molecular mechanism of this regulation still remains unclear. To address this problem, we have determined the binding affinities of eIF4E specifically mutated at position 209 or 159 for a series of novel mono- and dinucleotide cap analogues by a fluorometric time-synchronized titration method. A 1.5-3-fold reduction in the affinity of cap for the S209E mutant and a 1-2-fold increase in the affinity of cap for the S209K mutant, depending on the negative charge of phosphate chains, indicate that phosphorylation at Ser209 creates electrostatic repulsion between the protein and the negatively charged cap structure. The inhibition of the ability to bind cap analogues by the K159A mutant and its phosphorylated counterpart shows significant participation of Lys159 in the binding of the capped mRNA. Both structural modifications, phosphorylation and the replacement of lysine with alanine, result in an increase in the negative Gibbs free energy of association that is proportional to the length of the cap phosphate chain and additive, i.e., equal to the sum of the individual destabilizing changes of DeltaG degrees. The possible implication of these results for the mechanism of control of eIF4E by phosphorylation, especially for the "clamping model", is discussed.  相似文献   

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