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-

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-

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

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
,

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+

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

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

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

(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-

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

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

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

). 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

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

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