Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.     

Glutamatergic regulation of the p70S6 kinase in primary mouse neurons

Brief glutamatergic stimulation of neurons from fetal mice, cultured in vitro for 6 days, activates the mTOR-S6 kinase, ERK1/2 and Akt pathways, to an extent approaching that elicited by brain-derived neurotrophic factor. In contrast, sustained glutamatergic stimulation inhibits ERK, Akt, and S6K. Glutamatergic activation of S6K is calcium/calmodulin-dependent and is prevented by inhibitors of calcium/calmodulin-dependent protein kinase 2, phosphatidylinositol 3-OH-kinase and by rapamycin. 2-Amino-5-phosphonovaleric acid, an inhibitor of N'-methyl-D-aspartate receptors, abolishes glutamatergic activation of ERK1/2 but not the activation of mTOR-S6K; the latter is completely abolished by inhibitors of voltage-dependent calcium channels. Added singly, dopamine gives slight, and norepinephrine a more significant, activation of ERK and S6K; both catecholeamines, however, enhance glutamatergic activation of S6K but not ERK. After 12 days in culture, the response to direct glutamatergic activation is attenuated but can be uncovered by suppression of gamma-aminobutyric acid interneurons with bicuculline in the presence of the weak K(+) channel blocker 4-aminopyridine (4-AP). This selective synaptic activation of mTOR-S6K is also resistant to APV and inhibited by Ca(2+) channel blockers and higher concentrations of glutamate. Elongation factor 2 (EF2) is phosphorylated and inhibited by the eEF2 kinase (CaM kinase III); the latter is inhibited by the S6K or Rsk. Bicuculline/4-AP or KCl-induced depolarization reduces, whereas higher concentrations of glutamate increases, EF2 phosphorylation. Thus the mTOR-S6K pathway in neurons, a critical component of the late phase of LTP, is activated by glutamatergic stimulation in a calcium/calmodulin-dependent fashion through a calcium pool controlled by postsynaptic voltage-dependent calcium channels, whereas sustained stimulation of extrasynaptic glutamate receptors is inhibitory.     

Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.     

Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investigated the role of AMPK activation in regulating protein synthesis during both phenylephrine- and Akt-induced cardiac hypertrophy. Metformin and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside were used to activate AMPK in neonatal rat cardiac myocytes. Activation of AMPK significantly decreased protein synthesis induced by phenylephrine treatment or by expression of constitutively active Akt. Activation of AMPK also resulted in decreased p70S6 kinase phosphorylation and increased phosphorylation of eEF2, suggesting that inhibition of protein synthesis involves the eEF2 kinase/eEF2 axis and/or the p70S6 kinase pathway. Together, our data suggest that the inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism by which hypertrophic growth can be controlled.     

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.     

Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation

Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of eukaryotic mRNAs in the cell. The elongation stage of mRNA translation requires a substantial amount of energy and also eukaryotic elongation factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up-regulated by various mechanisms in the eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain elongation in eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the elongation factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology.     

Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.     

Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain

The pathogenesis of formation of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains is unknown. One of the possibilities might be that translation of tau mRNA is aberrantly regulated in AD brains. In the current study, levels of various translation control elements including total and phosphorylated (p) forms of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), eukaryotic elongation factor 2 (eEF2), and eEF2 kinase were investigated in relationship with tau in homogenates of the medial temporal cortex from 20 AD and 10 control brains. We found that levels of p-mTOR (Ser2481), and p-4E-BP1 (Thr70 and Ser65) dramatically increase in AD, and are positively significantly correlated with total tau and p-tau. Levels of p-eEF2K were significantly increased, and total eEF2 significantly decreased in AD, when compared to controls. The changes of p-mTOR (2481), p-4E-BP1, and p-eEF2 were immunohistochemically confirmed to be in neurons of AD brains. This suggested that there are obvious abnormalities of elements related with translation control in AD brain and their aberrant changes may up-regulate the translation of tau mRNA, contributing to hyperphosphorylated tau accumulation in NFT-bearing neurons.     

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.     

Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90(RSK1) (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90(RSK1).     

Coordination of changes in expression and phosphorylation of eukaryotic elongation factor 2 (eEF2) and eEF2 kinase in hypertrophied cardiomyocytes

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is one of the Ca²⁺/calmodulin-dependent protein kinases. Activated eEF2K phosphorylates its specific substrate, eEF2, which results in inhibition of protein translation. We have recently shown that protein expression of eEF2K was specifically increased in hypertrophied left ventricles (LV) from spontaneously hypertensive rats (SHR). However, phosphorylation state of eEF2K and eEF2 in hypertrophied LV is not determined. In the present study, we examined expression and phosphorylation of eEF2K and eEF2 in LV from SHR as well as the pressure overload (transverse aortic constriction: TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy model. In LV from TAC mice, eEF2K expression was increased as determined by Western blotting. In LV from TAC mice and SHR, eEF2K phosphorylation at Ser366 (inactive site) was decreased. Consistently, eEF2 phosphorylation at Thr56 was increased. In LV from ISO rats, while eEF2K phosphorylation was decreased, eEF2K expression and eEF2 phosphorylation were not different as determined by Western blotting. In the results obtained from immunohistochemistry, however, total eEF2K and phosphorylated eEF2 (at Thr56) localized to cardiomyocytes were increased in LV cardiomyocytes from ISO rats. Accordingly, the increased expression and the decreased phosphorylation of eEF2K and the increased phosphorylation of eEF2 in hypertrophied LV were common to all models in this study. The present results thus suggest that cardiac hypertrophy may be regulated at least partly via eEF2K-eEF2 signaling pathway.     

Cytoplasmic complex of p53 and eEF2

We have shown previously that cytoplasmic p53 is covalently linked to 5.8S rRNA. The covalent complex is associated with a small subset of polyribosomes, which includes polyribosomes translating p53 mRNA. Because 5.8S rRNA resides in or near the ribosomal P site, our findings suggested involvement of p53 in translational regulation. Ninety-seven kiloDaltons eEF2 was found to coimmunoprecipitate in a salt-stable complex with p53. The 97 kDa species was identified as eEF2, because it was (1) recognized by a polyclonal antiserum specific for eEF2, (2) ADP-ribosylated by diphtheria toxin (DT), and (3) radiolabeled by gamma-32P-azido-GTP and UV-irradiation. p53 and eEF2 sedimented in sucrose gradients in both polyribosomal and subribosomal fractions. Subribosomal p53 can bind eEF2 without the mediation of ribosomes, because (1) it binds subribososomal eEF2, (2) it binds phosphorylated eEF2, and (3) subribosomal p53-bound eEF2 can be ADP-ribosylated by DT. No effect of p53 activation was found on eEF2 expression or phosphorylation. However, the binding of eEF2 to p53 decreased when cytoplasmic p53 migrated to the nucleus. Renaturation of temperature sensitive A135V mutant p53 (ts-p53) was found to alter the sensitivity of p53 mRNA translation, but not bulk mRNA translation, to the translocation-specific elongation inhibitor, cycloheximide (Cx). The association of p53 with two translational components involved in ribosomal translocation, eEF2 and 5.8S rRNA, and the effect of p53 on sensitivity to the translocation inhibitor, Cx, as well as the known molecular interactions of these components in the ribosome suggest involvement of p53 in elongation.     

A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin

Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.     

Pharmacological eEF2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV‐PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti‐viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir‐resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir‐mediated anti‐tumoral activity is severely compromised in eEF2K‐deficient engrafted tumors in vivo. Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti‐tumoral properties of HIV‐PIs.     

Thermal injury impairs cardiac protein synthesis and is associated with alterations in translation initiation

The purpose of the present study was to determine whether burn injury decreases myocardial protein synthesis and potential contributing mechanisms for this impairment. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied 24 h late. Burn decreased the in vivo-determined rate of myocardial protein synthesis and translation efficiency by 25% but did not alter the protein synthetic rate in skeletal muscle. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, we examined several eukaryotic initiation factors (eIFs) and elongation factors (eEFs). Burn failed to alter eIF2B activity or the total amount or phosphorylation status of either eIF2 alpha or eIF2B epsilon in heart. In contrast, hearts from burned rats demonstrated 1) an increased binding of the translational repressor 4E-BP1 with eIF4E, 2) a decreased amount of eIF4E associated with eIF4G, and 3) a decreased amount of the hyperphosphorylated gamma-form of 4E-BP1. These changes in eIF4E availability were not seen in gastrocnemius muscle where burn injury did not decrease protein synthesis. Furthermore, constitutive phosphorylation of mTOR, S6K1, the ribosomal protein S6, and eIF4G were also decreased in hearts from burned rats. Burn did not appear to adversely affect elongation because there was no significant difference in the myocardial content of eEF1 alpha or eEF2 or the phosphorylation state of eEF2. The above-mentioned burn-induced changes in mRNA translation were associated with an impairment of in vitro myocardial performance. Finally, 24 h postburn, the cardiac mRNA content of IL-1 beta, IL-6, and high-mobility group protein B1 (but not TNF-alpha) was increased. In summary, these data suggest that thermal injury specifically decreases cardiac protein synthesis in part by decreasing mRNA translation efficiency resulting from an impairment in translation initiation associated with alterations in eIF4E availability and S6K1 activity.     

Hypoxia-induced energy stress regulates mRNA translation and cell growth

Oxygen (O2) deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators, including eIF2alpha, eEF2, and the mammalian target of rapamycin (mTOR) effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMP-activated protein kinase (AMPK) activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2alpha, eEF2, and mTOR inhibition represent important HIF-independent mechanisms of energy conservation that promote survival under low O2 conditions.