mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E

The mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. The immunosuppressive drug rapamycin inhibits cell cycle progression via inhibition of mTOR; however, the signaling pathways by which mTOR regulates cell cycle progression have remained poorly defined. Here we demonstrate that restoration of mTOR signaling (by using a rapamycin-resistant mutant of mTOR) rescues rapamycin-inhibited G(1)-phase progression, and restoration of signaling along the mTOR-dependent S6K1 or 4E-BP1/eukaryotic translation initiation factor 4E (eIF4E) pathways provides partial rescue. Furthermore, interfering RNA-mediated reduction of S6K1 expression or overexpression of mTOR-insensitive 4E-BP1 isoforms that block eIF4E activity inhibit G(1)-phase progression individually and additively. Thus, the activities of both the S6K1 and 4E-BP1/eIF4E pathways are required for and independently mediate mTOR-dependent G(1)-phase progression. In addition, overexpression of constitutively active mutants of S6K1 or wild-type eIF4E accelerates serum-stimulated G(1)-phase progression, and stable expression of wild-type S6K1 confers a proliferative advantage in low-serum-containing media, suggesting that the activity of each of these pathways is limiting for cell proliferation. These data demonstrate that, as for the regulation of cell growth and cell size, the S6K1 and 4E-BP1/eIF4E pathways each represent critical mediators of mTOR-dependent cell cycle control.     

Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.     

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 anticancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nanomolar 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 hyperphosphorylation 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.Key words: rapamycin, mTOR, 4E-BP1, eIF4E, Akt, apoptosis     

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.     

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.     

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.     

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

BACKGROUND: The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. RESULTS: Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. CONCLUSIONS: Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.     

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.     

Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase

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.     

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.     

Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.     

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.     

Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.     

Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins

Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.     

Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor

Mammalian hibernation involves cessation of energetically costly processes typical of homeostatic regulation including protein synthesis. To further elucidate the mechanisms employed in depressing translation, we surveyed key eukaryotic initiation factors [eIF2, eIF4B, eIF4E, eIF4GI and -II, and 4E-binding protein-1 (4E-BP1), -2, and -3] for their availability and phosphorylation status in the livers of golden-mantled ground squirrels (Spermophilus lateralis) across the hibernation cycle. Western blot analyses indicated only one significant locus for regulation of translational initiation in ground squirrel liver: control of eIF4E. We found seasonal variation in a potent regulator of eIF4E activity, 4E-BP1. Summer squirrels lack 4E-BP1 and apparently control eIF4E activity through direct phosphorylation. In winter, eIF4E is regulated through binding with 4E-BP1. During the euthermic periods that separate bouts of torpor (interbout arousal), 4E-BP1 is hyperphosphorylated to promote initiation. However, during torpor, 4E-BP1 is hypophosphorylated and cap-dependent initiation of translation is restricted. The regulation of cap-dependent initiation of translation may allow for the differential expression of proteins directed toward enhancing survivorship.     

Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation

Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4G.eIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G.eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G(Ser(1108)) and a decreased assembly of inactive 4E-BP1.eIF4E complex. The reduced assembly of 4E-BP1.eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the gamma-form during feeding. Phosphorylation of S6K1 on Ser(789) was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser(2448) or Ser(2481), an upstream kinase responsible for phosphorylating both S6K1 and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating mTOR, was elevated only after 0.5 h of meal feeding for Thr(308), whereas phosphorylation Ser(473) was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G.eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E.     

Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle

Muscle protein synthesis rates decrease during contraction/exercise, but rapidly increase post-exercise. Previous studies mainly focused on signaling pathways that control protein synthesis during post-exercise recovery, such as mTOR and its downstream targets S6K1 and 4E-BP1. In this study, we investigated the effect of high-frequency electrical stimulation on the phosphorylation state of signaling components controlling protein synthesis in rat skeletal muscle. Electrical stimulation increased S6K1 Thr389 phosphorylation, which was unaffected by Torin1, a selective mTOR inhibitor, suggesting that S6K1 phosphorylation by contraction was mTOR-independent. Phosphorylation of eIF4B Ser422 was also increased during electrical stimulation, which was abrogated by inhibition of MEK/ERK/RSK1 activation. Moreover, although phosphorylation of conventional mTOR sites in 4E-BP1 decreased during contraction, mTOR-independent phosphorylation was also apparent, which was associated with the release of 4E-BP1 from eIF4E. The results indicate mTOR-independent phosphorylation of S6K1 and 4E-BP1 and suggest MEK/ERK/RSK1-dependent phosphorylation of eIF4B during skeletal muscle contraction. These phosphorylation events would keep the translation initiation machinery “primed” in an active state so that protein synthesis could quickly resume post-exercise.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.