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.     

Shiga toxins activate translational regulation pathways in intestinal epithelial cells

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.     

Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts

Thephosphorylation states of three proteins implicated in the action ofinsulin on translation were investigated, i.e., 70-kDa ribosomalprotein S6 kinase (p70^S6k),eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein4E-BP1. Addition of insulin caused a stimulation of protein synthesisin L6 myoblasts in culture, an effect that was blocked by inhibitors ofphosphatidylinositide-3-OH kinase (wortmannin), p70^S6k (rapamycin), andmitogen-activated protein kinase (MAP kinase) kinase (PD-98059). Thestimulation of protein synthesis was accompanied by increasedphosphorylation of p70^S6k, aneffect that was blocked by rapamycin and wortmannin but not PD-98059.Insulin caused dephosphorylation of eIF-4E, an effect that appeared tobe mediated by the p70^S6kpathway. Insulin also stimulated phosphorylation of 4E-BP1 as well asdissociation of the 4E-BP1 · eIF-4E complex. Bothrapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G · eIF-4E complex, aneffect that was not prevented by any of the inhibitors. Overall, theresults suggest that insulin stimulates protein synthesis in L6myoblasts in part through utilization of both thep70^S6k and MAP kinase signaltransduction pathways.

     

Molecular cross-talk between MEK1/2 and mTOR signaling during recovery of 293 cells from hypertonic stress

To investigate the role for initiation factor phosphorylation in de novo translation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. As de novo translation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge upon de novo translation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuated de novo protein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promote de novo translation.     

Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.     

Identification of a novel Comamonas testosteroni gene encoding a steroid-inducible extradiol dioxygenase

Insulin-like growth factor-1 (IGF-1) both promotes survival and activates protein synthesis in neurons. In the present paper, we investigate the effect of IGF-1 treatment on cap-dependent translation in primary cultured neuronal cells. IGF-1 treatment increased the phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein 1 (4E-BP1), exclusively at Thr-36 and Thr-45 residues, and eIF-4G phosphorylation at Ser-1108. In contrast, a significant eIF-4E dephosphorylation was found. In parallel, increased eIF-4E/4G assembly and protein synthesis activation in response to IGF-1 treatment were observed. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, but not the mitogen-activated protein kinase (MAPK)-activating kinase (MEK) inhibitor PD98059, reversed the IGF-1-induced effects observed on eIF-4E/4G assembly and phosphorylation status of 4E-BP1, eIF-4E, and eIF-4G. Therefore, our findings show that the IGF-1-induced regulation of cap-dependent translation is largely dependent on the PI-3K and mTOR pathway in neuronal cells.     

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.     

Acute treatment with TNF-alpha attenuates insulin-stimulated protein synthesis in cultures of C2C12 myotubes through a MEK1-sensitive mechanism

Insulin and TNF-alpha exert opposing effects on skeletal muscle protein synthesis that are mediated in part by the rapamycin-sensitive mammalian target of rapamycin (mTOR) pathway and the PD-98059-sensitive, extracellular signal-regulated kinase (ERK)1/2 pathway. The present study examined the separate and combined effects of insulin (INS), TNF, PD-98059, or dnMEK1 adenovirus on the translational control of protein synthesis in C(2)C(12) myotubes. Cultures were treated with INS, TNF, PD-98059, dnMEK1, or a combination of INS + TNF with PD-98059 or dnMEK1. INS stimulated protein synthesis, enhanced eIF4E.eIF4G association, and eIF4G phosphorylation and repressed eIF4E.4E-BP1 association vs. control. INS also promoted phosphorylation of ERK1/2, S6K1, and 4E-BP1 and dephosphorylation of eIF4E. TNF alone did not have an effect on protein synthesis (vs. control), eIF4E.eIF4G association, or the phosphorylation of eIF4G, S6K1, or 4E-BP1, although it transiently increased ERK1/2 and eIF4E phosphorylation. When myotubes were treated with TNF + INS, the cytokine blocked the insulin-induced stimulation of protein synthesis. This appeared to be due to an attenuation of insulin-stimulated eIF4E.eIF4G association, because other stimulatory effects of INS, e.g., phosphorylation of ERK1/2, 4E-BP1, S6K1, eIF4G, and eIF4E and eIF4E.4E-BP1 association, were unaffected. Finally, treatment of myotubes with PD-98059 or dnMEK1 adenovirus before TNF + INS addition resulted in a derepression of protein synthesis and the association of eIF4G with eIF4E. These findings suggest that TNF abrogates insulin-induced stimulation of protein synthesis in myotubes through a decrease in eIF4F complex assembly independently of S6K1 and 4E-BP1 signaling and dependently on a MEK1-sensitive signaling pathway.     

Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.     

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.     

Regulation of Host Translational Machinery by African Swine Fever Virus

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host''s defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.     

Phosphatidylinositol 4-phosphate is required for translation initiation in Saccharomyces cerevisiae

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2alpha. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.     

N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways

During the oxidative stress generated by hydrogen peroxide (H2O2) in nerve growth factor (NGF)-differentiated PC12 cells, eIF4E binding protein (4E-BP1) and initiation factor 4E (eIF4E) phosphorylated levels decrease significantly, and an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreases eIF4F formation is observed. The treatment with N-acetyl-cysteine (NAC) completely abolishes the H2O2-induced decrease in eIF4E phosphorylated levels, whereas the decrease in 4E-BP1 phosphorylated levels and eIF4F activity inhibition are significantly but not fully reversed. Rapamycin, the mammalian target of rapamycin (FRAP/mTOR) inhibitor, prevents the effect of NAC on H2O2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H2O2. However, rapamycin has no effect on the NAC-induced recovery in phosphorylated eIF4E levels. Neither the MAP kinase inhibitors, PD98056 and SB203580, or the protein phosphatase 2A inhibitor, okadaic acid, mimic NAC effect on the H2O2-induced eIF4E dephosphorylation. Altogether our findings suggest that the effects caused by oxidative stress on eIF4s factors depends on two MAP kinase-independent signal transduction pathways, being at least one of them rapamycin-dependent.     

Brain-derived neurotrophic factor enhances neuronal translation by activating multiple initiation processes: comparison with the effects of insulin

The effects of neurotrophic factors on translational activation were investigated in cortical neurons. Brain-derived neurotrophic factor (BDNF) increased protein synthesis within 30 min, whereas insulin produced a weaker enhancement of protein synthesis. BDNF-triggered protein synthesis was inhibited by LY294002, PD98059, and rapamycin, whereas the effect of insulin was unaffected by PD98059. To explore the mechanisms underlying this effect, the protein phosphorylation cascades that lead to the activation of translation initiation in neurons were examined. BDNF induced the phosphorylation of both eukaryote initiation factor (eIF) 4E and its binding protein (eIF4E-binding protein-1). The former reaction was inhibited by PD98059, whereas the latter was inhibited by LY294002 or rapamycin. In agreement, BDNF induced the phosphorylation of mammalian TOR (target of rapamycin) and enhanced its kinase activity toward eIF4E-binding protein-1. In contrast, insulin failed to activate MAPK and did not induce the phosphorylation of eIF4E. Since BDNF and insulin increased the activity of eIF2B and eIF2, the only difference between them was eIF4E phosphorylation. Thus, this may explain the lower activity of insulin in potentiating neuronal protein synthesis. These results suggest strongly that BDNF simultaneously activates multiple signaling cascades consisting of phosphatidylinositol 3-kinase, mammalian TOR, and MAPK to enhance translation initiation in neurons.     

Signal pathways involved in activation of p70S6K and phosphorylation of 4E-BP1 following exposure of multiple myeloma tumor cells to interleukin-6

Interleukin-6 (IL-6) is a prominent tumor growth factor for malignant multiple myeloma cells. In addition to its known activation of the Janus tyrosine kinase-STAT and RAS-MEK-ERK pathways, recent work suggests that IL-6 can also activate the phosphatidylinositol 3-kinase (PI3-K)/AKT kinase pathway in myeloma cells. Because activation of the PI3-K/AKT as well as RAS-MEK-ERK pathways may result in downstream stimulation of the p70(S6K) (p70) and phosphorylation of the 4E-BP1 translational repressor, we assessed these potential molecular targets in IL-6-treated myeloma cells. IL-6 rapidly activated p70 kinase activity and p70 phosphorylation. Activation was inhibited by wortmannin, rapamycin, and the ERK inhibitors PD98059 and UO126, as well as by a dominant negative mutant of AKT. The concurrent requirements for both ERK and PI3-K/AKT appeared to be a result of their ability to phosphorylate p70 on different residues. In contrast, IL-6-induced phosphorylation of 4E-BP1 was inhibited by rapamycin, wortmannin, and dominant negative AKT but ERK inhibitors had no effect, indicating ERK function was dispensable. In keeping with these data, a dominant active AKT mutant was sufficient to induce 4E-BP1 phosphorylation but could not by itself activate p70 kinase activity. Prevention of IL-6-induced p70 activation and 4E-BP1 phosphorylation by the mammalian target of rapamycin inhibitors rapamycin and CCI-779 resulted in inhibition of IL-6-induced myeloma cell growth. These results indicate that both ERK and PI3-K/AKT pathways are required for optimal IL-6-induced p70 activity, but PI3-K/AKT is sufficient for 4E-BP1 phosphorylation. Both effects are mediated via mammalian target of rapamycin function, and, furthermore, these effects are critical for IL-6-induced tumor cell growth.     

The rapid activation of protein synthesis by growth hormone requires signaling through mTOR

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.