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Coping with stress: eIF2 kinases and translational control R.C. Wek*1 , H.-Y. Jiang* and T.G. Anthony† *Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A., and †Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Evansville, IN 47712, U.S.A.
Abstract In response to environmental stresses, a family of protein kinases phosphorylate eIF2 (eukaryotic initiation factor 2) to alleviate cellular injury or alternatively induce apoptosis. Phosphorylation of eIF2 reduces global translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions. Accompanying this general protein synthesis control, eIF2 phosphorylation induces translation of specific mRNAs, such as that encoding the bZIP (basic leucine zipper) transcriptional regulator ATF4 (activating transcription factor 4). ATF4 also enhances the expression of additional transcription factors, ATF3 and CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 (growth arrest and DNA-damage-inducible protein), that assist in the regulation of genes involved in metabolism, the redox status of the cells and apoptosis. Reduced translation by eIF2 phosphorylation can also lead to activation of stress-related transcription factors, such as NF-κB (nuclear factor κB), by lowering the steady-state levels of short-lived regulatory proteins such as IκB (inhibitor of NF-κB). While many of the genes induced by eIF2 phosphorylation are shared between different environmental stresses, eIF2 kinases function in conjunction with other stress-response pathways, such as those regulated by mitogen–activated protein kinases, to elicit gene expression programmes that are tailored for the specific stress condition. Loss of eIF2 kinase pathways can have important health consequences. Mice devoid of the eIF2 kinase GCN2 [general control non-derepressible-2 or EIF2AK4 (eIF2α kinase 4)] show sensitivity to nutritional deficiencies and aberrant eating behaviours, and deletion of PEK [pancreatic eIF2α kinase or PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase) or EIF2AK3] leads to neonatal insulin-dependent diabetes, epiphyseal dysplasia and hepatic and renal complications.
The family of eIF2 (eukaryotic initiation factor 2) kinases regulate translation during different stress conditions Eukaryotic cells recognize and process diverse stress signals to elicit programmes of gene expression that are designed to remediate cellular damage, or alternatively induce apoptosis. An important contributor to stress adaptation is a family of protein kinases that phosphorylate the α subunit of eIF2. Four different eIF2 kinases have been identified in mammals, and each contains unique regulatory regions that recognize a different set of stress conditions (Scheme 1). For example, the eIF2 kinase GCN2 [general control non-derepressible-2 or EIF2AK4 (eIF2α kinase 4)] is induced during amino acid deprivation by a mechanism that involves uncharged tRNA binding to a regulatory region homologous with HisRS (histidyl-tRNA synthetase) enzymes [1,2]. Interestingly, Key words: apoptosis, eukaryotic initiation factor 2 (eIF2), protein kinase, stress response, translational control, UV irradiation. Abbreviations used: ATF, activating transcription factor; bZIP, basic leucine zipper; CHOP, CCAAT/enhancer-binding protein homologous protein; eIF2, eukaryotic initiation factor 2; EIF2AK, eIF2α kinase; ER, endoplasmic reticulum; GADD153, growth arrest and DNA-damage-inducible protein 153; GCN2, general control non-derepressible-2; HisRS, histidyl-tRNA synthetase; IκB, inhibitor of NF-κB; IKK, IκB kinase; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamcyin; NF-κB, nuclear factor κB; PEK, pancreatic eIF2α kinase; PERK, RNA-dependent protein kinase-like ER kinase; PKR, RNA-dependent protein kinase; uORF, upstream open reading frame; WRS, Wolcott–Rallison syndrome. To whom correspondence should be addressed (email
[email protected]).
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GCN2 is also activated by other stresses that are not directly related to nutritional deprivation, including UV irradiation and proteasome inhibition, and genetic studies suggest a role for the HisRS-related domain and uncharged tRNA in response to diverse stresses. Phosphorylation of eIF2 impedes recycling of eIF2 to its active GTP-bound form (Scheme 1), and the accompanying reduction in the levels of eIF2– GTP reduces global translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions. Additional mammalian eIF2 kinases include PEK {pancreatic eIF2α kinase or PERK [PKR (RNA-dependent protein kinase)-like ER (endoplasmic reticulum) kinase] or EIF2AK3} that is activated in response to misfolded protein in the ER (ER stress) [3], PKR which participates in an anti-viral defence mechanism that is mediated by interferon [4], and HRI (haem-regulated inhibitor or EIF2AK1) that is activated by haem deprivation, and oxidative and heat stresses in erythroid tissues [5,6] (Scheme 1).
ATF4 (activating transcription factor 4) is integral to the eIF2 kinase pathway Phosphorylation of eIF2 reduces global protein synthesis concomitant with induced translation of selected mRNAs, such as those encoding the bZIP (basic leucine zipper) C 2006
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Scheme 1 Protein kinases, PKR, HRI (haem-regulated inhibitor),
Figure 1 Phosphorylation of eIF2 enhances ATF4, a
PEK and GCN2 are activated by different stress conditions to regulate the levels of eIF2–GTP via eIF2 phosphorylation
transcriptional activator of stress-responsive genes (A) The ATF4 mRNA is shown as a line with uORFs 1 and 2 and the
eIF2 associates with initiator Met-tRNAi Met (aminoacylated initiator methionyl-tRNA) and GTP, and participates in the ribosomal selection of the start codon [36]. As a prelude to the joining of the small and
ATF4-coding regions are illustrated as boxes. The shading of the small ribosomal subunits indicates that it is associated with eIF2–GTP bound with initiator Met-tRNAi Met (aminoacylated initiator methionyl-tRNA).
large ribosomal subunits, GTP complexed with eIF2 is hydrolysed to GDP, and eIF2–GDP is released from the translational machinery. The GDP-bound eIF2 is recycled to the active eIF2–GTP by a reaction catalysed
Phosphorylation of eIF2 reduces eIF2–GTP levels and facilitates the bypass of the inhibitory uORF2, thereby enhancing translation of the ATF4-coding region in response to stress conditions. (B) Phospho-
by the guanine nucleotide-exchange factor, eIF2B. Four different protein kinases have been identified in mammalian cells that phosphorylate the α subunit of eIF2 at Ser-51 in response to different environmental
rylation of eIF2 induces ATF4, ATF3 and CHOP, and each of these transcription factors regulate gene expression in response to environmental stresses. Additional stress pathways, such as those regulated by
stresses. Phosphorylation of eIF2 alters this translation factor from a substrate to an inhibitor of eIF2B. The ensuing reduction in eIF2–GTP levels lowers general translation, allowing cells sufficient time to correct
mitogen-activated protein kinase pathways, can contribute to the activity of ATF3 and CHOP [37–39].
the stress damage, and selectively enhance gene-specific translation that is important for stress remediation.
transcriptional activator ATF4 [7–10]. Enhanced ATF4 expression involves two uORFs (upstream open reading frames) located in the 5 -leader of the ATF4 mRNA that facilitate translation of this transcriptional activator in response to eIF2 phosphorylation (Figure 1A). The 5 -proximal uORF1 encodes a polypeptide only three amino acid residues in length, and uORF2 is 59 residues long and overlaps the ATF4 coding region. To determine the contribution of the 5 -leader sequence in ATF4 translation, a segment including this leader sequence and the ATF4 initiation codon was fused to a luciferase reporter gene [9]. The ATF4–luciferase fusion was expressed from a minimal TK (thymidine kinase) promoter and transfected into MEF (mouse embryonic fibroblast) cells containing wild-type eIF2α (S/S) or a mutant version containing alanine substituted for the phosphorylated Ser-51 residue (A/A). The roles of the two uORFs were analysed by mutating the start codons, individually or in combination, and studying the consequence of these changes for ATF4 translational control. Our studies indicate that the two uORFs contribute differentially to ATF4 expression [9]. The uORF1 is a positive-acting element that facilitates ribosome scanning and re-initiation at downstream coding C 2006
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regions in the ATF4 mRNA (Figure 1A). When eIF2–GTP is plentiful in non-stressed cells, ribosomes scanning downstream of uORF1 re-initiate at the next coding region: uORF2, an inhibitory element that blocks ATF4 expression. Under stress conditions, phosphorylation of eIF2, and the resulting reduction in the levels of eIF2–GTP, increases the time required for the scanning ribosomes to become competent to re-initiate translation. This delayed re-initiation allows for ribosomes to scan through the inhibitory uORF2, and instead re-initiate at the ATF4-coding region. Elevated levels of ATF4 lead to induction of additional bZIP transcriptional regulators, ATF3 and CHOP (CCAAT/ enhancer-binding protein homologous protein)/GADD153 (growth arrest and DNA-damage-inducible protein 153), which together induce a programme of gene expression
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important for cellular remediation and apoptosis (Figure 1B). Analysis of ATF4−/− and ATF3−/− MEF cells subjected to amino acid depletion or ER stress indicates that there is a sequential induction of transcription factors, whereby ATF4 induces ATF3, and ATF3 is required for enhanced expression of CHOP [7]. These transcriptional regulators can form heterodimers with other bZIP proteins, and directly or indirectly regulate expression of a large number of genes involved in metabolism and amino acid transport, the redox status of the cell, signalling and transcription, and apoptosis [11]. ATF3 and CHOP also enhance expression of GADD34, which is required for feedback control of the eIF2α kinase response by targeting the type 1 serine/threonine protein phosphatase to eIF2 [7,12–15]. While the cascade of bZIP transcription factors appears to be a conserved feature among many different cellular stress responses, there are certain stress arrangements whereby eIF2 phosphorylation does not enhance ATF4 expression. For example, there is significant GCN2 phosphorylation of eIF2 following UV irradiation, yet there is no induction of ATF4 expression [16,17]. Our studies suggest that ATF4 mRNA is substantially reduced during UV stress and therefore not amenable to translation regulation in response to eIF2 phosphorylation [17]. However, ATF3 expression was increased between 5 and 20 J/m2 , with minimal expression at 40 J/m2 . ATF3 induction was independent of GCN2, indicating that there are certain stress conditions that lead to enhanced expression of ATF3 independently of eIF2 phosphorylation. These observations suggest that there is versatility in the regulatory proteins induced by eIF2 phosphorylation, allowing for programmes of gene expression to be tailored to a given stress condition.
Phosphorylation of eIF2 activates NF-κB (nuclear factor κB) in response to diverse stresses NF-κB co-ordinates the transcription of genes involved in immune and inflammatory responses, cell growth and apoptosis in response to a range of environmental stresses, including UV irradiation and amino acid depletion [18–20]. To address the role of eIF2 phosphorylation in the induction of NF-κB in response to different stress conditions, the activity of this transcription factor was measured by using EMSA (electrophoretic mobility-shift assay) and nuclear lysates prepared from MEF cells deficient in eIF2 kinase function or their wild-type counterparts. GCN2 phosphorylation of eIF2 was required for activation of NF-κB in response to amino acid starvation or UV irradiation [17,21] (Scheme 2). Furthermore, PEK enhanced NF-κB transcriptional activity in response to ER stress [21]. Together, these studies indicate that eIF2 kinases facilitate NF-κB activity in response to a range of stress conditions. In its inert state, NF-κB is present in the cytoplasm in association with proteins known as inhibitors of NF-κB (IκBs), the most prominent and well-studied being IκBα. Exposure of cells to cytokines leads to IκBα phosphorylation
Scheme 2 GCN2 activates NF-κB in response to UV irradiation via reducing the levels of IκBα Phosphorylation of eIF2 by GCN2 reduces general translation and the synthesis of IκBα, leading to the lowered IκBα levels in MEF cells subjected to UV exposure. Mitogen-activated protein kinase p38 is proposed to direct CK2 phosphorylation of the PEST (Pro-Glu-Ser-Thr) region of IκBα, facilitating ubiquitination and proteasome-mediated degradation of IκBα in response to UV irradiation [23]. The resulting lowered levels of IκBα would release this inhibitory protein from NF-κB, allowing NF-κB to be translocated into the nucleus. Upon nuclear entry, NF-κB binds at DNA elements in the promoters of regulated genes, including those involved in remediation of stress damage and protection from apoptosis. Phosphorylation and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases in MEF cells occurs in the absence of GCN2 eIF2 kinase activity, as highlighted by the broken lines [16].
by IKK (IκB kinase) [18]. Phosphorylation of IκBα targets it for ubiquitination and proteasome-mediated degradation. Reduced levels of IκBα release NF-κB from this repressor, facilitating translocation of NF-κB into nucleus to regulate expression of its target genes. Activation of NF-κB in response to eIF2 phosphorylation does not involve IKK phosphorylation of IκBα [17,21]. Therefore eIF2 kinases represent a new pathway for activation of NF-κB. Lowered levels of the inhibitory protein IκBα are central to mechanisms activating NF-κB in response to UV irradiation [17,22] (Scheme 2). Reduced general protein synthesis in response to UV irradiation is directed by GCN2. Translation of IκBα mRNA is very sensitive to eIF2 phosphorylation, with a significant reduction in IκBα synthesis as compared with translation of the NF-κB subunit RelA/p65 (Scheme 2). Lowered synthesis of IκBα, coupled with the p38 mitogen-activated protein kinase-mediated turnover of this inhibitory protein [23], is suggested to trigger the nuclear translocation of NF-κB (Scheme 2). It is noted that while PEK phosphorylation of eIF2 is required for activation of NF-κB in response to ER stress there was no significant reduction in IκBα levels, suggesting that there are differences between the mechanisms inducing NF-κB in response to these two stress conditions [21]. This is best exemplified by the observation C 2006
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that pre-treatment of MEF cells with proteasome inhibitors, which would stabilize IκBα, blocked activation of NF-κB in response to UV irradiation, but did not diminish induction of this transcription factor in response to ER stress [17]. Co-immunoprecipitation studies suggest that the mechanism by which eIF2 phosphorylation activates NF-κB during ER stress involves release of IκBα from NF-κB, facilitating NFκB entry into the nucleus to enhance transcription of its target genes [21].
Phosphorylation of eIF2 can direct pro-survival and pro-apoptotic cellular pathways eIF2 kinase and NF-κB pathways regulate the expression of genes that are involved in stress remediation and apoptosis. To determine their roles in apoptosis, survival of MEF cells deleted for GCN2 or RelA/p65 was measured following UV irradiation. Deletion of either GCN2 or the subunit of NF-κB increased activation of caspases 3 and 8 and enhanced apoptosis in response to UV stress [17]. This result suggests that GCN2 modulation of NF-κB activity is important for protection against UV-induced apoptosis. GCN2 also induces eIF2 phosphorylation in response to treatment with different proteasome inhibitors that have been shown to be effective anti-cancer agents [24]. In this case, deletion of eIF2 kinase activity blocked caspase activation and delayed apoptosis. Furthermore, deletion of CHOP lowered apoptosis by 50% following 16 h of proteasome inhibitor treatment [24]. Therefore, under certain stress conditions, phosphorylation of eIF2 can elicit pro-apoptotic pathways, and eIF2 kinases may be central to the efficacy of anticancer drugs that target the ubiquitin–proteasome pathway. GCN2 phosphorylation of eIF2 in response to UV irradiation facilitates activation of NF-κB, but does not lead to accumulation of the pro-apoptotic factor CHOP. We propose that this regulatory pattern contributes to the prosurvival function of GCN2 in response to UV stress [17]. There is no induction of NF-κB by proteasome inhibition. In fact, pre-treatment with proteasome inhibitors can block NF-κB activation in response to exposure to cytokines or UV irradiation by stabilizing the labile IκBα protein. Failure of eIF2 phosphorylation to activate NF-κB in response to proteasome inhibition, combined with induced expression of CHOP, may be an important underlying reason for the pro-apoptotic function of GCN2 in response to proteasome inhibitors [24].
Physiological consequences of loss of GCN2 and PEK The importance of the eIF2 kinase pathway for normal physiology and growth is exemplified by data from mice carrying null mutations in the genes encoding GCN2 or PEK. Mice deficient for PEK show severe, but proportional, growth retardation that manifests within the first few days of post-natal development [25,26]. A defect in insulin pro C 2006
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duction from the β-cells of the pancreas produces a neonatal or early infancy insulin-dependent diabetes. These combined features are characteristic of a rare autosomal recessive disorder in humans known as WRS (Wolcott– Rallison syndrome) that is caused by mutations in the PEK/EIF2AK3 gene [27–29]. Other clinical manifestations of WRS include epiphyseal dysplasia, osteoporosis, hepatic and kidney dysfunction, exocrine pancreatic disorders, and neutropenia. Patients with the most severe clinical manifestations have nonsense or missense mutations in the catalytic/ kinase domain of the protein, resulting in partial or complete loss of PEK function [29]. Yet, overall, the disease displays a heterogeneous clinical profile, due to either variation in other genes that regulate ER stress responses or other environmental conditions that modulate ER stress. Initial examination of mice deleted for GCN2 revealed an unremarkable phenotype, with animals growing and reproducing normally under freely fed conditions [30]. Challenged with a leucine-free diet for 6 days, GCN2−/− mice lose more body mass than wild-type comparisons and become sickly in appearance [31]. For reasons not entirely clear, a subset (∼40%) dies or requires mercy-killing after 3–5 days. While all mice fed a leucine-devoid diet for several days demonstrate low plasma leucine and insulin, surviving GCN2−/− mice also display high circulating concentrations of isoleucine, valine and alanine, and disproportionate reductions in hind-limb muscle mass, suggestive of elevated protein catabolism. Interestingly, while liver protein synthesis is initially inhibited in response to a leucine-free diet, this reduction is not maintained in GCN2−/− mice, resulting over time in the sparing of liver mass, at the apparent expense of muscle mass [31]. Furthermore, normal down-regulation of mTOR (mammalian target of rapamcyin) signalling in response to amino acid deprivation is abated in GCN2−/− mice, suggesting that GCN2 contributes to mTOR regulation in response to amino acid depletion. The combination of studies in situ and in vivo confirms that GCN2 is the primary eIF2 kinase in response to essential amino acid limitation, and this regulatory pathway is important for managing nutritional stress. GCN2 also participates in nutritional stress management that guides food selection for survival. Animals faced with a diet lacking in essential amino acids quickly reject the imbalanced food and forage for complete or complementary sources of protein [32]. The rejection of the insufficient meal is linked to increased phosphorylation of eIF2 in the anterior piriform cortex of the brain [33]. This event requires sensing of deacylated tRNA by functional GCN2. Rats injected with an (L- but not D-) alcohol derivative of an essential amino acid reject a complete diet, and mice deleted for GCN2 fail to phosphorylate eIF2 and do not display the feeding-aversion behaviour [34,35].
This study was supported in part by grants R01GM49164 and R01GM64350 (to R.C.W.) from the National Institutes of Health.
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