Báo cáo khóa học: The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex pptx

The proteasome inhibitor, MG132, promotes the reprogramming
of translation in C2C12 myoblasts and facilitates the association
of hsp25 with the eIF4F complex
Joanne L. Cowan and Simon J. Morley
Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton, UK
The eukaryotic translation i nitiation f actor (eIF) 4E, is
regulated by modulating both its phosphorylation and its
availability to interact with the scaffold protein, eIF4G, to
form the mature eIF4F complex. Here we show that treat-
ment of C2C12 myoblasts with the proteasomal inhibitor,
MG132 (N-carbobenzoxyl-Leu-Leu -leucinal), resulted in an
early decrease in protein s ynthesis rates followed by a p artial
recovery, reflecting the reprogramming of translation. The
early inhibition of protein synthesis was preceded by a
transient increase in eIF2a phosphorylation, followed by a
sustained increase in eIF4E phosphorylation. Inhibition of
eIF4E phosphorylation with CGP57380 failed to prevent
translational reprogramming or the moderate decrease in
eIF4F complexes at later times. P rolonged incubation with
MG132 resulted i n t he increased e xpression of heat shock
protein ( hsp)25, aB-c rystallin and hsp70, w ith a population
of hsp25 associating with the eIF4F complex in a p38
mitogen-activated protein kinase-dependent manner. Under
these conditions, eIF4GI, and to a lesser extent eIF4E,
re-localized from a predominantly cytoplasmic distribution
to a more perinuclear and granular staining. Although
MG132 had little e ffect on the c olocalization o f eIF4E and
eIF4GI, it promoted the SB2 03580-sensitive a ssociation of
eIF4GI and h sp25, an effect not observed with aB-crystallin.
Addition of recombinant hsp25 to an in vitro translation
assay resulted i n stimulation of on-going translation and a

moderate decrease in de novo translation, indicating that
this modified eIF4F complex containing hsp25 has a r ole to
play in recovery of mRNA translation following cellular
stress.
Keywords: eIF4G; MG132; C2C12; translation; hsp25.
1
Stressful stimuli often result in the reversible inhibition of
translation, a process regulated by complex interactions
between a large number of protein initiation factors (eIFs)
and RNA molecules [1]. During the initiation phase of
translation, the presence of a cap structure on eukaryotic
mRNA facilitates the recruitment o f initiation f actors to
allow ribosome binding and initiation at the correct start
site (for a review, see [1]). eIF4E interacts directly with the
cap via its concave surface [2,3] a nd forms mutually
exclusive complexes on its c onvex surface with e ither
inhibitory regulatory proteins (4E b inding proteins, 4E-BPs
[4–8]); or with the scaffold proteins, eIF4GI and eIF4GII
[1,4,5]. In vivo eIF4G exists partly in the form of a complex
with eIF4E and the ATP-dependent RNA helicase eIF4A,
constituting the initiation factor eIF4F (for reviews, see
[1,9]). Within the sequences of eIF4GI and e IF4GII there
are domains that interact with eIF4E [8], eIF4A [9], eIF3
[1,9,10]), the poly(A) binding protein (PABP [11]); and the
kinases, Mnk1/2, which modulate the phosphorylation of
eIF4E on Ser209 [12,13]. Mnk1 and Mnk2, which act at
the convergence point of extracellular-signal regulated
kinase (ERK) and stress-activated p38 m itogen-activated
protein kinase (p38MAPK), phosphorylate eIF4E at the
physiological site in vitro and in vivo (for reviews, see

[1,5,7]). In contrast, association of 4E-BPs with eIF4E is
modulated by phosphorylation e vents controlled via the
Target of Rapamycin (mTOR) signalling pathway (for
reviews, see [1,5]), integrating s ignals from mitogens, nutri-
ents and energy availability with the translational a pparatus.
Current models suggest that hypophosphorylated 4E-BP1
binds to eIF4E to inhibit cap-dependent translation, a
process readily reversed following its phosphorylation.
The dissociation o f hyperphosphorylated 4E-BP1 from
eIF4E leads to the b inding of eIF4G to eIF4E an d the
initiation of protein s ynthesis (for r eviews, see [1,5]).
The heat shock protein (hsp), hsp25 can associate with
the central domain of eIF4G following a severe heat shock
in HeLa cells, a pro cess associated with the d issociation of
eIF4F complexes and the reversible formation of heat-
shock granules [14]. Exposure of cells to a wide variety of
different physical, chemical and biological stresses [15]
induces or enhances the expression of the heat shock
proteins, hsp25 and aB-crystallin [14,16–18]. These small
oligomeric p roteins are highly expre ssed in skeletal muscle
[19] and a number o f tumour cell lines [20], fulfilling diverse
Correspondence to S. J. Morley, Department of Biochemistry, School
of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG,
UK. Fax: +44 1273 678433, Tel.: +44 1273 678544,
E-mail:
Abbreviations:DAPI,4¢,6¢-diamidino-2-phenylindole hydrochloride;
eIF, eukaryotic initiation factor; ERK, extracellular-signal regulated
kinase; hsp, heat shock protein; FITC, fluorescein isothiocyanate;
m
7

GTP, 7-methyl guanosine triphosphate; MG132, N-carbobenz-
oxyl-Leu-Leu-leucinal; mTOR, target of Rapamycin; p38MAPK, p38
mitogen-activated protein kinase; PVDF, poly(vinylidine difluoride);
TRITC, tetramethylrhodamine isothiocyanate; VSIEF, vertical slab
isoelectric focusing.
(Received 2 June 2004, revised 27 July 2004, accepted 27 July 2004 )
Eur. J. Biochem. 271, 3596–3611 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04306.x
functions in the cell. These include functioning as a
chaperone, by binding to and sequestering unfolded
proteins [21], s tabilizing the cytoskeleton [22–24] a nd
conferring re sistance to oxidative stress and TNFa-induced
cytotoxicity [25]. The induction of h sp25 and aB-crystallin is
required f or the differentiation of cardiomyo cytes [26] and
for neuronal survival following growth factor withdrawal
or axotomy [27,28]. This most probably reflects the ability
of hsp25 and aB-crystallin to promote t he inhibition of
apoptosis by binding to and inhibiting pro-apoptotic
proteins that are often activated under these conditions
[16,17,20,29–34]. Hsp25 has also been reported t o interact
with eIF4G, mediating the inhibition of protein synthesis
in HeLa cells during severe heat shock [14]. However, in
mouse myoblast cells over-expressing aB-crystallin or
hsp25, cap-dependent initiation of translation was main-
tained following heat shock [35]. For this thermotolerance,
aB-crystallin needed to be in its nonphosphorylated state to
give protection, whereas phosphorylated hsp25 was more
potent in protection than the unphosphorylatable form.
These d ata suggest that chaperone a ctivity is not a
prerequisite for protection of t ranslation by small hsps after
heat shock [35].

The ubiquitin-proteasome pathway is a nother mech-
anism that contributes to cell protection f rom stressful
stimuli through the elimination of unfolded proteins [36].
Composed of an ubiquitin-conjugating system and the
26S proteasome, this multicatalytic proteinase complex is
responsible for intracellular protein degradation in mam-
malian cells [36]. I nhibition of the p roteasome with
MG132 (N-carbobenzoxyl-Leu-Leu-leucinal) or lactacys-
tin blocks the rapid degradation of short-lived regulatory
proteins and abnormal polypeptides and reportedly
promotes the re-localiz ation of hsp 25 and aB-crystallin
to the actin cytoskeleton [15,37,38]. By binding to, and
capping the F -actin barbed ends [39], h sp25 results i n a
reorganization of the a ctin filament s ystem, playing a r ole
in the migration of endothelial cells and i n recovery of
cells from wounding [40]. In a variety of cells, in a ddition
to the induction of hsp25 and aB-crystallin expression,
inhibition of the p roteasome induces aggresome forma-
tion [38], activation of stress kinases and programmed
cell death [16,17,20,25,29,30]. Aggresomes, which may be
associated with intermediate filaments [38], contain
misfolded p roteins, hsp25, aB-crystallin and c omponents
of the degradation machinery. As such, they are thought
to provide a cytoprotective role by clearing the cytoplasm
of potentially t oxic aggregates [36].
As part of an on-going investigation into the regulation of
protein synthesis in mammalian cells, we have investigated
the e ffects of inhibition of the p roteasome o n the localiza-
tion and integrity of eIF4G in C2C12 myoblasts. We show
that, in contrast to Jurkat T cells [1,41,42], MG132 does not

promote the degradation o f eIF4GI o r a loss of cell viability.
Rather, MG132 promoted a transient inhibition of protein
synthesis followed by a re-programming of the translational
apparatus, events associated with the efficient expression of
hsp25, aB-crystallin and hsp70. In addition, treatment of
cells with MG132 resulted in the activation of signalling
pathways responsible for the phosphorylation of eIF2a [1],
eIF4E [1,5] and those associated with cell survival [5]. While
aB-crystallin did not bind to eIF4F, biochemical and
immunofluorescence analyses showed that a population of
hsp25 w as associated with the e IF4F complex in a
p38MAPK-dependent manner. Furthermore, in vitro stud-
ies showed that hsp25 does not inhibit either cap-dependent
or IRES-driven translation, suggesting a role for the
association o f hsp25 with eIF4F in the recovery of
translation rates following cellular stress.
Materials and methods
Chemicals and biochemicals
Materials for tissue culture were from Invitrogen, fetal
bovine serum was from Labtech International (UK) and
MG132, SB203580, microcystin and UO126 were f rom
Alexis Corporation. RAD001 [43] and C GP57380 [44] were
gifts f rom Novartis (Basel, Switzerland). Antisera t o hsp25,
aB-crystallin and hsp70 were from Stressgen Biotec hnolo-
gies Inc.,
2
(San Diego, CA, USA) and antisera to phospho-
S6, phospho-ERK, phospho-p38MAPK, phospho-Akt,
phospho-eIF2a, total 4E-BP1, phospho-4E-BP1 (Ser65),
and total ERK were from Cell Signalling Technology.

Antiserum t o t otal eI F2a was a gift from the late
E. Henshaw (Rochester, NY, USA)
3
and antiserum to
eIF4GII was provided by N. Sonenberg (Montreal,
Canada). [
35
S]Methionine was from ICN Biomedicals,
Immobilon poly(vinylidene difluoride) (PVDF) was from
Millipore or Amersham Biosciences and unless otherwise
stated, all other chemicals were from Sigma.
Tissue culture
C2C12 cells, provided by the ECACC (Salisbury, UK)
4
,were
cultured in DMEM supplemented with 20% (v/v) fetal
bovine serum at 37 °C in a humidified atmosphere
containing 5% CO
2
.
Preparation of cell extracts
Following treatment, cells were isolated in a cooled
centrifuge a nd washed with 0.5 mL ice-cold NaCl/P
i
containing 40 m
M
b-glycerophosphate and 2 m
M
benzami-
dine. Pellets were resuspended in 200 lLice-coldBufferA

[20 m
M
MOPS/KOH,pH7.2,10%(v/v)glycerol,20m
M
sodium fluoride, 1 l
M
microcystin, 75 m
M
KCl, 2 m
M
MgCl
2
,2m
M
benzamidine, 2 m
M
Na
3
VO
4
, complete
protease inhibitor mix)EDTA
5
(Roche)] and lysed by
vortexing following the a ddition of 0.5% (v/v) Igepal and
0.5% (v/v) d eoxycholate. Cell debris was removed by
centrifugation in a microfuge f or 5 m in at 4 °Candthe
resultant supernatants frozen in liquid N
2
.

SDS/PAGE, vertical slab iso-electric focusing
and immunoblotting
Samples containing equal amounts of p rotein were resolved
by SDS/PAGE or vertical slab iso-electric focusing (VSIEF)
and processed as described previously [45,46]. Anti-peptide
serum specific for the C-terminal domain of eIF4GI
[RTPATKRSFSKEVEERSR(1179–1206)], eIF4E [TAT-
KSGSTTKNRFVV(203–217)], eIF4A, 4E-BP1 and poly(A)
binding protein (PABP) were a s described previously
Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3597
[41,42,46]. In all cases, care was taken to ensure that detection
was within the linear response of the individual antiserum to
the protein. All rabbit antisera were i solated from c rude
serum by a ffinity chromatography with the c orresponding
peptide using the SulfoLink kit (Perbio Science UK Ltd,
Cheshire, UK) according to the manufacturer’s instructions.
Measurement of protein synthesis
C2C12 myoblasts were incubated in the presence of
10 lCiÆmL
)1
[
35
S]methionine and either 0 .1% or 20% (v/v)
fetal bovine serum in comple te medium, as indicated. Cells
were recovered and washed once in NaCl/P
i
prior to lysis in
0.3
M
NaOH, o r extracts were prepared as d escribed above

following a period of labelling as described in the figure
legends. Incorporation of radioactivity into aliquots con-
taining equal amounts of protein was determined by
precipitation with trichloroacetic acid.
m
7
GTP-Sepharose affinity isolation of eIF4E
and associated factors
For the isolation of eIF4E and associated proteins, cell
extracts of equal protein concentration were subjected to
m
7
GTP-Sepharose chromatography and the resin washed
twice with Buffer B (20 m
M
Mops/KOH pH 7.4, 75 m
M
KCl, 2 m
M
MgCl
2
,1l
M
microcystin, 10 m
M
NaF, 2 m
M
benzamidine, 7 m
M
2-mercaptoethanol, 0.1 m

M
GTP).
Recovered protein was e luted either directly into sample
buffer or eluted with 0 .2 m
M
m
7
GTP in Buffer B for e ither
SDS/PAGE or VSIEF [44,45,47–49].
Immunoprecipitation of eIF4GI
Extracts containing equal amounts of protein were diluted
in Buffer C [50 m
M
Tris/HCl, pH 8, 20 m
M
NaF, 50 m
M
KCl, 2 m
M
EDTA, 2 m
M
benzamidine, 2 m
M
Na
3
VO
4
,
complete protease inhibitor mix)EDTA (Roche, UK),
0.5% (v/v) Igepal and 0.5% (v/v) deoxycholate] and

incubated for 2 h with protein G magnetic beads (Promega;
previously presaturated with 2 mgÆmL
)1
BSA) coated with
either purified anti-eIF4GI IgG or nonimmune rabbit IgG.
Beads were isolated, washed five times w ith 0.5 mL Buffer C
and recovered protein eluted with SDS/PAGE sample
buffer without 2-mercaptoethanol or boiling.
Immunolocalization of initiation factors, hsp25
and aB-crystallin
To enable antibodies raised in the same species to be
detected in the same cell during immunofluorescence
studies, affinity purified primary rabbit antibodies to eIF4E
and Hsp25 were labelled with Alexa Fluor dye 488 using the
Protein Labelling Kit from Molecular Probes according to
Fig. 1. The proteasome inhibitor MG132 decreases translation rates in C2C12 myoblasts without the cleavage of eIF4GI. (A ) C2 C12 ce lls were either
serum fed (unfilled bars) or s erum-starved (filled bars) for 24 h before incubation for 6 h with the indicated concentrations of MG132. To measure
the rate of protein sy nthesis, cells were incubated w ith 1 0 lCiÆmL
)1
[
35
S]methionine during the l ast 15 min, so lubilized in 0.3
M
NaOH an d
incorporation of radioactivity into protein d etermined b y t richloroacetic acid precipitation. The presented data are the means + SD (bars) of three
separate experiments, each performed in triplicate. (B) Serum-fed C2C12 cells were incubated as in (A), extracts were prepared and equal amounts
of protein (15 lg) were resolved by SD S/PAGE. Th e integrit y of eIF4G I and e IF4E w as visualized by immunoblotting using antiserum specific to
the C-terminal domains of each protein [41,42,46]. The phosphorylation of eIF2a and eIF4E was monitored using either phospho-specific
antiserum or VSIEF and immunoblotting, as described in Materials and methods. Results are from a single experiment but are r epresentative of
those obtained on t hree separate occasions. (C) Q uantification of the data p resented in (B). The p resented data for e IF4E was quantified by

densitometric scanning from the VS IEF analysis; all a re the m eans + SD ( bars) of three s eparate experiments.
3598 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
the manufacturer’s instructions. Anti-(C-terminal eIF4GI)
was labelled i n the same way with Alexa Fluor 555.
Antibodies were diluted as follows into NaCl/P
i
containing
1% BSA: anti-(C-terminal eIF4GI), 1 : 300; anti-eIF4E,
1 : 200; anti-Hsp25, 1 : 50; anti-(aB-crystallin), 1 : 300.
Goat anti-(mouse IgG) conjugated to fluorescein isothio-
cyanate (FITC) (DakoCytomation Ltd, Ely, UK)
6
was
used as a secondary antibody to detect the monoclonal
aB-crystallin antibody, diluted 1 : 100. The actin cytoske-
leton was visualized with a phalloidin-tetramethylrhodamine
isothiocyanate ( TRITC) (1 : 8000) or phalloidin-FITC
(1 : 500) conjugate (DakoCytomation).
Immunofluorescence microscopy
C2C12 cells were seeded on 5 cm plates ( 300 000 cells per
plate). F ollowing incubation with or without SB203580 or
MG132, cells were washed once in NaCl/P
i
,thenfixedin
4% (w/v) paraformaldehyde in NaCl/P
i
, pH 7.4 for 15 min.
After a single rinse with NaCl/P
i
, cells were permeabilized in

NaCl/P
i
containing 0.1% (v/v) Triton X-100 for 5 min,
rinsed once in NaCl/P
i
then incubated with 0.1% (w/v)
NaBH
4
for 5 min prior to three w ashes with NaCl/P
i
. Non-
specific binding was blocked by adding 1% BSA in NaCl/P
i
for 30 min. Cells were incubated in the primary antibody
solution for 60 min, w ashed extensively and then incubated
with the appropriate secondary antibody and phalloidin–
FITC (or TRITC)
7
conjugate f or 45 min. Following further
extensive washing, nuclei were stained with 12.5 ngÆmL
)1
4¢,6¢-diamidino-2-phenylindole hydrochloride (DAPI)
(Sigma) for 5 min. After a further two washes, coverslips
were mounted directly onto the 5 c m plates with mowiol
mounting s olution [ 0.2
M
Tris, pH 8.5, 33% (w/v) glycerol,
13% (w/v) mowiol, 2.5% (w/v) 1,4-diazobicyclo [2,2,2]-
octane (DABCO)] and sealed with clear nail polish. Cells
were analysed using a Zeiss A xioscop 2 microscope

equipped with a 63· oil immersion objective and fitted
with the appropriate filter sets. Images were captured with a
Photometrics ÔQuantixÕ digital camera. Images were proc-
essed using
METAMORPH
imaging software ( Universal Ima-
ging Corp., Downingtown, PA, U SA)
8
. Greyscale images
were pseudo-coloured to correspond to the red (TRITC/
Alexa Fluor 555), green (FITC/Alexa Fluor 488) or blue
minutes
0
20000
40000
60000
[
35
S] m
ethionine incorpor
ation (cp
m)
A
360
240
6040200 120
1 765432
B
221
17

32
43
82
128
eIF4E
0
min
360240120604020
eIF4E
1 65432
C
Mnk1-P
eIF2
Mnk1
p38MAPK-P
S6-P
ERK-P
ERK
eIF4E-P
0min1206030155
eIF2
-P
Fig. 2. MG132 promotes the re-programming of translation and the
activation of multiple intracellular signalling pathways. (A) Serum-fed
C2C12 cells were incubated with 50 l
M
MG132 f or the times indicated
and the rate of protein synthesis measured as in Fig. 1A. The presented
data are the means + SD (bars) o f two s epar ate experiments, each
performed in triplicate. (B) In parallel cultures, cells were treated as in

(A) but in the presence of 100 lCiÆmL
)1
[
35
S]methionine and cell
extracts were prepared as described. Aliquots containing equal
amounts of radioactive counts (54 000 c.p.m.) were r esol ved by SDS/
PAGE and visualized by autoradiography (upper panel). In addition,
aliquots containing e qual amounts of protein (15 lg) were resolved by
SDS/PAGE ( middle panel) o r V SIEF (lower panel) and eIF4E visu-
alized by immunoblotting. Results are from a single experiment but are
representative of t hose obtained on f our separate occasions. (C) Ser-
um-fed C2C12 cells were incubated with 50 l
M
MG132 for the times
indicated and cell extracts were prepared. Aliquots containing equal
amounts of protein (15 lg) were resolved by SDS/PAGE and P VDF
membranes were pro bed with a ntiserum specific for the proteins
indicated in t he figure. Results are representative of those obtained in
three separate e xperiments.
Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3599
(DAPI) fluorescence and the digital images were merged.
Preparation of i mages for publication used Adobe
PHOTO-
SHOP
v5.5. All images are to the same scale.
In vitro
binding of hsp25 with eIF4GI
His-tagged or His/FLAG-tagged initiation factors were
expressed in Sf9 insect cells and isolated by s equential affinity

chromatography and gel filtration [64]. Purified initiation
factors ( 1 lg) were bo und to nitrioltriacetic acid–nickel–
agarose (Qiagen) for 30 min at 4 °CinBufferE(50m
M
Mops/KOH,pH7.4,300m
M
KCl, 2 m
M
MgCl
2
,2m
M
benzamidine, 3.5 m
M
2-mercaptoethanol, complete protease
inhibitor mix – EDTA), the resin was washed and further
incubated f or 20 min on ice in Buffer F ( Buffer E, but
containing 75 m
M
KCl and supplemented with 1 mgÆmL
)1
BSA). Isolated resin was then resuspended i n 1 0 lLBufferF
and incubated with continuous mixing in th e presence of 3 lg
hsp25 for 30 min at 4 °C. Beads w ere isolated by c entrifu-
gation, washed twice with 0.2 mL Buffer F (without BSA)
and recovered protein eluted w ith 100 m
M
EDTA for SDS/
PAGE analysis.
In vitro

translation
Reticulocyte lysates were p repared in-house; incubation s
for on-going protein synthesis and luciferase reporter assays
were as described previously [50,51].
Results
The proteasome inhibitor, MG132, promotes
an inhibition of protein synthesis without the cleavage
of eIF4GI or eIF4GII
In a number of different cell types, inhibition of the 26S
proteasome induces activation of stress kinases, promotes
apoptosis [16,17,20,25,29–31,38] or potentiates the clea-
vage of eIF4GI [1,42,52]. In addition, treatment with
MG132 can also result in the re-localization o f h sp25 and
aB-c rystallin to the actin cytoskeleton [15,38] and result
in cell cycle arrest. T o address whether MG132 caused
similar effects in C2C12 myoblasts, starved or fed cells
were incubated with different concentrations of MG132
for 6 h. Figure 1A shows that treatment of cells with
10 l
M
MG132 for 6 h resulted in a 40% inh ibition of
translation i rrespective of the nutrit ional s tatus o f the cell.
Increasing the concentration of MG132 up to 50 l
M
did
not further inhibit t ranslation and did not decrease cell
viability even following prolonged incubation times (data
not shown).
Previously, we h ave shown that e IF4GI is cleaved during
anti-Fas-mediated apoptosis [52] and that MG132 and

lactacystin promoted the cleavage of eIF4G in Jurkat cells
[41]. In contrast, Fig. 1B (lanes 2–4 vs. lane 1 ) shows that i n
C2C12 myoblasts, MG132 treatment for 6 h did not affect
A
B
C
Fig. 3. MG132 promotes the upregulation of hsp25 and aB-crystallin in
C2C12 myoblasts. (A) Serum-fed C2C12 cells wereincubatedinthe
presence of dimethylsulfoxide alon e (lane 1) or 50 l
M
MG132 (lanes
2–5) for th e times indicated a nd cell extracts were prep ared. Aliquots
containing equal amounts of protein (15 lg) were reso lved by SDS/
PAGE, protein transferred to PVDF and membranes probed with
antiserum specific for the proteins indicated. Results shown are from a
single experiment but are representative of those obtained on four
separate occasions. (B ) S erum-fed C2C12 cells were incu bated in the
presence of dimethylsulfoxide alone (lanes 5 and 6) o r 50 l
M
MG132
(lanes 1–4) for the times indicated and cell extracts were prepared and
resolved as in (A). Membranes were pro bed with antiserum specific for
PABP (loading co ntrol) or a phospho-specific an tiserum f or 4E -BP1
phosphorylated o n Se r64. Results are from a single experiment but are
representative of th ose obtained on two separate occasions. (C) Serum-
fed C2C12 cells were incubated with 50 l
M
MG132 for the times
shown and the expression of aB-crystallin determined by Western
blotting. Results are from a single experiment but are representative of

those obtained o n four s eparate occasions.
3600 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
the integrity o f eIF4GI or eIF4E, as visualized by immuno-
blotting. Furthermore, all initiation factors analysed (inclu-
ding e IF4GII) remained intact even after 24 h of treatme nt
with high levels of MG132 ( data not shown ). Together,
these d ata suggest that in contrast to Jurkat cells [41],
incubation of C2C12 cells with MG132 did not generate
active caspase-3 or caspase-8-like proteases that target
eIF4G. However, immunoblot analysis of extracts indicated
that MG132 treatment did result in up to a twofold increase
in the phosphorylation of eIF2a even at 10 l
M
MG132
(Fig. 1 B, lane 2 v s. lane 1; quantified in C), an e vent often
associated with a global inhibition of translation rates [1]. In
addition, the phosphorylation of e IF4E, an event often
correlated with increased rates of translation [1,5], was
also increased fo llowing MG132 treatment for 6 h, with
maximal effects only observed at the highest doses
(Fig. 1 B,C).
MG132 causes a re-programming of translation
and the activation of multiple signalling pathways
in C2C12 myoblasts
To further i nvestigate the e ffect of c ell stress o n
translation rates in C2C12 myoblasts, cells were exposed
to 50 l
M
MG132 and pulse-labelled with [
35

S]methionine
at different times. Figure 2A shows that the rate of
eIF4GI
8h 50
M
MG132
actin merge
eIF4E
actin
merge
A
B
untreated
cells
untreated
cells
8h 50
M
MG132
Fig. 4. Intracellular loc alization of eIF4GI a nd e I F4E. C2C12 cells were left untreated (upper panels) or incubated with 5 0 l
M
MG132 for 8 h
(lower panels) before being fixed in 4% (v/v) paraformaldehyde for 15 min and permeabilized in NaCl/P
i
containing 0.1% (v/v) Triton X-100 for
5 min. Rabbit antisera recognizing the C-terminus of eIF4GI (A), eIF4E (B), total hsp25 protein (C) or monoclonal anti-(aB-crystallin) (D) were
used to visualize the localization of the endogenous proteins within these cells. Anti-eIF4GI was labelled with Alexa Fluor 555 (pseudocoloured in
red), whereas anti-hsp25 or anti-eIF4E were labelled with Alexa Fluor 488 (pseudocoloured green). G oat anti-(mouse IgG) co njugated to F ITC
(green) detected the unlabelled mAb aB-crystallin. Actin was visualized with phalloidin conjugated to FITC or TRITC (pseudocoloured green or
red, respectively) and nuclei with D API (pseudocoloured blue). White bars represent 20 lm for all panels.

Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3601
translation decreased b etween 40 and 60 min after
exposure to MG132, with rates further d ecreasing to
20% of control levels within 2 h. Analysis of cell extracts
by SDS/PAGE indicated that t here was a dramatic
re-programming of translation (Fig. 2B upper panel,
lanes 5–7 vs. lane 1), concomitant with increased levels
of eIF4E phosphorylation (Fig. 2B, lower panel). A more
detailed time course ( Fig. 2C) showed that the increase in
eIF4E phosphorylation w as preceded by a transient
activation of ERK (lanes 2–4 vs. lane 1) but coincided
with the sustained activation o f p38MAP kinase and
increased phosphorylation of Mnk1 (lanes 5 and 6 v s.
lane 1), signalling molecules which are functionally
upstream of eIF4E [3]. In addition, MG132 promoted
the phosphorylation of ribosomal protein S6 (Thr421/
Ser424;Figs2Cand3A)andAkt(Fig.3A)at1–2h,
consistent with activation of the mTOR signalling
pathway and the maintenance of cell survival under
these assay conditions.
However, these events were all preceded by a biphasic
increase in eIF2a phosphorylation. The early phase of
eIF2a phosphorylation was evident within 1 5 min of
treatment (Fig. 2C, lane 3 vs. l ane 1), declined at 1 h (lane
5) and increased again at later times (Fig. 2C, lane 6 and
Fig. 3A). While prolonged incubation of cells with MG132
resulted in a partial recovery of translation rates t o 50% of
the control by 6 h ( Fig. 2A), this occurred despite elevated
levels of eIF2a phosphorylation (Figs 1 and 3A).
MG132 treatment induces expression of hsp25

and its increased association with eIF4F
Cell stress, includ ing MG132 treatment, has been reported
to increase the expression of the s tress-related proteins,
hsp25, aB-crystallin and hsp70 [14,17,38]. To determine
whether the MG132-induced expression of hsps occurr ed in
C2C12 cells, extracts were prepared from cells incubated for
various times with MG132. Both hsp25 (Fig. 3A) and
C
D
8h 50 M
MG132
8h 50 M
MG132
untreated
cells
untreated
cells
merge
actin
Hsp25
mergeactin
B-crystallin
Fig. 4. (Continued).
3602 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
aB-crystallin levels (Fig. 3C) increase d dramatically after
2–6 h , with a lesser induction of hsp70 (Fig. 3A). This
increased expression of hsp25 at 2 h occurred before any
detectable dephosphorylation of 4E-BP1 ( Fig. 3A, lane 5 vs.
lane 1) and prior to the recovery of translation r ates. U sing
antiserum specific for 4E-BP1 phosphorylated on Ser64 we

have confirmed t hat f ollowing MG132 treatment of cells, a
modest decrease in 4E-BP1 phosphorylation occurs between
2 and 6 h (Fig. 3B), after the robust expression of hsp25 was
evident.
We have also investigated the subcellular localization of
initiation factors in C2C12 cells by immunofluoresence
using affinity-purified, AlexaFluor-labelled antibodies.
Figure 4A and B show that both eIF4E and eIF4GI are
predominantly cytoplasmic (left panel), with neither pro-
tein associated directly with the actin cytoskeleton to any
great e xtent (see m erged images in right panels and [55,56]).
In untreated cells, hsp25 staining was d iffuse, b eing
localized,inparttotheperinuclearregionandtoareas
of more intense s taining at the cell p eriphery (Fig. 4C).
Similarly, aB-crystallin was detectable in resting cells, but
showed a largely diffuse, and grainier cytoplasmic staining
(Fig. 4 D). Following tre atment of cells with MG132 for
8 h , stress fibres were more pronounced (Fig. 4A), and
eIF4GI (Fig. 4A), and to a lesser extent eIF4E (Fig. 4 B),
showed a m ore granular appearance, bein g generally
concentrated in the perinuclear region. Levels of both
hsp25 (Fig. 4C) and aB-crystallin (Fig. 4D) were increased
with MG132, each showing a distinct, reproducible local-
ization; hsp25 appeared to be mainly perinuclear while
aB-crystallin was more cytoplasmic and granular in
appearance. These studies are consistent with reports using
a r elated myoblast cell line, H9c2, w hich suggested that
inhibition of proteasom al activity resulted in a re-localiza-
tion of aB-crystallin to aggresomes [15]. However, in our
cells, these were not associated with th e cytoskeleton t o any

large extent (Fig. 4C,D).
In light of published data [14], we have also investigated
the effects of MG132 on the eIF4F complex in C2C12 cells.
Isolation of eIF4E and associated factors w ith m
7
GTP-
Sepharose (Fig. 5A) indicated that MG132 caused a partial
dissociation of eIF4GI, PABP and eIF4A from eIF4E at
later times of incubation (Fig. 5 A, lane 5 vs. lane 1 and
Fig. 5B, lane 7 vs. lane 1). As eIF4G exists in two forms
(eIF4GI a nd eIF4GII [1,53]), we a lso monitored t he associ-
ation of e IF4GII with eIF 4E under t hese conditio ns.
Figure 5B shows that as with eIF4GI, MG132 also caused
a partial dissociation of e IF4GII and Mnk1 from eIF4E after
4 h of MG132 t reatment (lane 7 vs. lane 1), indicative of a
general decrease in eIF4F complex levels. As predicted from
accepted models [1,5], th e partial dissociation of eIF4GI and
eIF4GII from eIF4E occurs concomitantly with a moderate
increase in binding of 4E-BP1 to eIF4E. In contrast, the
association of hsp25 (and hsp70; data not shown) with eIF4F
increased dramatically at later times of incubation (Fig. 5A,
lane 5 v s. lane 1; Fig. 5B, lane 7 vs. lane 1). However, aB-
crystallin did not associate with eIF4F under t hese conditions
(Fig. 5 A), even though it was induced to high levels by
MG132 (Fig. 3C). These data suggest th at, as with h eat
shock [14,17,35], hsp25 may have a role in regulating eIF4F
activity following the inhibition o f proteasome activity.
eIF4E and ERK phosphorylation are not required
for translational re-programming in response to MG132
To investigate the r ole of eIF4E phosphorylation or t he

activation of ERK in the translational response to M G132,
we have used the c ell permeable inhibitors, CGP57380 and
UO126, respectively [44,46,54]. Figure 6 A shows that p re-
treatment of cells with CGP57380 (lane 4 vs. l ane 2 ) had little
effect on the translational re-programming in response to
MG132. Under these assay c onditions, while the i ncrease in
eIF4E phosphorylation w as prevented ( Fig. 6B, lane 4 vs.
lane 2), CGP57380 did not influence the accumulation of
hsp25, the phosphorylation of p38MAPK, ribosomal
protein S6 or eIF2a (Fig. 6B, upper panel). Furthermore,
inhibition of eIF4E phosphorylation did not prevent the
coisolation of hsp25 with the eIF4F complex (lower p anel).
4E-BP1
eIF4E
m7GTP-
Sepharose
A
123 45
eIF4GI
eIF4GII
PABP
Mnk1
eIF4E
hsp25
0 min240120603015 360
m7GTP-
Sepharose
B
12345 6 7
αB crystallin

60126
MG132
hsp25
PABP
eIF4A
eIF4G
Fig. 5. Hsp25 associates with the eIF4F complex. (A) A liquots of
extract (75 lg protein) were subjected to m
7
GTP-Sepharose chroma-
tography to isolate eIF4E and associated proteins. Recovered proteins
were resolved by SDS/ PAGE and v isualiz ed by i mmunob lotting.
Results are representative of those obtained in three separate experi-
ments. (B) Serum-fed C2C12 cells were incubated in t he presence of
50 l
M
MG132 for the times indicated and cell extracts were prepared.
Aliquots o f extract (75 lg protein) were subjected to m
7
GTP-Seph-
arose chromatography to isolate eIF4E and associated proteins.
Recovered p roteins were resolved by SDS /PAGE and visualize d by
immunoblotting . Resu lts are r epresen tative o f t hose obtained in two
separate experiments.
Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3603
Similarly, inhibition of ERK signalling with U O126 had no
effect on any of the MG132-ind uced responses (Fig. 6A,B,
lanes 3 vs. lanes 1) even though i t prevented the transient
activation of ERK (data not shown).
In a similar manner, we have also investigated the role

of the p38MAPK and mTOR signalling p athways using
specific, cell permeable inhibitors. Figure 7A (lane 3 vs.
lane 2, upper panel) shows that inhibition of p38MAPK
signalling with SB203580 reduced, but did not completely
prevent the reprogramming of translation in these cells.
The s pecificity of t his inhibitor was demonstrated by its
ability to reduce both the expression of hsp25 and eIF4E
phosphorylation, without affecting t he phosphorylation
status of ribosomal protein S6, 4E-BP1 or eIF2 a (Fig. 7A,
lower panels). Consistent with these findings, inhibition of
signalling downstream of p38MAPK with SB203580
prevented the coisolation of h sp25 with eIF4F o n
m
7
GTP-Sepharose (Fig. 7B, lane 3 vs. lane 2) or following
immunoprecipitation of eIF4G from extracts (Fig. 7C,
lane 3 vs. lane 2). In c ontrast, inhibition of mTOR by
RAD001 [43] had no influence on the MG132-induced
translational re-programming or the expression of hsp25,
but reduced levels of phosphorylation of ribosomal
protein S6 a nd 4E-BP1 (Fig. 7A, lane 4 vs. lane 2).
Furthermore, RAD001 did not prevent the association of
hsp25 with eIF4F isolated by either affinity chromato-
graphy (Fig. 7B, lane 4 vs. lane 2) or immunoprecipitation
(Fig. 5 C, lane 4 vs. lane 2), but did, as predicted, promote
increased association of 4E-BP1 w ith eIF4E (Fig. 7B). As
pretreatment of cells with any of these inhibitors alone, or
in combination w as unable t o prevent the g lobal inhibi-
tion of protein synthesis observed with MG132, t hese data
suggest a central role for the phosphorylation of eIF2a in

translational reprogramming events.
Colocalization of hsp25 and eIF4GI following MG132
treatment of C2C12 myoblasts
The association of hsp25 with eIF4G has been suggested to
inhibit t ranslation in cell extracts following severe heat shock
by promoting stress granule formation, sequestering eIF4G
into inactive c omplexes [14]. To determ ine whether such
events occur during t ranslational re-programming of C2C12
myoblasts in response to MG132, we have examined the
intracellular colocalization of eIF4E, eIF4GI, aB-crystallin
and hsp25 in the absence or presence of SB203580.
Figure 8A (upper panel) i ndicates t hat eIF4E and eIF4GI
showed extensive colocalization i n untreated cells. These
findings are in agreement with previous wo rk [55,56] and the
biochemical data presented in Fig. 5. Treatment of cells with
MG132 (Fig. 8A, middle panel) reduced the general level of
costaining of these p roteins w hile promoting distinct a reas of
intense cytoplasmic colocalization. Pre-treatment of cells
with 20 l
M
SB203580 largely prevented this redistribution
of factors, with colocalization p redominant in the perinuclear
region (lower panel). In a similar manner, we have also
covisualized eIF4GI and hsp25 (Fig. 8B). T hese data show
that although MG132 promoted a distinct nuclear staining of
hsp25, a substantial population of the protein colocalized
with eIF4GI in the perinuclear region (middle panel). This
colocalization of eIF4GI and hsp25 was prevented by
SB203580 (lower panel), consistent with the data presented
in Fig. 7 showing decreased levels of hsp25 expression and

association with eIF4G. In contrast, F ig. 8C shows that
B
A
Fig. 6. eIF4E phosphorylation is not required for translational re-programming in response to M G132. (A) S erum-fed C2C12 cells were preincubated
for 30 min with dimethylsulfoxide a lone (lanes 1 and 2), 10 l
M
UO126 (lane 3) or 20 l
M
CGP57380 (lane 4), prior to the addition of 50 l
M
MG132
(lanes 2–4) for 6 h. Cells were labelled as described in Fig. 2B and aliquots containing equal am ounts of radioactive counts (45 000 c.p.m.) w ere
resolved by SDS/ PAGE and v isualiz ed by a utorad iography. (B, u pper panels) Aliquots of extract containing equal amounts of protein (15 lg) were
resolved by VSIEF (top panel) and the phospho rylation status of eIF 4E visualized by immunoblotting. A liquots were also resolved by SDS/PAGE
(remaining panels), protein transferred t o PVDF and membranes probed with antiserum specific for the p roteins indicated. R esults are from a single
experiment but a re representative of those obtained on three separate occasions. (B, lower pane ls) Aliquots of extract (75 lg protein) were subjected
to m
7
GTP-Sepharose chromatography to isolate eIF4E and associated proteins. Recovered proteins were resolved by SDS/PAGE and visualized
by immunoblotting. Results are representative of those obtained in three separate experiments.
3604 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
although MG132 treatment resulted in an SB203580-sensi-
tive re-distribution o f aB-crystallin to granules (compare
middle and lower panels), t here was little or no detectable
colocalization of aB-crystallin with eIF4G under these
conditions. These data are in agreement with those presented
in Fig. 5A which demonstrated the biochemical coisolation
of hsp25 with eIF4GI, but no association of aB-crystallin
with the eIF4F complex.
Hsp25 does not inhibit cap-dependent or IRES-driven

translation
in vitro
To investigate m ore directly the effects of h sp25 on protein
synthesis, we have added purified, recombinant hsp25 to the
reticulocyte lysate translation system. Rela tive to buf fer
alone, or to an unrelated protein (GST), Fig. 9A shows that
addition of hsp25 actually resulted in a dose-dependent
stimulation of on-going translation. As hsp25 levels
increased in C2C12 cells at a time when translational
re-programming was evident, we also assessed the effect of
hsp25 on de novo translation by repeating these experiments
in the presence of low concentratio ns of added capped or
IRES-driven luciferase reporter m RNAs. Figure 9 B shows
that relative to GST, low concentrations of added hsp25 did
not influence reporter m RNA translation; only at the
highest concentrations of added hsp25 was a modest
inhibition of both cap-dependent and EMCV-IRES-driven
reporter translation observed. To ensure that the hsp25 was
functional in these assays, we monitored its association with
the eIF4F complex and its ability to interact directly with
eIF4G in vitro. Under our conditions, hsp25 was specifically
incorporated into the eIF4F complex (Fig. 9C, lane 4 vs.
lanes 3 and 2) and was able t o bind directly to intact eIF4G
in vitro (Fig. 9D, lane 2 vs. lane 1). We also f ound that hsp25
had the ability to interact directly with a fragment of eIF4G
(M-FAG [48,49,52]); containing the e IF4E binding site and
central domain of eIF4 G (lane 4 vs. l ane 1), less w ith the
N-terminal domain of eIF4G (N-FAG; lane 3), and not t o
the C-terminal domain of eIF4G (C-FAG; lane 5) or to
eIF4E ( lane 6). These data suggest that the purified hsp25

was biologically active and that eIF4F associated with
hsp25 is functional in protein synthesis.
A
B
C
Fig. 7. Inhibition of p38MAP kinase a ctivity attenuates the induction of hsp25 and prevents its interaction with the eIF4F complex. (A) Serum-fed
C2C12 cells were preincubated for 30 min with dimethylsulfoxide alone (lanes 1 and 2), 20 l
M
SB203580 (lane 3) o r 100 n
M
RAD001 (lane 4), prior
to the addition of 50 l
M
MG132 (lanes 2 –4) f or 6 h. Cells were labelled as described in Fig. 2B and aliquots containing eq ual a mounts of
radioactive counts (42 500 c.p.m.) were resolved by SDS/PAGE and visualized by autoradiography. In addition, aliquots containing equal
amounts of protein (15 lg) were resolved by SDS/PAGE, protein transfer red t o PVDF and membranes prob ed with antise rum specific f or the
proteins indicated. Results are fro m a single experiment but are representative of those o btained on t hree separate o ccasions. (B) Aliq uots of extract
(75 lg protein) were subjected to m
7
GTP-Sepharose chromatography to isolate eIF4E and associated proteins. Recovered proteins were resolved
by SDS/PAGE and visualized by immunoblotting. Results are representative of t hose obtained in three separate experiments. (C) Se rum-fed C2C12
cells were preincubated fo r 30 min with dimethylsulfoxide alone (lanes 1, 2 and 5), 20 l
M
SB203580 (lane 3) or 100 n
M
RAD001 (lane 4), prior to
the add it io n o f 5 0 l
M
MG132 (lanes 2–5) for 6 h. Aliquots of extract (200 lg protein) were subjected to immunoprecipitation using none-immune
serum (lane 5) or anti-eIF4G serum (lanes 1–4) to isolate eIF4G an d associated proteins. Recovered proteins were resolved b y SDS/PAGE and

recovered eIF4E and hsp25 visualiz ed by immunoblotting. Results arerepresentativeofthoseobtainedinthreeseparateexperiments.
Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3605
B
A
Fig. 8. MG132 treatment of C2C12 myoblasts relocalizes eIF4GI and hsp25 to the perinuclear region. C2C12 cells were left untreated ( upper panels),
incubated w ith 50 l
M
MG132 for 8 h (middle panels) or incubated for 1 h with 20 l
M
SB203580 then 8 h with 50 l
M
MG132 ( lower panels) before
being processed as in Fig. 4. The localization of the endogenous proteins within these cells was visualized using combinations of a ntisera: (A) Rabbit
anti-eIF4GI labelled with Alexa Fluor 555 (red) and rabbit anti-eIF4E labelled with Alexa Fluor 488 (green); (B) Rabbit anti-eIF4GI (Alexa Fluor
555) and rabbit anti-hsp25 ( Alexa Fluor 488); (C) Rabbit anti-eIF4GI (Alexa Fluor 555) and mouse anti-(aB-crystallin) Ig d etected w ith goat anti-
(mouse IgG) conjug ated to FITC. White bars represent 20 lmforallpanels.
3606 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
This study demonstrates that in C2C12 myoblasts,
inhibition of the proteasome with MG132 or lactacystin
(data n ot shown) promotes a transient, incomplete
inhibition of the rate of protein synthesis, followed by a
re-programming of the t ranslational apparatus (Figs 1A
and 2A). In contrast to our previous findings using
Jurkat cells [41], MG132 did not result in a loss of cell
viability (data not shown) or facilitate the cleavage of
eIF4GI and eIF4GII (Figs 1 , 3 and 5B), but did promote
the re-localization of eIF4GI to the perinuclear region
(Fig. 4 A). Even after exposure to MG132 or cisplatin for
24 h, eIF4GI and eIF4GII remained intact and cells were

viable. These data suggest that incubation of C2C12 cells
with MG1 32 did not generate active caspase-3 or
caspase-8-like proteases that target eIF4G for cleavage
[41,48,49]. Rather, inhibition of the proteasome resulted
in the induction of hsp25, aB-crystallin and hsp70
(Figs 3–5), protecting cells against a poptosis [16,17,20,
29–34], and activating signalling pathways associated
with cell s urvival (Fig. 3). Furthermore, MG132 promo-
ted the colocalization of e IF4GI and hsp25 to t he
perinuclear region, in a n SB203580-sensitive manner
(Fig. 8 B); this effect was not observed with aB-c rystallin
(Fig. 8 C). In p ublished studies, M G132 has b een shown
to promote the transcriptional activation of heat shock
genes as a consequence of hyper-phosphorylation of t he
transcription factor, HSF1 [57,58]. The induction of
hsp25 and aB-crystallin is likely to be central to the
observed survival of our cells under these stressful
conditions as they can d irectly modulate the activity of
Akt30 and prevent apoptotic cell death by binding to,
and inactivating caspase-3, caspase-9 and c ytochrome c
[29,59].
Prior to the induction of hsp25, we have shown that
MG132 promotes a transient increase in the level of eIF2a
phosphorylation and a later, sustained increase in eIF4E
phosphorylation (Figs 1–3). Phosphorylation an d thus
inhibition of eIF2 a activity is commonly fou nd during cell
stress [60]. One possible explanation for the early increase in
eIF2a phosphorylation is that it i s required to disaggregate
polysomes allowing a different population of mRNA access
to the ribosomes. Similar suggestions have been made to

explain the early, transient a ctivation of eIF2a phosphory-
lation during the i nduction of apoptosis [42]. Enhanced
phosphorylation of eIF2a may reflect transient changes in
the activity of a number o f protein kinases, including PKR
and PERK [61,62]. However, using immunoblotting with
available phospho-specific a ntisera or i mmunocomplex
kinase assays, w e have been unab le to find evidence for
the activation of either PERK or PKR in response to
MG132 in our cells (data not shown). Therefore, at this
time, it is unclear whether the biphasic increase in eIF2a
phosphorylation reflects activation of kinase(s) or inhibition
of eIF2a phosphatase(s), or the temporal m odulation of
both. Clearly, further work is required to unravel the
mechanisms involved in this response.
In an attempt to understand the relative importance of
the observed signalling events i n promoting translational
C
Fig. 8. (Continued).
Ó FEBS 2004 MG132 and the reprogramming of translation (Eur. J. Biochem. 271) 3607
re-programming, w e have investigated the role of eIF4E
phosphorylation, ERK, mTOR and p 38MAP kinase activ-
ity in this response. The data presented in F ig. 6 shows that
inhibition of either Mnk1 or ERK activity with CGP57380
or UO126, respectively, did not affect MG132-induced
changes in t ranslation rates or t he interaction of hsp25 with
eIF4F. Similarly, inhibition of mTOR activity with
RAD001, rapamycin or LY294002 (data not shown) was
without effect (Fig. 7). As predicted [57], inhibition of
p38MAP kinas e activity with SB203580 prevented the
accumulation of hsp25 and its association with eIF4F

(Fig. 7 ). It is widely accepted that following activation of
p38 MAP kinase [63], hsp25 is phosphorylated by MAPK-
activated protein kinases 2 and 3 [64] and Akt. While
phosphorylation m ay not always be required for its
protective function [16], it often results in a redistribution
of hsp25 oligomers (600–800 kDa) into both tetramers and
dimers that associate with the actin cytoskeleton [15,65] and
eIF4F (Fig. 3 [14]);. Our data shows that hsp25 and eIF4GI
can b e coisolated by biochemical means (Figs 5–7) and b e
visualized in the s ame cellular c ompartment (Fig. 8). In
mouse cardiac myoblasts, human glioma cells and HeLa
0
50000
100000
[
35
S] methionine incorporation (cpm
)
0
20 40
60
time (min)
150
75
37.5
0
225
GST
A
eIF4G

N-FAG
M-FAG
C-FAG
eIF4E
143256
input
D
037.5
75
150
GST
0
50
100
Luciferase ( of control)
µg/ml hsp25
B
1432
eIF4G
C
eIF4E
hsp25
Sepharose m7GTP-
Sepharose
++
hsp25
Fig. 9. Hsp25 interacts with eIF4G in the reticulocyte lysate without an inhibition of translation. (A) Rabbit reticulocyte lysate was incubated in the
absence (0) or presence of 37.5 lgÆmL
)1
,75 lgÆmL

)1
,150 lgÆmL
)1
, 225 lgÆmL
)1
hsp25 or GST (120 lgÆmL
)1
), for the tim es shown. I ncorporatio n
of [
35
S]methionine into protein was d etermined by trichloroacetic acid precipitation. The presented data are the means and SD (bars) of t wo
separate experiments, each performed in triplicate. (B) Rabbit reticulocyte lysate, supplemented with either 3.125 lgÆmL
)1
capped luciferase
reporter mRNA (unfilled bars) or 6.25 lgÆmL
)1
uncapped, EMCV-IRES-luciferase mRNA (filled bars) was incubated for 90 min in the absence or
presence of hsp25 at the final concentrations indicated, or with 120 lgÆmL
)1
GST. The production of l uciferase is expressed as the percentage o f that
obtained in the absence of ad ded protein. T he presented data are the means + SD (bars) of two separate experiments, each performed in triplicate.
(C) Aliquots of lysate (10 lL) derived from incubations shown in (A) (in the absence or presence 150 lgÆmL
)1
hsp25) were subjected to m
7
GTP-
Sepharose or S epharose affinity chromatography to isolate e IF4E and a ssociated proteins. Recovered proteins were eluted with m
7
GTP and
resolved by SDS/PAGE and visualized by immunoblotting. Results are representative of those obtained in three separate experiments. (D) Buffer

alone (lane 1) or purified eIF4G (lane 2), N-FAG (lane 3), M-FAG (lane 4), C-FAG (lane 5) or eIF4E (lane 6) were prebound to nickel–agarose
before the a ddition of recombinant hsp25, as described in Materials and methods. Following isolation a nd washing of the resin, p rotein w as e luted
and v isualized by SDS/PAGE a nd Coomassie staining. Th e upper p anel shows the input p roteins and the lower p anel, the recovere d hsp25. Results
are representative of those obtained in three s eparate experiments.
3608 J. L. Cowan and S. J. Morley (Eur. J. Biochem. 271) Ó FEBS 2004
cells [16,38], M G132 treatment promoted the formation of
aggresomes containing hsps, associated with the a ctin
cytoskeleton. In C2C12 myoblasts (Fig. 8), hsp25 was
colocalized with actin, albeit to a lesser extent than reported
with H9c2 cells [15]. The reasons for these differences are
unclear, but may reflect cell-specific effects, the relative
abundance of stress fibres or that a specific type of actin
assembly is required for efficient hsp25 binding.
In a different study with HeLa cells where the stress was a
severe heat shock, protein synthesis was inhibited, eIF4G
was sequestered into perinuclear stress granules by hsp27
(the human equivalent of hsp25), eIF4F was disaggregated
and eIF4E was dephosphorylated [14]. This is distinct to the
response w e s ee with C2C12 cells upon proteasome
inhibition where Mnk1 and eIF4E become phosphorylated
within 1–2 h (Figs 1–3), indicating that cell and stress-
specific effects can impinge upon translation initiation. To
investigate further any role for hsp25 a ssociation with the
eIF4F complex, we have used purified, recombinant protein
and the reticulocyte lysate translation system. As shown in
Fig. 9, bacterially expressed hsp25 was able t o bind d irectly
to eIF4G and to a central domain of eIF4G in vitro,
associating with the eIF4F c omplex to stimulate on-going
translation to a modest level. In agreement with published
studies [16], these data suggest that phosphorylation of

hsp25 is not absolutely required for its function. These data
contrast the findings o f Cuesta et al. [14] who showed that
hsp27 formed an insoluble complex with eIF4G in the
reticulocyte lysate to inhibit translation. How ever, this effect
was only observed at heat shock temperatures. It has
recently been re ported that over-expression o f hsp27 i n
C2C12 cells during r ecovery from heat shock increased
translation of a luciferase reporter gene producing a cap-
dependent transcript [35]. While hsp27 was without effect
on IRES-driven translation, over-expression of hsp70 under
these assay c onditions stimulated both cap-dependent and
IRES-driven reporter gene expression. However, by using
phosphorylation site mutan ts of hsp27, these authors have
shown t hat there was no c orrelation between chaperone
activity and translational thermotolerance. Our d ata, com-
bined with published studies ([14,17,35]) suggests a model
whereby hsp25 interacts with eIF4G to stabilize the pro tein
during recovery f rom stress. Further w ork will be required
to determine whether our observations reflect the chaperone
activity of hsp25 and to delineate the exact site(s) of
interaction of hsp25 on eIF4G.
Acknowledgements
We would like to thank all members of the lab for helpful discussions.
This research was supported by a We ll come Trust Prize Studentship to
J.L.C and by grants from The Wellcome Trust (0 408 00, 050703,
045619, 056778). S.J.M. is a Senior Research Fellow of The Wellcome
Trust.
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