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: 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; 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 [...]... before the addition of recombinant hsp25, as described in Materials and methods Following isolation and washing of the resin, protein was eluted and visualized by SDS/PAGE and Coomassie staining The upper panel shows the input proteins and the lower panel, the recovered hsp25 Results are representative of those obtained in three separate experiments re-programming, we have investigated the role of eIF4E... and the reprogramming of translation (Eur J Biochem 271) 3607 C Fig 8 (Continued) Discussion This study demonstrates that in C2C12 myoblasts, inhibition of the proteasome with MG132 or lactacystin (data not shown) promotes a transient, incomplete inhibition of the rate of protein synthesis, followed by a re-programming of the translational apparatus (Figs 1A and 2A) In contrast to our previous findings... phosphorylated within 1–2 h (Figs 1–3), indicating that cell and stressspecific effects can impinge upon translation initiation To investigate further any role for hsp25 association 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 to bind directly to eIF4G and to a central domain of eIF4G in vitro,... Rather, inhibition of the proteasome resulted in the induction of hsp25, aB-crystallin and hsp70 (Figs 3–5), protecting cells against apoptosis [16,17,20, 29–34], and activating signalling pathways associated with cell survival (Fig 3) Furthermore, MG132 promoted the colocalization of eIF4GI and hsp25 to the perinuclear region, in an SB203580-sensitive manner (Fig 8B); this effect was not observed with. .. 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 luciferase is expressed as the percentage of that obtained in the absence of added protein The 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)... mTOR and p38MAP kinase activity in this response The data presented in Fig 6 shows that inhibition of either Mnk1 or ERK activity with CGP57380 or UO126, respectively, did not affect MG132-induced changes in translation rates or the 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... by binding to, and inactivating caspase-3, caspase-9 and cytochrome 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 and thus inhibition of eIF2a activity is commonly found during cell stress [60] One possible explanation for the. .. in vitro, associating with the eIF4F complex 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 of Cuesta et al [14] who showed that hsp27 formed an insoluble complex with eIF4G in the reticulocyte lysate to inhibit translation However,... that there was no correlation between chaperone activity and translational thermotolerance Our data, combined with published studies ([14,17,35]) suggests a model whereby hsp25 interacts with eIF4G to stabilize the protein during recovery from stress Further work will be required to determine whether our observations reflect the chaperone activity of hsp25 and to delineate the exact site(s) of interaction... 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 we see with C2C12 cells upon proteasome inhibition where Mnk1 and eIF4E become . The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex Joanne. [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