The proteasome inhibitor, MG132, promotes the reprogrammingof translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex Joanne L.. Prolonged incubati
Trang 1The 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 initiation factor (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 synthesis rates followed by a partial
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 Prolonged incubation with
MG132 resulted in the increased expression of heat shock
protein (hsp)25, aB-crystallin and hsp70, with 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 effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with aB-crystallin Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role 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 of initiation factors 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] and forms mutually
exclusive complexes on its convex surface with either
inhibitory regulatory proteins (4E binding 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 eIF4GII 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 mitogen-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 events controlled via the Target of Rapamycin (mTOR) signalling pathway (for reviews, see [1,5]), integrating signals from mitogens, nutri-ents and energy availability with the translational apparatus Current models suggest that hypophosphorylated 4E-BP1 binds to eIF4E to inhibit cap-dependent translation, a process readily reversed following its phosphorylation The dissociation of hyperphosphorylated 4E-BP1 from eIF4E leads to the binding of eIF4G to eIF4E and the initiation of protein synthesis (for reviews, 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 process associated with the dissociation 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 proteins are highly expressed in skeletal muscle [19] and a number of 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: s.j.morley@sussex.ac.uk
Abbreviations: DAPI, 4¢,6¢-diamidino-2-phenylindole hydrochloride;
eIF, eukaryotic initiation factor; ERK, extracellular-signal regulated
kinase; hsp, heat shock protein; FITC, fluorescein isothiocyanate;
m7GTP, 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)
Trang 2functions in the cell These include functioning as a
chaperone, by binding to and sequestering unfolded
proteins [21], stabilizing the cytoskeleton [22–24] and
conferring resistance to oxidative stress and TNFa-induced
cytotoxicity [25] The induction of hsp25 and aB-crystallin is
required for the differentiation of cardiomyocytes [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 the 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 to 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 data suggest that chaperone activity is not a
prerequisite for protection of translation by small hsps after
heat shock [35]
The ubiquitin-proteasome pathway is another
mech-anism that contributes to cell protection from 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] Inhibition of the proteasome 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-localization of hsp25 and aB-crystallin
to the actin cytoskeleton [15,37,38] By binding to, and
capping the F-actin barbed ends [39], hsp25 results in a
reorganization of the actin filament system, playing a role
in the migration of endothelial cells and in recovery of
cells from wounding [40] In a variety of cells, in addition
to the induction of hsp25 and aB-crystallin expression,
inhibition of the proteasome induces aggresome
forma-tion [38], activaforma-tion of stress kinases and programmed
cell death [16,17,20,25,29,30] Aggresomes, which may be
associated with intermediate filaments [38], contain
misfolded proteins, hsp25, aB-crystallin and components
of the degradation machinery As such, they are thought
to provide a cytoprotective role by clearing the cytoplasm
of potentially toxic aggregates [36]
As part of an on-going investigation into the regulation of
protein synthesis in mammalian cells, we have investigated
the effects of inhibition of the proteasome on 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 of eIF4GI or 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 was associated with the eIF4F 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 of 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 from Alexis Corporation RAD001 [43] and CGP57380 [44] were gifts from Novartis (Basel, Switzerland) Antisera to hsp25, aB-crystallin and hsp70 were from Stressgen Biotechnolo-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 to total eIF2a was a gift from the late
E Henshaw (Rochester, NY, USA)
eIF4GII was provided by N Sonenberg (Montreal, Canada) [35S]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)
cultured in DMEM supplemented with 20% (v/v) fetal bovine serum at 37C in a humidified atmosphere containing 5% CO2
Preparation of cell extracts Following treatment, cells were isolated in a cooled centrifuge and washed with 0.5 mL ice-cold NaCl/Pi containing 40 mMb-glycerophosphate and 2 mM benzami-dine Pellets were resuspended in 200 lL ice-cold Buffer A [20 mMMOPS/KOH, pH 7.2, 10% (v/v) glycerol, 20 mM sodium fluoride, 1 lM microcystin, 75 mM KCl, 2 mM MgCl2, 2 mM benzamidine, 2 mM Na3VO4, complete protease inhibitor mix)EDTA
vortexing following the addition of 0.5% (v/v) Igepal and 0.5% (v/v) deoxycholate Cell debris was removed by centrifugation in a microfuge for 5 min at 4C and the resultant supernatants frozen in liquid N2
SDS/PAGE, vertical slab iso-electric focusing and immunoblotting
Samples containing equal amounts of protein 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 as described previously
Trang 3[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 isolated from crude
serum by affinity chromatography with the corresponding
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[35S]methionine and either 0.1% or 20% (v/v)
fetal bovine serum in complete medium, as indicated Cells
were recovered and washed once in NaCl/Piprior to lysis in
0.3MNaOH, or extracts were prepared as described 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
m7GTP-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
m7GTP-Sepharose chromatography and the resin washed
twice with Buffer B (20 mM Mops/KOH pH 7.4, 75 mM
KCl, 2 mMMgCl2, 1 lMmicrocystin, 10 mMNaF, 2 mM
benzamidine, 7 mM 2-mercaptoethanol, 0.1 mM GTP) Recovered protein was eluted either directly into sample buffer or eluted with 0.2 mMm7GTP in Buffer B for either SDS/PAGE or VSIEF [44,45,47–49]
Immunoprecipitation of eIF4GI Extracts containing equal amounts of protein were diluted
in Buffer C [50 mMTris/HCl, pH 8, 20 mM NaF, 50 mM KCl, 2 mM EDTA, 2 mM benzamidine, 2 mM Na3VO4, 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)1BSA) coated with either purified anti-eIF4GI IgG or nonimmune rabbit IgG Beads were isolated, washed five times with 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) C2C12 cells were either serum fed (unfilled bars) or serum-starved (filled bars) for 24 h before incubation for 6 h with the indicated concentrations of MG132 To measure the rate of protein synthesis, cells were incubated with 10 lCiÆmL)1[ 35 S]methionine during the last 15 min, solubilized in 0.3 M NaOH and incorporation of radioactivity into protein determined by trichloroacetic 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 SDS/PAGE The integrity of eIF4GI and eIF4E was 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 representative of those obtained on three separate occasions (C) Quantification of the data presented in (B) The presented data for eIF4E was quantified by densitometric scanning from the VSIEF analysis; all are the means + SD (bars) of three separate experiments.
Trang 4the manufacturer’s instructions Anti-(C-terminal eIF4GI)
was labelled in the same way with Alexa Fluor 555
Antibodies were diluted as follows into NaCl/Picontaining
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)
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) Following incubation with or without SB203580 or
MG132, cells were washed once in NaCl/Pi, then fixed in
4% (w/v) paraformaldehyde in NaCl/Pi, pH 7.4 for 15 min
After a single rinse with NaCl/Pi, cells were permeabilized in
NaCl/Pi containing 0.1% (v/v) Triton X-100 for 5 min,
rinsed once in NaCl/Pi then incubated with 0.1% (w/v)
NaBH4for 5 min prior to three washes with NaCl/Pi
Non-specific binding was blocked by adding 1% BSA in NaCl/Pi
for 30 min Cells were incubated in the primary antibody
solution for 60 min, washed extensively and then incubated
with the appropriate secondary antibody and phalloidin–
FITC (or TRITC)
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 cm plates with mowiol
mounting solution [0.2MTris, 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 Axioscop 2 microscope
equipped with a 63· oil immersion objective and fitted
with the appropriate filter sets Images were captured with a
Photometrics Quantix digital camera Images were
proc-essed using imaging software (Universal
Ima-ging Corp., Downingtown, PA, USA)
were pseudo-coloured to correspond to the red (TRITC/ Alexa Fluor 555), green (FITC/Alexa Fluor 488) or blue
minutes 0
20000 40000 60000
A
360 240 60
40 20
1 7 2 3 4 5 6 B
221
17
32 43 82 128
eIF4E
eIF4E
C
Mnk1-P
eIF2
Mnk1 p38MAPK-P S6-P
ERK-P ERK
eIF4E-P
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 for the times indicated
and the rate of protein synthesis measured as in Fig 1A The presented
data are the means + SD (bars) of two separate experiments, each
performed in triplicate (B) In parallel cultures, cells were treated as in
(A) but in the presence of 100 lCiÆmL)1[35S]methionine and cell
extracts were prepared as described Aliquots containing equal
amounts of radioactive counts (54 000 c.p.m.) were resolved by SDS/
PAGE and visualized by autoradiography (upper panel) In addition,
aliquots containing equal amounts of protein (15 lg) were resolved by
SDS/PAGE (middle panel) or VSIEF (lower panel) and eIF4E
visu-alized by immunoblotting Results are from a single experiment but are
representative of those obtained on four 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 PVDF
membranes were probed with antiserum specific for the proteins
indicated in the figure Results are representative of those obtained in
three separate experiments.
Trang 5(DAPI) fluorescence and the digital images were merged.
Preparation of images for publication used Adobe
PHOTO-SHOPv5.5 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 sequential affinity
chromatography and gel filtration [64] Purified initiation
factors ( 1 lg) were bound to nitrioltriacetic acid–nickel–
agarose (Qiagen) for 30 min at 4C in Buffer E (50 mM
Mops/KOH, pH 7.4, 300 mM KCl, 2 mM MgCl2, 2 mM
benzamidine, 3.5 mM2-mercaptoethanol, complete protease
inhibitor mix – EDTA), the resin was washed and further
incubated for 20 min on ice in Buffer F (Buffer E, but
containing 75 mMKCl and supplemented with 1 mgÆmL)1 BSA) Isolated resin was then resuspended in 10 lL Buffer F and incubated with continuous mixing in the presence of 3 lg hsp25 for 30 min at 4C Beads were isolated by centrifu-gation, washed twice with 0.2 mL Buffer F (without BSA) and recovered protein eluted with 100 mMEDTA for SDS/ PAGE analysis
In vitro translation Reticulocyte lysates were prepared in-house; incubations 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 of hsp25 and aB-crystallin to the actin cytoskeleton [15,38] and result
in cell cycle arrest To 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 lM MG132 for 6 h resulted in a 40% inhibition of translation irrespective of the nutritional status of the cell Increasing the concentration of MG132 up to 50 lM did not further inhibit translation and did not decrease cell viability even following prolonged incubation times (data not shown)
Previously, we have shown that eIF4GI 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 in 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 were incubated in the presence of dimethylsulfoxide alone (lane 1) or 50 l M MG132 (lanes 2–5) for the times indicated and cell extracts were prepared Aliquots containing equal amounts of protein (15 lg) were resolved 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) Serum-fed C2C12 cells were incubated in the presence of dimethylsulfoxide alone (lanes 5 and 6) or 50 l M MG132 (lanes 1–4) for the times indicated and cell extracts were prepared and resolved as in (A) Membranes were probed with antiserum specific for PABP (loading control) or a phospho-specific antiserum for 4E-BP1 phosphorylated on Ser64 Results are from a single experiment but are representative of those 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 on four separate occasions.
Trang 6the integrity of eIF4GI or eIF4E, as visualized by
immuno-blotting Furthermore, all initiation factors analysed
(inclu-ding eIF4GII) remained intact even after 24 h of treatment
with high levels of MG132 (data not shown) Together,
these data 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 lM MG132
(Fig 1B, lane 2 vs lane 1; quantified in C), an event often
associated with a global inhibition of translation rates [1] In
addition, the phosphorylation of eIF4E, an event often
correlated with increased rates of translation [1,5], was also increased following MG132 treatment for 6 h, with maximal effects only observed at the highest doses (Fig 1B,C)
MG132 causes a re-programming of translation and the activation of multiple signalling pathways
in C2C12 myoblasts
To further investigate the effect of cell stress on translation rates in C2C12 myoblasts, cells were exposed
to 50 lM MG132 and pulse-labelled with [35S]methionine
at different times Figure 2A shows that the rate of
eIF4GI
8h 50 M
MG132
A
B
untreated
cells
untreated
cells
8h 50 M
MG132
Fig 4 Intracellular localization of eIF4GI and eIF4E C2C12 cells were left untreated (upper panels) or incubated with 50 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) Goat anti-(mouse IgG) conjugated to FITC (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 DAPI (pseudocoloured blue) White bars represent 20 lm for all panels.
Trang 7translation decreased between 40 and 60 min after
exposure to MG132, with rates further decreasing to
20% of control levels within 2 h Analysis of cell extracts
by SDS/PAGE indicated that there 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 was preceded by a transient
activation of ERK (lanes 2–4 vs lane 1) but coincided
with the sustained activation of p38MAP kinase and
increased phosphorylation of Mnk1 (lanes 5 and 6 vs
lane 1), signalling molecules which are functionally
upstream of eIF4E [3] In addition, MG132 promoted
the phosphorylation of ribosomal protein S6 (Thr421/
Ser424; Figs 2C and 3A) and Akt (Fig 3A) at 1–2 h,
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 15 min of treatment (Fig 2C, lane 3 vs lane 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 to 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, including MG132 treatment, has been reported
to increase the expression of the stress-related proteins, hsp25, aB-crystallin and hsp70 [14,17,38] To determine whether the MG132-induced expression of hsps occurred 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
merge
actin B-crystallin
Fig 4 (Continued).
Trang 8aB-crystallin levels (Fig 3C) increased 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 rates Using
antiserum specific for 4E-BP1 phosphorylated on Ser64 we
have confirmed that following 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 extent (see merged images in right panels and [55,56])
In untreated cells, hsp25 staining was diffuse, being localized, in part to the perinuclear region and to areas
of more intense staining at the cell periphery (Fig 4C) Similarly, aB-crystallin was detectable in resting cells, but showed a largely diffuse, and grainier cytoplasmic staining (Fig 4D) Following treatment 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 4B), showed a more granular appearance, being 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 related myoblast cell line, H9c2, which suggested that inhibition of proteasomal activity resulted in a re-localiza-tion of aB-crystallin to aggresomes [15] However, in our cells, these were not associated with the cytoskeleton to 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 with m7 GTP-Sepharose (Fig 5A) indicated that MG132 caused a partial dissociation of eIF4GI, PABP and eIF4A from eIF4E at later times of incubation (Fig 5A, lane 5 vs lane 1 and Fig 5B, lane 7 vs lane 1) As eIF4G exists in two forms (eIF4GI and eIF4GII [1,53]), we also monitored the associ-ation of eIF4GII with eIF4E under these conditions Figure 5B shows that as with eIF4GI, MG132 also caused
a partial dissociation of eIF4GII and Mnk1 from eIF4E after
4 h of MG132 treatment (lane 7 vs lane 1), indicative of a general decrease in eIF4F complex levels As predicted from accepted models [1,5], the 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 vs lane 1; Fig 5B, lane 7 vs lane 1) However, aB-crystallin did not associate with eIF4F under these conditions (Fig 5A), even though it was induced to high levels by MG132 (Fig 3C) These data suggest that, as with heat shock [14,17,35], hsp25 may have a role in regulating eIF4F activity following the inhibition of proteasome activity
eIF4E and ERK phosphorylation are not required for translational re-programming in response to MG132
To investigate the role of eIF4E phosphorylation or the activation of ERK in the translational response to MG132,
we have used the cell permeable inhibitors, CGP57380 and UO126, respectively [44,46,54] Figure 6A shows that pre-treatment of cells with CGP57380 (lane 4 vs lane 2) had little effect on the translational re-programming in response to MG132 Under these assay conditions, while the increase in eIF4E phosphorylation was 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 panel)
4E-BP1 eIF4E
m7GTP-Sepharose
eIF4GI eIF4GII PABP Mnk1 eIF4E hsp25
m7GTP-Sepharose
αB crystallin
MG132
hsp25
PABP eIF4A eIF4G
Fig 5 Hsp25 associates with the eIF4F complex (A) Aliquots of
extract (75 lg protein) were subjected to m7GTP-Sepharose
chroma-tography 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
experi-ments (B) Serum-fed C2C12 cells were incubated in the presence of
50 l M MG132 for the times indicated and cell extracts were prepared.
Aliquots of extract (75 lg protein) were subjected to m7
GTP-Seph-arose 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 two
separate experiments.
Trang 9Similarly, inhibition of ERK signalling with UO126 had no
effect on any of the MG132-induced responses (Fig 6A,B,
lanes 3 vs lanes 1) even though it 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 pathways 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 specificity of this inhibitor was demonstrated by its
ability to reduce both the expression of hsp25 and eIF4E
phosphorylation, without affecting the phosphorylation
status of ribosomal protein S6, 4E-BP1 or eIF2a (Fig 7A,
lower panels) Consistent with these findings, inhibition of
signalling downstream of p38MAPK with SB203580
prevented the coisolation of hsp25 with eIF4F on
m7GTP-Sepharose (Fig 7B, lane 3 vs lane 2) or following
immunoprecipitation of eIF4G from extracts (Fig 7C,
lane 3 vs lane 2) In contrast, 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 and 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 5C, lane 4 vs lane 2), but did, as predicted, promote
increased association of 4E-BP1 with eIF4E (Fig 7B) As
pretreatment of cells with any of these inhibitors alone, or
in combination was unable to prevent the global
inhibi-tion of protein synthesis observed with MG132, these 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 translation in cell extracts following severe heat shock
by promoting stress granule formation, sequestering eIF4G into inactive complexes [14] To determine whether such events occur during translational 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) indicates that eIF4E and eIF4GI showed extensive colocalization in untreated cells These findings are in agreement with previous work [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 proteins while promoting distinct areas of intense cytoplasmic colocalization Pre-treatment of cells with 20 lMSB203580 largely prevented this redistribution
of factors, with colocalization predominant in the perinuclear region (lower panel) In a similar manner, we have also covisualized eIF4GI and hsp25 (Fig 8B) These 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, Fig 8C shows that
B A
Fig 6 eIF4E phosphorylation is not required for translational re-programming in response to MG132 (A) Serum-fed C2C12 cells were preincubated for 30 min with dimethylsulfoxide alone (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 amounts of radioactive counts (45 000 c.p.m.) were resolved by SDS/PAGE and visualized by autoradiography (B, upper panels) Aliquots of extract containing equal amounts of protein (15 lg) were resolved by VSIEF (top panel) and the phosphorylation status of eIF4E visualized by immunoblotting Aliquots were also resolved by SDS/PAGE (remaining panels), protein transferred to PVDF and membranes probed with antiserum specific for the proteins indicated Results are from a single experiment but are representative of those obtained on three separate occasions (B, lower panels) 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.
Trang 10although MG132 treatment resulted in an
SB203580-sensi-tive re-distribution of aB-crystallin to granules (compare
middle and lower panels), there 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
translationin vitro
To investigate more directly the effects of hsp25 on protein
synthesis, we have added purified, recombinant hsp25 to the
reticulocyte lysate translation system Relative to buffer
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 concentrations of added capped or IRES-driven luciferase reporter mRNAs Figure 9B shows that relative to GST, low concentrations of added hsp25 did not influence reporter mRNA 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 to bind directly to intact eIF4G
in vitro(Fig 9D, lane 2 vs lane 1) We also found that hsp25 had the ability to interact directly with a fragment of eIF4G (M-FAG [48,49,52]); containing the eIF4E binding site and central domain of eIF4G (lane 4 vs lane 1), less with the N-terminal domain of eIF4G (N-FAG; lane 3), and not to 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 activity 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) or 100 n M RAD001 (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 amounts 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 transferred to PVDF and membranes probed with antiserum specific for the proteins indicated Results are from a single experiment but are representative of those obtained on three separate occasions (B) Aliquots of extract (75 lg protein) were subjected to m7GTP-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 (C) Serum-fed C2C12 cells were preincubated for 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 addition of 50 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 and associated proteins Recovered proteins were resolved by SDS/PAGE and recovered eIF4E and hsp25 visualized by immunoblotting Results are representative of those obtained in three separate experiments.