Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors Jashmin Patel 1 , Laura E. McLeod 2 , Robert G. J. Vries 1 , Andrea Flynn 1 , Xuemin Wang 1,2 and Christopher G. Proud 1,2 1 Department of Biosciences, University of Kent at Canterbury, Canterbury, UK; 2 Division of Molecular Physiology, School of Life Sciences, University of Dundee, UK We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( 30 min) caused a profound inhibition of protein syn- thesis. In each case this was accompanied by dephosphory- lation of the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and increased binding of this repressor protein to eIF4E. Binding of 4E-BP1 to eIF4E correlated with loss of eIF4F complexes. Sorbitol and hydrogen per- oxide each caused inhibition of the 70-kDa ribosomal pro- tein S6 kinase, while arsenite activated it. The effects of stresses on the phosphorylation of eukaryotic elongation factor 2 also differed: oxidative stress elicited a marked increase in eEF2 phosphorylation, which is expected to contribute to inhibition of translation, while the other stresses did not have this effect. Although all three proteins (4E-BP1, p70 S6 kinase and eEF2) can be regulated through the mammalian target of rapamycin (mTOR), our data imply that stresses do not interfere with mTOR function but act in different ways on these three proteins. All three stresses activate the p38 MAP kinase pathway but we were able to exclude a role for this in their effects on 4E-BP1. Our data reveal that these stress-inducing agents, which are widely used to study stress-signalling in mammalian cells, exert multiple and complex inhibitory effects on the translational machinery. Keywords: stress; initiation; elongation factor; mRNA translation; S6 kinase. The control of mRNA translation in mammalian cells involves the regulation of a range of components of the translational machinery, principally by changes in their phosphorylation, leading to modulation of their activities or their abilities to interact with one another [1,2]. Initiation factor 4E (eIF4E) plays a key role in mRNA translation and its control in eukaryotic cells. eIF4E binds to the 5¢ cap structure (containing 7-methylguanosine triphosphate; m 7 GTP) which is present at the 5¢ end of all cellular cytoplasmic mRNAs [3,4]. eIF4E can be regulated by its own phosphorylation (which occurs at a single major site (Ser209) [5,6]; and by binding proteins (4E-BPs) that modulate its availability for initiation complex formation (reviewed in [7]). eIF4E forms a complex termed eIF4F, which also contains the translation factors eIF4G (formerly called p220) and eIF4A. eIF4A has ATP-dependent RNA helicase activity thought to be required to unwind regions of self-complementary secondary structure in the 5¢ UTRs of certain mRNAs [4,8]. Such secondary structure inhibits translation and therefore mRNAs with 5¢ UTRs that contain significant secondary structure are often poorly translated. In contrast to many other cellular mRNAs, translation of heat shock protein mRNAs appears to be relatively cap-independent (reviewed in [9–11]), and trans- lation of the mRNA for the stress-protein BiP/grp78 occurs by a cap-independent mechanism [12]. The eIF4E binding proteins (4E-BPs) 1 and 2 interact with eIF4E and inhibit cap-dependent mRNA translation [13–15]. 4E-BP1 (also termed PHAS-I) competes with eIF4G for binding to eIF4E, preventing formation of the eIF4F complex and thus potentially inhibiting the recruit- ment of eIF4A to the initiation complex on the 5¢ end of the mRNA [16,17]. 4E-BP1 does not block the translation of mRNAs that contain features allowing cap-independent initiation to occur, e.g. internal ribosome-entry elements derived from picornaviral mRNAs [13,15]. 4E-BP1 is a phosphoprotein whose state of phosphorylation increases in response to insulin and other agents that activate translation (reviewed in [7]). This causes its dissociation from eIF4E. Studies on 4E-BP2 (PHAS-II) show that its phosphoryla- tion is also enhanced by insulin and that this causes it to dissociate from eIF4E [18]. The main signalling pathway that regulates 4E-BP1 phosphorylation is inhibited by the immunosuppressant rapamycin (reviewed in [7]), a specific inhibitor of the FRAP/TOR signalling pathway, which also leads to activation of p70 ribosomal protein S6 kinase Correspondence to C. Proud, Division of Molecular Physiology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. Fax: + 44 1382322424; Tel.: + 44 1382344919; E-mail: c.g.proud@dundee.ac.uk Abbreviations: 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; EF2, elongation factor 2; mTOR, mammalian target of rapamycin; eIF4E, initiation factor 4E; m 7 GTP, 7-methylguanosine triphosphate; CaM, calmodulin; eIF, eukaryotic initiation factor; rpS6, ribosomal protein S6. (Received 11 March 2002, revised 24 April 2002, accepted 2 May 2002) Eur. J. Biochem. 269, 3076–3085 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02992.x (p70 S6k), although 4E-BP1 and p70 S6k appear to lie on separate branches of this pathway [19]. p70 S6k is activated by insulin and growth factors (reviewed in [20]) and appears to play a role in up-regulating the translation of mRNAs that are characterized by possessing 5¢ terminal tracts of pyrimidines (and are thus termed 5¢-TOP mRNAs [21,22]). Agents that activate p70 S6k up-regulate the otherwise low basal translation of these mRNAs, and evidence has been presented suggesting a causal link between these events, although this remains to be confirmed [21]. A third component of the translational machinery that can be regulated through mTOR is elongation factor eEF2, the protein that mediates the translocation step of elongation [23,24]. Phosphorylation of eEF2 inhibits its activity, apparently by inhibiting its ability to interact with the ribosome [25] (reviewed in [24]). Insulin induces the dephosphorylation of eEF2 and this is blocked by rapamy- cin, demonstrating a requirement for mTOR dependent signalling. The effect of insulin appears to involve a decrease in the activity of the kinase that acts on eEF2, an unusual calcium/calmodulin (Ca/CaM)-dependent enzyme termed eEF2 kinase [26–28]. We recently showed that eEF2 kinase is phosphorylated and inactivated by p70 S6k, thus estab- lishing a molecular mechanism for the regulation of eEF2 kinase by insulin via mTOR [28]. The initiation factor eIF2 is required to recruit the initiator methionyl-tRNA (Met-tRNA i ) to the 40S ribo- somal subunit [29]. eIF2 is only active when bound to GTP and an additional protein factor, eIF2B, is required under physiological conditions to promote recycling of eIF2 to this form [29,30]. The activity of eIF2B can be modulated in a variety of ways [29,31] including by its own phosphoryla- tion, and through phosphorylation of the a subunit of its substrate, eIF2, at a conserved site (Ser51 in mammals [32]). Control of eIF2B activity is thought to play a key role in regulating overall mRNA translation [30]. Here we have investigated the effects of a range of stressful conditions that are widely employed to study the roles of stress-activated signalling pathways on cell func- tion. We find that these stresses rapidly and profoundly inhibit protein synthesis and markedly alter the phos- phorylation and/or activity of proteins involved in regula- ting mRNA translation. These stressful agents exert effects on several components of the translational machinery, the common feature being that they cause dephosphorylation of 4E-BP1 and thus inhibition of eIF4E. In addition to providing further information on the regulation of a number of components involved in mRNA translation, our data show that it is very important, when evaluating the effects of these stressful agents on cell function, to take into account their marked effects on translation and on the translational machinery. EXPERIMENTAL PROCEDURES Chemicals, biochemicals and other reagents m 7 GTP–Sepharose was from Pharmacia Biotech Inc. [c- 32 P]ATP and 35 S-labelled methionine/cysteine were pur- chased from Amersham Corp. Chinese hamster ovary (CHO.K1) cells were kindly provided by L. Ellis (Hannover School of Medicine, Houston, TX, USA). Materials for tissue culture were obtained from Gibco. Microcystin-LR and rapamycin were from Calbiochem. Recombinant mouse hsp25 was kindly provided by M. Gaestel (Texas A & M University, Berlin, Germany). The antiserum to rodent eIF4E has been described previously [33] and that to 4E-BP1 was raised against a synthetic peptide correspond- ing to residues 101–113 of the human protein and has also been described earlier [34]. The antisera against eIF4G were generously provided by S. J. Morley (University of Sussex, Brighton, UK) or was raised against a synthetic peptide based on part of the C-terminus of eIF4G 1 [35]. The antibody for phosphorylated eEF2 was raised against a synthetic peptide corresponding to the region around Thr56 of mammalian eEF2 and has been described previously [36]. The loading of eEF2 was assessed using an antibody that reacts with the protein irrespective of its state of phos- phorylation [37]. Cell culture and stress treatment Chinese hamster ovary (CHO.K1) cells were cultured as described previously [38]. Cells were grown to near-conflu- ence prior to exposure to arsenite, hydrogen peroxide or sorbitol at the concentrations and for the times indicated. Where applicable, cells were preincubated with signalling inhibitors (as described in the text) prior to exposing cells to stress conditions. In all cases, cell extracts were prepared as described previously [38] and clarified by centrifugation at 4 °C (13 000 g, 10 min). To assess cell viability, CHO.K1 cells were left untreated or exposed to stress conditions for specific times. After this, cells were washed with NaCl/P i , removed from the plate by trypsin treatment in a volume of 0.5mL,andtrypanbluewasaddedtoaconcentrationof 0.4% (w/v) to the cell suspension. Cells were transferred to a haemocytometer and inspected visually for their ability to exclude the stain. Viability (%) was scored as number of clear cells/total number of cells · 100. Analysis of eIF4E and associated proteins eIF4E and associated proteins were isolated from cell extracts by affinity chromatography on m 7 GTP–Sepharose and bound proteins were subjected to SDS/PAGE and Western blotting as described previously [34,39] (any minor modi- fications are noted in the text). Gel electrophoresis, isoelectric focusing and immunoblotting For most purposes, samples were subjected to electrophor- esis on SDS/polyacrylamide gels containing 15% acryla- mide/0.4% methylene bis-acrylamide. For analysis of eEF2, gels contained 10% (w/v) acrylamide plus 0.1% methylene bis-acrylamide. For analysis of the electrophoretically distinct forms of 4E-BP1, gels contained 13.5% (w/v) acrylamide and 0.36% (w/v) methylene bis-acrylamide. In all cases, proteins were transferred to poly(vinylidene difluoride) membrane (Millipore) and Western blotting was performed as described earlier [40] using the Enhanced Chemiluminescence (ECL) system (Amersham plc). Other assay procedures Rates of protein synthesis were assayed in CHO.K1 cells by measuring the incorporation of [ 35 S]methionine/cysteine into acid-insoluble material as described earlier [41]. Ó FEBS 2002 Modulation of translation factors by cellular stress (Eur. J. Biochem. 269) 3077 Approximately 20 lCi of radioisotope (> 1000 CiÆ mmol )1 ) was used per 60-mm dish of cells. p70 S6k activity was assayed, following immunoprecip- itation from cell extracts, using a synthetic peptide substrate based on the C-terminus of S6 [42]. This peptide binds to phosphocellulose paper and incorporated radioactivity was determined by the C ˇ erenkov method. Control assays were performed in each case from which the peptide substrate was omitted to correct for Ôself-incorporationÕ into the immunoprecipitated protein; the values thus obtained were subtracted from those obtained in duplicate assays contain- ing the peptide substrate. eEF2 kinase activity was assayed in CHO.K1 cell extracts using purified eEF2 as a substrate, measuring the incorporation of 32 P into the protein. The extracts were incubated with eEF2 (1 lg) for 20 min at 30 °Cinthe presence and absence of Ca 2+ /CaM. The Ca 2+ /CaM buffer contained 66 m M MgCl 2 ,1.2m M ATP, 4 m M CaCl 2 and 3 lgÆlL )1 CaM while the Ca 2+ /CaM-free buffer contained 66 m M MgCl 2 ,1.2m M ATP and 1 m M EGTA. These concentrations are for the stock solutions which were diluted sixfold in the assays. To terminate the reactions, SDS sample buffer was added and samples were incubated at 95 °C for at least 5 min. The denatured samples were analysed 10% SDS/PAGE and the results visualized by autoradiography. RESULTS AND DISCUSSION Stresses markedly inhibit protein synthesis in CHO cells Treatment of CHO.K1 cells with agents that induce chemical (arsenite), oxidative (hydrogen peroxide) or osmotic (sorbitol) stress led to a rapid and marked inhibition of protein synthesis (Fig. 1A). Each of the stresses employed inhibited protein synthesis by about 80% under the conditions used here. We have previously shown that a different stress-condition, heat shock, also inhibits protein synthesis in these cells [43]. These conditions arewidelyusedtostimulateÔstress-activatedÕ responses such as the stress activated protein kinases (p38 MAP kinases and c-Jun N-terminal kinases, JNKs). There is substantial interest in the roles of these kinases and signalling pathways in the transcriptional control of gene expression, although most of this work ignores possible effects or interference due to modulation of later stages in gene expression, such as mRNA translation. We also analysed the ability of these agents to inhibit protein synthesis over a range of concentrations. For arsenite, half-maximal inhibition occurred at 60 l M (Fig. 1B), while for hydrogen peroxide and sorbitol this degree of inhibition was observed at about 0.5 m M and 0.2 M , respectively (Fig. 1B). For arsenite or hydrogen peroxide, higher concentrations resulted in inhibition by >90%, while the effect of sorbitol was incomplete even at the highest concentration tested here, 0.4 M .Wewere concerned that these chemical stresses might cause a loss of cells, but in all cases we saw no evidence of this over the time periods examined. For example, there was no loss of cellular material as assessed by the protein content of the resulting lysates (data not shown). To assess cell viability more quantitatively, we assessed their ability to exclude trypan blue. As judged by this criterion, cell viability was > 99.5% after 25 min and > 99% after 2 h of treatment with the stress stimuli even up to the highest concentrations of these agents used in this study. Viability was 99% or higher after 4 h, except for the highest concentration of hydrogen peroxide tested (3 m M ) where it was about 94%. It therefore appears that the effects of the stress conditions on protein synthesis (and on translation factor phosphorylation, etc., see below) are not the consequences of toxic effects leading to a loss of cell viability. In view of this substantial inhibition of protein synthesis, it will be important to consider their effects on protein synthesis when studying the effects of these stress conditions on cell physiology. Other agents that inhibit protein synthesis (such as cycloheximide and anisomycin [44–46]) cause activation of stress-activated kinases. Although it may be that the ability of these conditions to inhibit protein synthesis underlies, or contributes to, their stimulation of the stress-activated kinases, the effects of anisomycin on stress-regulated kinases generally occur at concentrations where this agent has little effect on overall protein synthesis. Fig. 1. Cellular stresses inhibit protein synthesis. (A) CHO.K1 cells were incubated with sorbitol (0.4 M ), hydrogen peroxide (3 m M ), or arsenite (100 l M ) for 25 min prior to the addition of [ 35 S]methionine for a further 15 min. Cells were then extracted and samples processed to measure incorporation of label into trichloroacetic acid-precipitable material. Data are expressed as percentage of untreated control cells ± SEM (n ¼ 5, hydrogen peroxide; n ¼ 6, other conditions). (B) Triplicate plates of CHO.K1 cells were incubated with hydrogen peroxide (0.1, 0.2, 1, 3 m M )or sorbitol (0.2, 0.3, 0.4 M ) for 10 min prior to the addition of 20 lCi [ 35 S]methionine for 15 min. The cells were extracted and triplicate samples (60 lg of protein) were processed to measure the incorporation into trichloroacetic acid-precipitable material. Data are expressed as percentages of untreated control cells ± SD (for hydrogen peroxide and sorbitol), where n ¼ 9 for all conditions. For arsenite, data are the mean of triplicate determinations. Incorporation into control samples was typically about 10 000 d.p.m. 3078 J. Patel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Cellular stresses affect the association of eIF4E with 4E-BP1 and eIF4G We have previously shown that the inhibition of protein synthesis by heat shock was associated with increased binding of 4E-BP1 with eIF4E [43]. To examine whether this was a common cellular response to these varied stress conditions, CHO cells were treated for various times with the above agents, extracts were prepared and then subjected to affinity chromatography on m 7 GTP–Sepharose, which retains eIF4E and associated proteins. The bound proteins were analysed by SDS/PAGE and Western blotting. As shown in Fig. 2, treatment with any of the three stresses used above (arsenite, hydrogen peroxide or sorbitol) caused a time-dependent rapid increase in the binding of 4E-BP1 to eIF4E. For sorbitol or hydrogen peroxide, increased binding was seen as early as 5 min after application of the stress, and the effect was maximal at 15–20 min (Fig. 2A,B). For arsenite, the effect was slightly slower, a discernible increase first being visible at 15 min (Fig. 2C). Dose– response studies showed that the effect of sorbitol on the association of 4E-BP1 with eIF4E required 300–400 m M , while that of hydrogen peroxide was already maximal at 0.5 m M (Fig. 2D,E). For CHO cells, 4E-BP1 can be resolved into three electrophoretically distinct species termed a–c,ofwhicha is the least, and c the most, highly phosphorylated. 4E-BP1 undergoes phosphorylation at least six sites that have differing effects on its mobility and/or binding to eIF4E [7,47–50]. Each ÔbandÕ is therefore likely to contain a mixture of different species. In particular, the b form contains some species that bind to eIF4E and some that do not. This is evident from our earlier work [52,53] and from Fig. 2D,F, where in control cells both the b and c species are present but no 4E-BP1 is bound to eIF4E, while in cells treated with 300 m M sorbitol, the protein is mostly present as the b form, but this form is now bound to eIF4E. The main effect of the higher concentrations of sorbitol is to cause the loss of the most phosphorylated c-form, which is not found in association with eIF4E. Loss of this species coincides with the marked increase in binding of 4E-BP1 to eIF4E observed at 300 and 400 m M sorbitol (Fig. 2F). The further dephosphorylation of 4E-BP1 seen at the highest sorbitol concentration results in the appearance of the a species, which can be seen (Fig. 2D) to associate with eIF4E. Similarly, hydrogen peroxide and arsenite each caused a shift in the behaviour of 4E-BP1 to more mobile, less phosphorylated species (data not shown), consistent with the increased binding to eIF4E (Fig. 2A,B). We have previously shown that, as expected, increased binding of eIF4E to 4E-BP1 in CHO cells results in loss of eIF4F complexes in response, e.g. to amino-acid withdrawal [51,52] or heat shock [43]. As anticipated from these earlier studies, treatment of CHO cells with sorbitol, arsenite or higher concentrations of hydrogen peroxide resulted in the loss of eIF4F complexes, as shown by the loss of eIF4G bound to eIF4E that occurred concomitantly with the increased binding of eIF4E to 4E-BP1 (Fig. 3A). In a few experiments, arsenite was observed to cause an increase in the binding of eIF4G to eIF4E when used at concentrations up to 150 l M , even though binding of 4E-BP1 to eIF4E was also enhanced (data not shown). Such findings are hard to reconcile with the simple model where 4E-BP1 and eIF4G Fig. 2. Cellular stresses increase the binding of 4E-BP1 to eIF4E. (A–E) CHO.K1 cells were treated with the indicated reagents for the times and/or using the concentrations shown. After treatment, cell extracts were prepared and samples were subjected to affinity chromatography on m 7 GTP– Sepharose, and the bound material was then analysed by SDS/PAGE followed by immunoblotting using antibodies for eIF4E and 4E-BP1. The positions of migration of eIF4E and 4E-BP1 are indicated. 4E-BP1 generally appears as two bands corresponding to the a and b species of 4E-BP1. The signal for eIF4E serves as a ÔloadingÕ control and should be compared with the signal for 4E-BP1 in each lane. (F) Extracts of cells treated with the indicated concentrations of sorbitol for 25 min were analysed directly by SDS/PAGE and Western blotting using gels containing 13.5% acrylamide/0.36% methylene bis-acrylamide. Arrows labelled a, b and c indicated the positions of these electrophoretically distinct species of 4E-BP1. Ó FEBS 2002 Modulation of translation factors by cellular stress (Eur. J. Biochem. 269) 3079 compete for binding to the same site in eIF4E. Scheper et al. [53] have also reported that arsenite (at similar concentra- tions to those used here) increased the binding of eIF4E to eIF4G. In their case, however, binding of eIF4E to 4E-BP1 was decreased, which is more in line with the expected reciprocal effects on the binding of these two proteins to eIF4E. The total cellular content of eIF4G was not affected by any of these treatments, ruling out the possibility that degradation of eIF4G was the cause of the loss of signal (Fig. 3B). Cellular stresses also affect p70 S6 kinase activity 4E-BP1 phosphorylation is mediated through the rapamy- cin-sensitive mTOR pathway. To assess whether these cellular stresses caused a generalized inhibition of mTOR signalling, we therefore studied their effect on the activity of p70 S6 kinase. Treatment of cells with hydrogen peroxide or sorbitol did indeed cause the inactivation of p70 S6 kinase in a dose-dependent manner (Fig. 4A). For sorbitol, concen- trations that induced dephosphorylation of 4E-BP1 also caused inactivation of p70 S6 kinase. For hydrogen perox- ide, changes in p70 S6 kinase activity were only observed at relative high concentrations of the compound. In contrast to the effects of these agents, arsenite had little effect on p70 S6 kinase activity and even caused modest activation at higher concentrations. This is rather reminiscent of the ability of arsenite to activate p70 S6 kinase in cardiomyocytes [54]. Activation of S6 kinase by all stimuli so far tested is inhibited by rapamycin [20]. The finding that arsenite does not inhibit p70 S6 kinase indicates that arsenite does not cause inhibi- tion of mTOR signalling, because if it did, p70 S6 kinase activity would have been decreased by arsenite. We have previously shown that the activation of p70 S6 kinase by arsenite in cardiomyocytes is blocked by rapamycin [54] indicating that arsenite activates p70 S6 kinase in a manner that still requires the input provided by mTOR. As an indication of intracellular p70 S6 kinase activity, we examined the phosphorylation state of ribosomal protein S6 (rpS6), using an antibody that detects this protein only when it is phosphorylated [28]. Decreases in rpS6 phos- phorylation were observed for cells treated with the higher concentrations of hydrogen peroxide or with sorbitol, where decreased p70 S6 kinase activity was also observed (Fig. 4B). Arsenite had little effect on the phosphorylation of rpS6. Oxidative stress also modulates the phosphorylation of elongation factor 2 A third target of mTOR signalling in CHO cells is eEF2 [26]. The phosphorylation of this protein plays an important role in regulating mRNA translation, by inhibiting the activity of eEF2 [23,25,55]. Given that cellular stresses inhibit translation, it was important to study whether they elicited an increase in the phosphorylation of eEF2. To assess whether cellular stresses affected the phosphorylation state of eEF2, we made use of an antibody that detects eEF2 only when it is phosphorylated at its main site of phosphorylation, Thr56 [36]. The basal level of phosphorylation of eEF2 depends upon the density of the cells: the more nearly confluent the cells, the higher the level of phosphorylation (Fig. 5A, top section, cf. middle section for loading control). The level of phosphorylation of ribosomal protein S6 was observed to fall with increasing cell density (Fig. 5C). Both effects are likely to contribute to a slow-down in protein synthesis [20,23], which is a logical response for cells approaching Fig. 4. Effects of cellular stresses on the activity of p70 S6 kinase. CHO.K1 cells were treated for 25 min with the indicated concentra- tions of the agents under study, and extracts were then prepared. p70 S6 kinase activity was measured, using a synthetic peptide sub- strate, following immunoprecipitation of p70 S6 kinase from the cells extract using an anti-(p70 S6) kinase serum. All assays were performed in duplicate. For arsenite, the data are mean ± SEM for four separate experiments. For hydrogen peroxide and sorbitol, the values shown are from one set of data that is representative of four to five separate experiments performed. Fig. 3. Effects of cellular stresses on the association of eIF4E with eIF4G and 4E-BP1. CHO.K1cellsweretreatedfor25minwiththe indicated concentrations of sorbitol, hydrogen peroxide or arsenite, and extracts were prepared. (A) Samples were subjected to affinity chromatography on m 7 GTP–Sepharose, and the bound material was then analysed by SDS/PAGE followed by immunoblotting using antibodies for eIF4E, eIF4G and 4E-BP1 (positions indicated). (B) Samples of cell lysate were subjected to SDS/PAGE followed by Western blotting with an antibody for eIF4G (position shown). 3080 J. Patel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 confluence. The phosphorylation states of S6 and eEF2 are regulated in opposing directions by mTOR signalling. It may therefore be that mTOR signalling is repressed at higher cell densities, although other explanations are possible. The basis of these effects is not known and further study of this falls outside the scope of this report. However, it is important to be aware of this effect when designing experiments to study the regulation of eEF2 phosphoryla- tion. For example, hydrogen peroxide elicited a marked increase in eEF2 phosphorylation in less dense cells where the initial level of eEF2 phosphorylation is lower, but had no discernible effect in denser cells where basal eEF2 phosphorylation is high (Fig. 5B). This effect was not blocked by an inhibitor of the p38a/b MAP kinase pathway, SB203580 [56], even though this compound effectively inhibits this pathway at the concentration used (see below). In fact, in some experiments, SB203580 actually caused a small increase in the phosphorylation of eEF2 (as seen in Fig. 5B, top section). eEF2 phosphorylation was sensitive to low doses of hydrogen peroxide, increases being seen at concentrations as low as 30 l M (Fig. 5C), with the maximal effect already being seen at about 100 l M . It is thus more sensitive to this agent than either 4E-BP1 or p70 S6 kinase. Treatment of low density cells with hydrogen peroxide led to a reproducible increase in the maximal activity of eEF2 kinase (i.e. when measured in the presence of saturating amounts of calcium ions and calmodulin, Fig. 5D). Neither sorbitol nor arsenite increased the level of eEF2 phosphorylation in low density cells (Fig. 5B). Sorbitol, but not arsenite, reproducibly caused a modest decrease in eEF2 phosphorylation in cells where this level is basally high (Fig. 5B). This appeared to be associated with a decrease in the activity of eEF2 kinase (Fig. 5E). Because only hydro- gen peroxide increases the phosphorylation of eEF2, while all three stresses inhibit translation, it seems that inhibition of protein synthesis by arsenite or sorbitol is not due changes in the phosphorylation state of this factor, but rather to other effects. Because the phosphorylation of eEF2 is regulated in an mTOR-dependent manner in CHO cells, the above data suggest that the cellular stress conditions used here are not acting to inhibit mTOR function. If this were the case, all Fig. 5. Effects of stresses on the phosphorylation of elongation factor 2. (A) One plate of confluent (80–90%) CHOK1 cells was trypsinized and then seeded into new dishes at the indicated approximate dilutions (1 : 2, i.e. 1 part trypsinized cell suspension and 1 part fresh medium, etc.). Each plate of cells was grown in medium containing serum for 24 h and the cells were then extracted and samples were subjected to 10% SDS/PAGE and Western blotted with the indicated antisera (probing with anti-eEF2 served as a loading control). (B) Upper and middle sections: CHO.K1 cells grown to subconfluence (approx. 60–70% confluence) were treated with sodium arsenite (100 l M ), hydrogen peroxide (3 m M ) or sorbitol (0.4 M ) for 25 min, prior to extraction. In some cases (+ SB203580), cells were pretreated with SB203580 (25 l M ) for 25 min prior to addition of the stress agent. Samples (30 lg protein) were analysed by SDS/PAGE and Western blotting using antisera specific for eEF2 phosphorylated at Thr56 (top) or an antibody that recognizes eEF2 irrespective of its state of phosphorylation, as a loading control (middle). The bottom section shows a similar analysis for cells at 80–90% confluence. Loading controls using anti-eEF2 again confirmed equal loading of cell protein (not shown). (C) CHO.K1 cells were treated for 25 min with a range of concentrations of hydrogen peroxide as indicated. Samples were analysed by SDS/PAGE and Western blotting using antisera specific for eEF2 phosphorylated at Thr56 (upper section) or an antibody that recognizes eEF2 irrespective of its state of phosphorylation, as a loading control (loading control, lower section). (D,E) Assays for eEF2 kinase activity. Samples of extracts (20 lgprotein)of low density (60–70% confluence, D) or higher density cells (80–90%, E) that had been treated with stressful agents as indicated (for 25 min) were assayed for eEF2 kinase activity using purified eEF2 as substrate. Samples were analysed by SDS/PAGE and autoradiography. The position of the radiolabelled eEF2 on the autoradiograph is indicated. Similar data were obtained in four (D) or three (E) experiments. Ó FEBS 2002 Modulation of translation factors by cellular stress (Eur. J. Biochem. 269) 3081 three stresses would be expected to have the same effect on eEF2 phosphorylation. It is thus unlikely that the dephosphorylation of 4E-BP1 and the inactivation of p70 S6 kinase caused by hydrogen peroxide and sorbitol are due to impairment of the function of mTOR itself, and perhaps more likely that these stresses interfere with signalling events downstream of mTOR that impinge on 4E-BP1 and p70 S6 kinase. Knebel et al. [57] have reported that eEF2 kinase can be phosphorylated and inactivated by the SB203580-insensitive d-form of p38 MAP kinase. Thus it is possible that, this enzyme may play a role in the dephosphorylation of eEF2 and the inactivation of eEF2 kinase caused by sorbitol. However, sorbitol has not been showntoactivatep38MAPkinase-d, and in the absence of an inhibitor for this enzyme, it is not possible to test its involvement. Other mechanisms may also be involved: for example, an earlier study concluded that osmotic stress activated a protein phosphatase acting on p70 S6 kinase, resulting in its inactivation [58]. These authors also reported that osmotic stress led to dephosphorylation of 4E-BP1. Stress regulation of 4E-BP1 is not mediated by the p38 MAP kinase pathway Dephosphorylation of 4E-BP1 is a common response to the cell stresses tested here. Sorbitol is known to activate the p38 MAP kinase a/b pathway in other cell types [59]. As assessed by measuring the activity of the downstream kinase, MAPKAP-K2 (using hsp27 as substrate), it also did so in CHO.K1 cells (Fig. 6A). The compound SB203580 inhibits the a and b isoforms of p38 MAP kinase (that activate MAPKAP-K2 [56]) and did indeed prevent the activation of MAPKAP-K2 in response to sorbitol, hydro- gen peroxide or arsenite in CHO.K1 cells (Fig. 6A). SB203580 did not however, prevent the increase in binding of 4E-BP1 to eIF4E caused by sorbitol or low concentra- tions of arsenite, indicating that this effect is not mediated through p38 MAP kinase a/b (Fig. 6B). SB203580 also failed to prevent the increase in the binding of 4E-BP1 to eIF4E induced by hydrogen peroxide (Fig. 6C). It therefore appears that the effects of stresses on 4E-BP1 phosphory- lation are not mediated by p38 MAP kinase a/b. Effects of stress conditions on other translation factors Other important regulatory proteins for mRNA translation are eIF2 and its guanine-nucleotide exchange factor, eIF2B. The activity of eIF2B is important in controlling translation initiation under a variety of conditions [20,29]. However, in multiple experiments using a range of concentrations of the agents studied here, we observed no change in eIF2B activity under any of the stress conditions tested here (data not shown), seemingly ruling out a role for this protein in the inhibitory effects of all three stresses on protein synthesis in these cells. Heat shock has been reported to inhibit eIF2B activity in vitro [60]. Concluding comments All three cell stresses used here cause profound inhibition of protein synthesis, as also seen for heat shock in these cells. The three stress conditions studied here have differing effects on the translation factors studied: these factors are all those thought to be important in the acute regulation of mRNA translation in mammalian cells, eIF4F, eIF2B, eEF2 and p70 S6 kinase. We have previously reported that osmotic, oxidative or heat stress cause the dephosphoryla- tion of eIF4E in CHO.K1 cells, while arsenite actually enhanced eIF4E phosphorylation [61]. None of the stresses studied here affected eIF2B activity, and they had differing effects upon p70 S6 kinase and the phosphorylation of eEF2. However, all these stresses, including, as described earlier, heat shock [43], caused increased binding of eIF4E to 4E-BP1 and the consequent loss of eIF4F complexes. These data suggest this is a common and rapid response of CHO cells to these stress conditions. Similar, but less complete, data were published previously for human embryonic kidney 293 cells [61]. Because no other transla- tion factor responds in the same way to all the stresses used, it seems likely that inhibition of eIF4E by increased binding to 4E-BP1 represents a major mechanism, possibly the primary mechanism, by which these stresses inhibit mRNA translation. Anderson and coworkers [62,63] have reported that certain cell stresses, such as hyperthermia, cause the formation of stress granules and that this may play an important role in the inhibition of translation under this condition. Their data indicate that the formation of such granules is driven by the phosphorylation of eIF2a [63]. These authors have argued that stress granule formation may be driven by loss of active eIF2, availability of which is Fig. 6. The stress-activated p38 MAP kinase pathway is not involved in the regulation of 4E-BP1 by stresses. CHO.K1 cells were left untreated (Con) or treated for 25 min with arsenite (50 l M ), hydrogen peroxide (1.5 m M ) or sorbitol (0.4 M ). In some cases, where indicated (+), cells were preincubated for 60 min with SB203580 (25 l M ) prior to addition of the stress stimulus. (A) samples were assayed for MAPKAPK-2 using recombinant hsp27 as substrate; position of radiolabelled hsp27 is shown (figure is an autoradiograph). (B) Samples were analysed directly by SDS/PAGE and Western blotting using gels containing 13.5% acrylamide/0.36% methylene bis-acrylamide. Positions of the three electrophoretically separable forms of 4E-BP1 are indicated. (C) Samples were subjected to affinity chromatography on m 7 GTP– Sepharose, and the bound material was then analysed by SDS/PAGE followed by immunoblotting using antibodies for eIF4E and 4E-BP1. The positions of migration of eIF4E and 4E-BP1 are indicated. 3082 J. Patel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 determined by the activity of eIF2B. In our studies, however, we saw no effect of the stresses tested upon eIF2B activity and only sorbitol caused significant phosphoryla- tion of eIF2, making it unlikely that this pathway is involved in the inhibition of translation under the other stress conditions studied here. The absence of an effect of arsenite on eIF2a phosphorylation, a consistent observation in these studies, differs from the finding of Anderson and colleagues that this agent elicited increased eIF2a phosphorylation in other cell-types. The loss of eIF4F complexes is expected to strongly impair de novo initiation of translation of the cap-dependent mRNAs [15], which are thought to represent the bulk of cellular mRNAs. Novoa & Carrasco [64] have presented evidence that reinitiation onto mRNAs that are already being translated is less dependent on the eIF4F complex than de novo initiation, consistent with the relatively small effect of rapamycin on protein synthesis in the short term [15]. Loss of such complexes should not impair translation of those cellular mRNAs that possess internal ribosome entry sequences, because translation of such mRNAs is independent of the cap and of eIF4E/4F [64] (reviewed in [65]). A number of cellular proteins are thought to be encoded by such mRNAs, including stress proteins such as grp78/BiP, a molecular chaperone whose expression is increased under stress conditions [66]. Other stress proteins whose expression rises in response to stressful conditions include the heat shock proteins. The translation of these mRNAs shows a low requirement for eIF4F [9] and, consistent with this, they continue to be translated in cells in which the level of eIF4E has been reduced by antisense techniques [67]. Taken together our data suggest that inactivation of eIF4E, by sequestration by 4E-BP1, is a common cellular response to stress. It may serve simulta- neously to impair general cellular translation under stressful conditions while allowing continued synthesis of stress proteins whose mRNAs possess internal ribosome entry sequences or have low requirements for eIF4F for other reasons. It was recently shown that the 5¢ UTR of the human hsp70 mRNA contains a potent enhancer of mRNA translation [68]. This may allow high levels of hsp70 synthesis in the absence of normal eIF4F function, although this idea remains to be tested. However, because inhibiting eIF4F formation by treating cells with rapamycin only has a small effect on the overall rate of protein synthesis in the short term [15], it is unlikely that the stress-induced dephosphorylation of 4E-BP1 and loss of eIF4F complexes is a major cause of the inhibition of protein synthesis caused by these agents. Indeed, it seems likely that this involves additional regulatory events, which remain to be identified, are also important in the stress- induced inhibition of protein synthesis. Further work will be required to characterize these events. Because the stress conditions we have studied have disparate effects upon the three targets of mTOR that we have studied (4E-BP1, p70 S6 kinase, eEF2), our data imply that these stresses do not exert a general inhibitory effect on mTOR signalling. For example, although hydrogen perox- ide and sorbitol cause inhibition of p70 S6 kinase and dephosphorylation of 4E-BP1, arsenite has opposite effects on these two proteins. In the case of eEF2, arsenite has little effect, while sorbitol and hydrogen peroxide have opposite effects. It is more likely therefore that these stress conditions intervene in different ways to regulate these target proteins, and that they probably do so by modulating the activities of the poorly understood signalling components that lie downstream of ÔdownstreamÕ of mTOR. This could, for example, involve inactivation of the kinases acting on 4E-BP1, or activation of the corresponding phosphatases. Lastly, our data reveal a multiplicity of effects of cell stresses on translation regulators, and their profound inhibitory effect on protein synthesis. These ÔartificialÕ stresses are widely used to activate the stress-activated protein kinases in order to study their roles, e.g. in the regulation of transcription. It is clearly essential to bear in mind their effects on mRNA translation and translation factors when using these agents, and when interpreting data obtained using them, especially where longer-term effects on gene expression are being evaluated. 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