essential role of pact-mediated pkr activation in tunicamycin-induced apoptosis

doi:10.1016/j.jmb.2008.10.068 J. Mol. Biol. (2009) 385, 457–468

Available online at www.sciencedirect.com

Essential Role of PACT-Mediated PKR Activation inTunicamycin-Induced Apoptosis

Madhurima Singh1, Vennece Fowlkes1, Indhira Handy1,Chandrashekhar V. Patel2 and Rekha C. Patel1⁎

1Department of BiologicalSciences, University of SouthCarolina, 700 Sumter Street,Columbia, SC 29208, USA2Department of Cell,Developmental Biology andAnatomy, University of SouthCarolina, Columbia, SC 29208,USA

Received 9 September 2008;received in revised form8 October 2008;accepted 22 October 2008Available online5 November 2008

*Corresponding author. E-mail [email protected] used: ER, endoplas

dsRNA, double-stranded RNA; dsRmotifs; CHOP, C/EBP homologous pembryonic fibroblasts; GRP, glucosePKR, dsRNA-activated protein kinaEGFP, enhanced green fluorescent pPKR-like ER resident kinase; eIF2α,eukaryotic initiation factor 2; GCN-2nonderepressible-2; DMEM, Dulbecmedium.

0022-2836/$ - see front matter © 2008 E

Cellular stresses such as disruption of calcium homeostasis, inhibition ofprotein glycosylation, and reduction of disulfide bonds result in accumula-tion of misfolded proteins in the endoplasmic reticulum (ER) and lead tocell death by apoptosis. Tunicamycin, which is an inhibitor of proteinglycosylation, induces ER stress and apoptosis. In this study, we examinedthe involvement of double-stranded RNA (dsRNA)-activated proteinkinase (PKR) and its protein activator PACT in tunicamycin-inducedapoptosis. We demonstrate for the first time that PACT is phosphorylated inresponse to tunicamycin and is responsible for PKR activation by directinteraction. Furthermore, PACT-induced PKR activation is essential fortunicamycin-induced apoptosis, since PACT as well as PKR null cells aremarkedly resistant to tunicamycin and show defective eIF2α phosphoryla-tion and C/EBP homologous protein (CHOP, also known as GADD153)induction especially at low concentrations of tunicamycin. Reconstitutionof PKR and PACT expression in the null cells renders them sensitive totunicamycin, thus demonstrating that PACT-induced PKR activation playsan essential function in induction of apoptosis.

© 2008 Elsevier Ltd. All rights reserved.

Edited by J. Karn
Keywords: interferon; PKR; PACT; apoptosis; eIF2α
Introduction

Double-stranded RNA (dsRNA)-activated proteinkinase (PKR) is an interferon (IFN)-induced serine/threonine kinase that is expressed ubiquitously.1–3

Although IFNs increase PKR's cellular abundance,PKR's kinase activity stays latent until it binds toone of its activators leading to its autophosphoryla-tion and activation.4,5 The well-known activator ofPKR is dsRNA, a replication intermediate for severalviruses.6 The best-characterized cellular substrate of

ess:

mic reticulum;BMs, dsRNA-bindingrotein; MEFs, mouse-regulated protein;se; wt, wild type;rotein; PERK,α subunit of, general controlco's modified Eagle's

lsevier Ltd. All rights reserve

PKR is the translation initiation factor, eIF2α, thephosphorylation of which results in an inhibition ofprotein synthesis.7,8 Although PKR's antiviral acti-vities are the most studied, PKR is also implicated inthe signal transduction pathways activated by cyto-kines, growth factors, dsRNA, and extracellularstresses.2,3,9,10 Optimal activation of p38, c-Jun N-terminal kinase, stress-activated protein kinases,and the downstream transcription factors inducedby these kinases, such as NF-κB, IRF-1, p53, STAT1,ATF, STAT3, and AP-1, have been shown to requirePKR activity.2,3,9,10 Thus, PKR is involved in multi-ple cellular processes such as differentiation, apop-tosis, proliferation, and oncogenic transformation.3

dsRNA binds to PKR via the two dsRNA-bindingmotifs (dsRBMs) present at the N terminus,11–15changing the conformation of PKR to expose theATP-binding site16,17 and consequent autophospho-rylation.18 In addition, the two dsRBMs can mediatedsRNA-independent protein–protein interactionswith other proteins that carry similar domains.19–24

Among these are proteins inhibitory for PKR acti-vity such as TRBP,23 Dus2L,24 and activator proteinPACT.25,26 PACT's association with PKR leads to theactivation of PKR in the absence of dsRNA.25,26

PACTcontains three identifiable copies of dsRBM, of

d.

458 PACT Activates PKR in Response to Tunicamycin

which motifs 1 and 2 are true dsRBMs and exhibitdsRNA-binding activity. In addition, these twoamino-terminal dsRBMs in PACT also bind tightlyto the N-terminal dsRBMs of PKR. The third, car-boxy-terminal motif shows significant homology toa consensus dsRBM but is not a functional dsRBMsince it does not bind dsRNA. However, this thirdmotif is essential for PKR activation and binds to aspecific region in the kinase domain of PKR withlow affinity.27,28

Although purified, recombinant PACTcan activatePKR by a direct interaction in vitro,25 PACT-depen-dent PKR activation in intact cells occurs in the pre-sence of a cellular stress signal.26,29,30 PACT-mediated activation of PKR occurs in response tocellular stresses, such as arsenite, peroxide, growthfactor withdrawal, thapsigargin, and actinomycin,and leads to phosphorylation of the translationinitiation factor eIF2α and cellular apoptosis.26,29,30

PACT (and its murine homolog RAX) is phos-phorylated in response to the stress signals and thisleads to its increased association with PKR, causingPKR activation.26,29–32 In addition, an overexpres-sion of a truncated form of PACT (PACT305) can alsolead to cellular apoptosis in the absence of a stresssignal.26,27 PACT305 is presumably in an active con-formation due to the truncation and thus may notrequire the stress-induced phosphorylation of thespecific serine residues32 to bring about PKR asso-ciation and activation.In this study, we investigated the possible in-

volvement of PACT-mediated activation of PKR inapoptosis induced by endoplasmic reticulum (ER)stressor tunicamycin. Tunicamycin inhibits N-glyco-sylation of proteins and thus results in an accumula-tion of misfolded proteins in the ER, a primary causeof ER stress. To counteract the adverse effects of ERstress, cells trigger compensatory responses, whichare collectively termed as unfolded protein res-ponse. This includes a generalized suppression oftranslation33 while inducing increased expression ofmolecular chaperones, such as glucose-regulatedprotein (GRP)94 and GRP78/Bip, which promoteproper folding of proteins,34 and ER-associateddegradation35,36 of misfolded proteins. These threeprotective responses act transiently to control theaccumulation of misfolded proteins within the ER.The inhibition of protein synthesis to cope with ERstress is mainly achieved by phosphorylation of theinitiation factor eIF2α.37 The expression of the trans-cription factor C/EBP homologous protein (CHOP,also known as GADD153) is induced in response toeIF2α phosphorylation.38,39 Although phosphoryla-tion of eIF2α causes a general block in translation, itparadoxically activates translation of the ATF4mRNA, which encodes a transcription factor thatbinds and activates the CHOP promoter. SustainedER stress leads to apoptosis, with the characteristicfragmentation of nuclei, condensation of chromatin,and shrinkage of cells.40 Cells lacking CHOP aresignificantly protected fromER stress-induced apop-tosis and thus, CHOP is thought to be one of themajorinducers of apoptosis in response to ER stress.41,42

Our results establish for the first time that PACT isphosphorylated in response to tunicamycin treat-ment, causing its increased association with PKRleading to PKR activation. PACT-dependent PKRactivation in response to tunicamycin is essential forinduction of apoptosis, since both PACT−/− andPKR−/− cells are resistant to tunicamycin-inducedapoptosis. The essential role of PACT-induced PKRactivation in this pathway is further underscored bythe fact that phosphorylation of eIF2α and inductionof CHOP were defective in PACT null cells. Thus,our results presented here uncover a novel func-tional role of PACT and PKR in the tunicamycin-induced stress response pathway.

Results

PKR null cells are defective intunicamycin-induced apoptosis

In order to test the involvement of PKR in tuni-camycin-induced apoptosis, we compared the apop-totic response in mouse embryonic fibroblasts(MEFs) derived from wild-type (wt) and PKR nullmice. The characteristic subdiploid peak represent-ing the apoptotic population was compared at 24,48, and 72 h after tunicamycin treatment by flowcytometry analysis. The subdiploid DNA content isa characteristic of apoptotic cells and these cellsshow a distinct peak before the G1 peak. In the wtMEFs, tunicamycin at 0.1 μg/ml concentrationinduced apoptosis in 17.8 %, 36.0%, and 38.7%cells at 24, 48, and 72 h, respectively (Fig. 1a). Incontrast to this, the PKR null MEFs showed noapoptosis above the control at any of these timepoints. Thus, the PKR null cells are markedly resis-tant to tunicamycin-induced apoptosis. To examinethe ability of tunicamycin to induce apoptosis inPKR null MEFs, we further investigated if higherconcentrations of tunicamycin could induce apop-tosis in the absence of PKR activity. The progressionof apoptosis was monitored by DNA fragmentationanalysis performed 48 h after tunicamycin treat-ment. Apoptosis is associated with the fragmenta-tion of chromosomal DNA into multiples of thenucleosomal units, known as DNA laddering. Asseen in Fig. 1b, the wt MEFs show a clear DNAfragmentation ladder at tunicamycin concentrationshigher than 0.025 μg/ml (lanes 3–8). In contrast, thePKR null cells showed DNA fragmentation only attunicamycin concentrations higher than 0.25 μg/ml(lanes 6–8). Thus, PKR null cells are markedly resis-tant to tunicamycin concentrations up to 0.1 μg/ml.Even at concentrations higher than 0.25 μg/ml, theDNA fragmentation in PKR null MEFs was sig-nificantly weaker than in wt MEFs. These resultsestablish that PKR null MEFs are significantlyresistant to tunicamycin and that PKR plays animportant functional role in tunicamycin-inducedapoptosis.

Fig. 1. PKR null MEFs are resis-tant to tunicamycin-induced apop-tosis. (a) Flow cytometry analysis ofcell-cycle distribution after tunica-mycin treatment. PKR+/+ andPKR−/− MEFs were treated with0.1 μg/ml tunicamycin. Cells wereharvested at 24, 48, and 72 h afterthe treatment and subjected to flowcytometry analysis after propidiumiodide staining. The sub-G0/G1 cellpopulation represents the apoptoticcells. The sub-G0/G1 percentagesare displayed in each panel. Theseexperiments were repeated twice,each time with duplicate samples.Themost representative profiles froma single experiment are shown. (b)DNA fragmentation analysis inresponse to tunicamycin. PKR+/+

and PKR−/− MEFs were treated withincreasing concentrations of tunica-mycin. Forty-eight hours after tunica-mycin treatment, the fragmented

DNAwas analyzed. The cell type is as indicated on the top of the lanes. Lane 1, untreated cells; lanes 2–8 cells treated withdifferent concentrations of tunicamycin. Lane 2, 0.01 μg/ml; lane 3, 0.025 μg/ml; lane 4, 0.05 μg/ml; lane 5, 0.1 μg/ml; lane 6,0.25 μg/ml; lane 7, 0.5 μg/ml, and lane 8, 1 μg/ml. M, 100-bp ladder DNA size markers.

459PACT Activates PKR in Response to Tunicamycin

PKR is activated in response to tunicamycin

We next examined if PKR activation takes placein response to tunicamycin treatment. As seen inFig. 2a, PKR is activated in response to tunicamycintreatment of the MEFs. AWestern blot analysis witha threonine 451 phosphospecific anti-PKR antibody,which specifically detects only the catalyticallyactive PKR, showed PKR activation 2 h after tunica-mycin treatment. PKR activation peaked at 4 h and

anti-PKRmonoclonal antibody to detect total PKR. The ratio oflane. (b) SK-N-SH: Western blot analysis with a phosphothreonwith 1 μg/ml tunicamycin and cell extracts were prepared at inwas examined by Western blot analysis with a phosphothreonreprobed with an anti-PKR monoclonal antibody to detect totarepresented below each lane. (c) SK-N-SH: PKR kinase actunicamycin and PKR activity was assayed from the ceimmunoprecipitated with a 71/10 monoclonal antibody anperformed with PKR attached to the protein A-Sepharosemarked +ve C are positive controls to show that activationpolyI–polyC (dsRNA) and 0.2 pmol of pure recombinant trassay reaction in vitro. (d) SK-N-SH: DNA fragmentation atreated with 1 μg/ml tunicamycin. At 24 and 48 h after tunLane 1, untreated cells; lane 2, 24 h treatment; lane 3, 48 h

showed a gradual decrease after that. We also exa-mined PKR activation in a human neuroblastomacell line SK-N-SH in response to tunicamycin. Asseen in Fig. 2b, PKR is activated in response totunicamycin in SK-N-SH cells with the same kineticsas in MEFs. To further ascertain that tunicamycintreatment activates PKR, we also performed PKRactivity assays with cell extracts prepared at varioustime points after the tunicamycin treatment in theabsence of any exogenously added PKR activator.

Fig. 2. PKR is activated in bothmurine and human cells in responseto tunicamycin. (a) PKR+/+ MEFs:Western blot analysis with a phos-phothreonine 451-specific antibody.TheMEFswere treatedwith 1μg/mltunicamycin and cell extracts wereprepared at indicated times afterthe treatments. Phosphorylation ofPKR was examined by Western blotanalysis with a phosphothreonine451-specific antibody. The same blotwas stripped and reprobed with an

phosphorylated PKR/total PKR is represented below eachine 451-specific antibody. The SK-N-SH cells were treateddicated times after the treatments. Phosphorylation of PKRine 451-specific antibody. The same blot was stripped andl PKR. The ratio of phosphorylated PKR to total PKR istivity assay. SK-N-SH cells were treated with 1 μg/mlll extracts prepared at the indicated times. PKR wasd protein A-Sepharose beads. PKR activity assay wasbeads without any exogenous activator added. Lanesoccurs with extract from untreated cells with 0.1 μg/mluncated PACT (amino acids 1–305) added to the kinasenalysis in response to tunicamycin. SK-N-SH cells wereicamycin treatment, the fragmented DNA was analyzed.treatment. M, 100-bp ladder DNA size markers.

Fig. 3. PACT activates PKR in response to tunicamy-cin. (a) PACT associates with PKR in response to tuni-camycin. SK-N-SH cells were treated with 1 μg/mltunicamycin. The cell extracts were prepared at indicatedtimes and 500 μg of protein from the cell extracts wasallowed to interact with 1 μg of purified PKR proteinimmobilized on Ni-agarose beads. The beads werewashed to remove unbound material and the PKR-associated PACT was analyzed by SDS-PAGE followedby immunoblotting using anti-PACT antibody. The same


As seen in Fig. 2c, PKR was activated 2 h aftertunicamycin treatment in SK-N-SH cells and itsactivity peaked 4 h after treatment. These resultsfurther confirm that PKR is activated by tunicamy-cin treatment of cells. At later time points of 8 and12 h, PKR activity gradually decreased. PKR activa-tion observed in response to tunicamycin is equallystrong compared with the activation observed withcell extract from untreated samples activated withexogenously added dsRNA or recombinant PACT(positive control lanes). Similar results wereobtained in several other cell types includingHeLa and human embryonic kidney (HEK) 293cells (data not shown). These results furtherestablish that tunicamycin treatment results inactivation of PKR in both murine and humancells. To confirm the apoptotic response of SK-N-SH cells to tunicamycin, the DNA laddering assaywas performed. As seen in Fig. 2d, the SK-N-SHcells showed characteristic DNA laddering inresponse to tunicamycin at 24 h, which furtherincreased at 48 h. This demonstrates that the SK-N-SH cells respond to tunicamycin in a mannersimilar to that of the MEFs.

blot was stripped and reprobed with anti-hexahistidinetag antibody to detect total PKR immobilized on Ni-agarose beads. To analyze total PACT amounts in theextracts, 100 μg of the total protein from extracts wasanalyzed by Western blot analysis with anti-PACT anti-body. (b) PACT phosphorylation and PKR association inresponse to tunicamycin. SK-N-SH cells were transfectedwith Flag-PACT/pCDNA3.1− (full-length/amino acids1–313) to achieve overexpression of Flag-PACT. Twenty-fours hours after transfection, the cells were treatedwith 1 μg/ml tunicamycin for 4 h in the presence of 0.5mCi/ml [32P]orthophosphate in phosphate-free medium.The phosphorylation status of PACT was examined byimmunoprecipitation with anti-Flag monoclonal antibodyfollowed by phosphorimager analysis (32P-FlagPACT). Inthe same gel, a weaker 32P-labeled band was observed at aposition corresponding to PKR (32P-PKR). Aliquots of celllysate with 100 μg total protein were also examined byWestern blot analysis for total Flag-PACT with anti-Flagantibody (Flag-PACT Western). A Western blot analysiswas performed with anti-PKR monoclonal antibody on1 mg of cell extracts immunoprecipitated with anti-FlagM2 monoclonal antibody (PKR Western). Subsequentautoradiographic analysis of theWestern blot also showedthe presence of 32P-labeling in the band corresponding toPKR.

PACT associates with PKR and activates itskinase activity in response to tunicamycin

Since PACT activates PKR in response to severalstress signals,26,29–32 we examined the involvementof PACT in PKR activation in response to tunicamy-cin. PACT-induced PKR activation occurs by a directassociation of PACTwith PKR in response to a stresssignal.26,32 Thus, we decided to test the associationof PACT with PKR in response to tunicamycin. Toassay this, we performed a PACT pull-down expe-riment with pure recombinant hexahistidine-taggedPKR bound to Ni-agarose. SK-N-SH cells weretreated with tunicamycin and cell extracts preparedat different time points were used to test for bindingof the endogenous PACT from cells to recombinantPKR protein immobilized in Ni-agarose beads. Asshown in Fig. 3a, tunicamycin treatment resulted ina time-dependent increase in association of PACTwith PKR. In untreated control cells, very littlePACT associated with PKR. At 2 and 4 h aftertunicamycin treatment, PACT association with PKRincreased significantly and then declined at 12 h.These results establish that PACT associates withPKR in response to tunicamycin treatment, leadingto its activation. Since it is established that stress-induced PACT phosphorylation triggers associationof PACT with PKR,26,32 we next examined if PACTis phosphorylated in response to tunicamycin treat-ment. We performed an in vivo labeling experimentwith [32P]orthophosphate during tunicamycintreatment. To detect PACT phosphorylation effi-ciently, we used Flag-PACT overexpression in SK-N-SH cells. As represented in Fig. 3b, tunicamycintreatment showed significant PACT phosphoryla-tion compared with the untreated cells. PACTphosphorylation was also detected at a low basal

level in untreated cells, and this may be due to theconstitutive phosphorylation of PACT at serine 246in the absence of cellular stress.32 In addition tophosphorylation of PACT, we also observed thatthe amount of phosphorylated PKR that co-immunoprecipitated with Flag-PACT alsoincreased significantly after tunicamycin treatment(32P-labeled PKR panel, Fig. 3b). These resultsclearly demonstrate that PACT is phosphorylatedin response to tunicamycin, leading to its in-creased association with PKR and causing PKRactivation.

Fig. 4. PKR is not activated in PACT null MEFs. ThePACT+/+ (a) and PACT−/− (b) MEFs were treated with1 μg/ml tunicamycin and cell extracts were prepared atindicated times after the treatments. Phosphorylation ofPKR was examined by Western blot analysis with a phos-phothreonine 451-specific antibody. The same blot wasstripped and reprobed with an anti-PKR monoclonal anti-body to detect total PKR. The ratio of phosphorylated PKRto total PKR is represented below each lane.


PACT null cells are defective in PKR activationin response to tunicamycin

To further evaluate if PACT is essential for PKRactivation in response to tunicamycin, we comparedthe PKR activation in response to tunicamycin in

Fig. 5. PACT null MEFs are resistant to tunicamycin-inddistribution after tunicamycin treatment. PACT+/+ and PACTwere harvested at 24, 48, and 72 h after the treatment and subjeG1 cell population represents the apoptotic cells. The sub-experiments were repeated twice, each time with duplicateexperiment are shown. (b) DNA fragmentation analysis in restreated with increasing concentrations of tunicamycin. FortyDNAwas analyzed. The cell type is indicated on the top of thedifferent concentrations of tunicamycin. Lane 2, 0.01 μg/ml; llane 6, 0.25 μg/ml; lane 7, 0.5 μg/ml; and lane 8, 1 μg/ml. M

PACT+/+ and PACT−/− MEFs. As seen in Fig. 4a,PKR activation was seen after 2 h of tunicamycintreatment in PACT+/+ MEFs similar to PKR+/+ MEFsand SK-N-SH cells (Fig. 2a and b). In contrast to this,the threonine 451 phosphospecific PKR antibody didnot detect any autophosphorylated PKR after tuni-camycin treatment in PACT−/− MEFs (Fig. 4b). Thus,PACT is essential for PKR activation in response totunicamycin treatment. These results establish thatPACT mediates PKR activation in response to tuni-camycin and that PKR activation is defective in theabsence of PACT.

PACT null cells are defective intunicamycin-induced apoptosis

Since PACTacts as the activator of PKR in responseto tunicamycin, we next compared tunicamycin-induced apoptosis in PACT wt and null cells usingthe flow cytometry technique to measure the per-centage of cells with hypodiploid DNA contentcharacteristic of apoptotic cells. In the wt MEFs,0.1 μg/ml tunicamycin induced apoptosis in 18.9%,36.2%, and 39.5% cells at 24, 48, and 72 h, respec-tively (Fig. 5a). In contrast to this, the PACT nullMEFs showed no apoptosis above the control at anyof these time points. Thus, the PACT null cells are

uced apoptosis. (a) Flow cytometry analysis of cell-cycle−/− MEFs were treated with 0.1 μg/ml tunicamycin. Cellscted to flow cytometry analysis as described. The sub-G0/G0/G1 percentages are displayed in each panel. Thesesamples. The most representative profiles from a singleponse to tunicamycin. PACT+/+ and PACT−/− MEFs were-eight hours after tunicamycin treatment, the fragmentedlanes. Lane 1, untreated cells; lanes 2–8, cells treated withane 3, 0.025 μg/ml; lane 4, 0.05 μg/ml; lane 5, 0.1 μg/ml;, 100-bp ladder DNA size markers.

Fig. 6. Reconstitution of PKR and PACT expressionrestores the apoptotic response in null MEFs. (a) Reconsti-tution of PKR expression. As indicated, PKR−/− or PKR+/+

MEFs grown on coverslips were transfected with 500 ng ofpCDNA3.1−+100 ng of pEGFPC1 (white bars) or 500 ng ofFlag-PKR/pCDNA3.1−+100 ng of pEGFPC1 (black bars)using Effectene. The cells were observed for EGFP fluores-cence 24 h after transfection and were treated with0.1 μg/ml tunicamycin. Themorphology of cells wasmoni-tored every 12 h. At 72 h after treatment, the cells were fixedandmounted in VectashieldwithDAPI nuclear stain.Whitebars represent empty vector transfected cells and black barsrepresent cells with PKR expression construct transfection.At least 300 fluorescent (EGFP-positive) cells were countedas live or dead based on their morphology and nuclearcondensation was indicated by intense DAPI fluorescence.Percent apoptosis=(fluorescent dead cells with intenseDAPI fluorescence/total fluorescent cells)×100. (b) Recon-stitution of PACT expression. As indicated, PACT−/− orPACT+/+ MEFs grown on coverslips were transfected with500 ng of pCDNA3.1−+100 ng of pEGFPC1 (white bars) or500 ng of Flag-PACT/pCDNA3.1− (encoding full-lengthPACT, amino acids 1–313)+100 ng of pEGFPC1 (black bars)using Effectene. The cells were subjected to similar treat-ment and analysis as in (a).


markedly resistant to tunicamycin-induced apopto-sis. In order to confirm that the hypodiploid peakrepresented the apoptotic population, we examinedthe apoptosis by DNA fragmentation analysis. Theprogression of apoptosis was monitored at 48 h aftertunicamycin treatments at different tunicamycinconcentrations. As seen in Fig. 5b, the wt MEFsshow a clear DNA fragmentation ladder character-istic only of apoptotic cells at tunicamycin concen-trations higher than 0.025 μg/ml (lanes 3–8) and aweak but detectable DNA ladder at 0.01 μg/ml(lane 2). In contrast to this, the PACT null cellsshowed a relatively weaker DNA fragmentationladder only at tunicamycin concentrations higherthan 0.1 μg/ml (lanes 5–8). At concentrations lowerthan 0.1 μg/ml, PACT null MEFs showed no DNAfragmentation. Thus, PACT null cells are markedlyresistant to tunicamycin concentrations up to0.1 μg/ml compared to wt MEFs, which show equi-valent DNA fragmentation even at 10-fold lower(0.01 μg/ml) tunicamycin concentrations (lane 2).These results establish that PACT is essential fortunicamycin-induced apoptosis.

Reconstitution of PKR and PACT expressionrescues the defective apoptosis in thecorresponding null cells

If the defective apoptotic response of PKR andPACT null cells to tunicamycin is specifically dueto lack of these proteins, a reconstitution of PKR orPACT expression is expected to rescue the pheno-type. We tested this by transfecting the PKR andPACT expression constructs in PKR and PACT nullMEFs, respectively. We co-transfected PKR nulland PACT null MEFs with Flag epitope-tagged wtPKR or PACT expression constructs along withpEGFPC1 (Clontech) that encodes enhanced greenfluorescent protein (EGFP). As a negative control,we used empty vector pcDNA3.1− co-transfectedwith pEGFPC1. Twenty-four hours after transfec-tion, the cells were treated with tunicamycin, and72 h after the treatment, the GFP-positive cellswere assessed for hallmark signs of apoptosis suchas cell shrinkage, membrane blebbing, and nuclearcondensation. Reconstitution of PKR expression inPKR null MEFs caused pronounced chromatincondensation seen as intense DAPI (4,6-diami-dino-2-phenylindole) fluorescence after tunicamy-cin treatment. In contrast, transfection of emptyvector showed no nuclear condensation aftertunicamycin treatment. To quantify this effect, wecounted the percentage of cells showing nuclearcondensation within the GFP-positive transfectedpopulation. It was seen that 33.5% of Flag-PKR-transfected PKR null cells showed nuclear con-densation after tunicamycin treatment (Fig. 6a,black bars). In contrast, this percentage was only6.6% in the pCDNA3.1– transfected cells (Fig. 6a,white bars). Thus, there was about a 5-fold increasein apoptosis in response to tunicamycin when PKRexpression was reconstituted in PKR null MEFs.The wt MEFs showed about 38% apoptosis in

response to tunicamycin with or without PKRreconstitution as expected. Similar results wereobtained with PACT null MEFs as seen in Fig. 6b.

Fig. 8. Tunicamycin dose response. PKR+/+, PACT+/+,PKR−/−, and PACT−/− MEFs were treated with 0.05, 0.1,0.25, 0.5, and 1.0 μg/ml tunicamycin as indicated on top of


It was seen that 35.3% of Flag-PACT-transfectedcells showed nuclear condensation after tunicamy-cin treatment (Fig. 6b, black bars). In contrast, thispercentage was only 9.1% in the pCDNA3.1– trans-fected cells (Fig. 6b, white bars). Thus, there was a3.8-fold increase in apoptosis in response to tuni-camycin when PACT expression was reconstitutedin PACT nullMEFs. ThewtMEFs showed about 30%apoptosis in response to tunicamycin with or with-out PACT expression construct. Western blot analy-sis confirmed the expression of the Flag-tagged PKRand PACT proteins (data not shown). These resultsestablish that the defective apoptosis observed inPKR and PACT null cells indeed is solely due to theabsence of PKR or PACT, respectively.

the lanes. The cell extracts were prepared 2 h after thetreatments and analyzed by Western blot analysis. Thesame blot was stripped and reprobed with both theantibodies. The numbers at the bottom of the panels repre-sent the ratio of phosphorylated eIF2α to total eIF2α,calculated using the Imagequant software and Stormphosphorimager. Since the results obtained from PKR+/+

and PACT +/+ MEFS were identical, only the PKR+/+ MEFresults are represented as wt panel.

Both PACT and PKR null cells are defective inCHOP induction in response to tunicamycin

To characterize the impaired apoptotic response ofPACT and PKR null MEFs, we examined the eIF2αphosphorylation and CHOP induction by Westernblot analysis. Since the difference in apoptosis wasmost pronounced at 0.1 μg/ml tunicamycin concen-tration (Figs. 1 and 5), we performed the treatmentsfor the Western blot analysis at the same concentra-tion. As shown in Fig. 7a and b, the PACT+/+ andPKR+/+ MEFs show rapid phosphorylation of eIF2αand CHOP induction in response to tunicamycin. Incontrast, eIF2α phosphorylation as well as CHOPinduction was absent in PACT−/− MEFS after tunica-mycin treatment. These results further demonstratethat the defective apoptosis observed in PACT nullcells is due to a lack of eIF2α phosphorylation andconsequent CHOP induction in these cells. The lackof CHOP induction and the eIF2α phosphorylationresponse of PKR−/− MEFs was similar to that ofPACT−/− MEFs. As seen in Fig. 7b, PKR−/− MEFsdid not show any significant eIF2α phosphorylation

phorimager. The analysis was repeated five times and best reafter tunicamycin treatment compared to the control samplestatistical analysis with the use of Student's t test. The observesignificant with all probability values being less than 0.05. (b)MEFs. PKR+/+ and PKR−/− MEFs were treated with 0.1 μg/mlwere analyzed as described in (a).

response, and since eIF2α phosphorylation leads toCHOP induction, the expression of CHOP wasbarely detectable. These results indicate that PACT-induced PKR activation plays a central role in tuni-camycin-induced apoptosis by regulating eIF2αphosphorylation and CHOP induction.To further investigate the response of PKR and

PACT null cells to tunicamycin treatment, westudied the eIF2α phosphorylation in response tovarying doses of tunicamycin. As represented inFig. 8, the wt MEFs showed a strong eIF2α phos-phorylation response at all concentrations of tuni-camycin that were examined. In contrast to this,PKR−/− as well as PACT−/− MEFs showed good

Fig. 7. Functional requirementof PACT and PKR for eIF2α phos-phorylation and CHOP expressionin response to tunicamycin. (a)eIF2α phosphorylation and CHOPinduction in PACT null MEFs.PACT+/+ and PACT−/− MEFs weretreated with 0.1 μg/ml tunicamy-cin. The cell extracts were preparedat indicated times and analyzed byWestern blot analysis. The same blotwas stripped and reprobed with allantibodies. The numbers at the bot-tom of the panels represent the ratioof phosphorylated eIF2α to totaleIF2α, calculated using the Image-quant software and Storm phos-

presentative blots are shown. The fold increases in ratioss were calculated at each time point and subjected to ad differences between +/+ and −/− MEFS are consideredeIF2α phosphorylation and CHOP induction in PKR nulltunicamycin. The cell extracts prepared at indicated times


eIF2α phosphorylation only at tunicamycin concen-trations of 0.25 μg/ml and above. At concentrationsof tunicamycin 0.1 μg/ml and below, there was noobserved eIF2α phosphorylation response in boththese cell types. These results also explain the lackof apoptotic response of these two cell types, asshown in Figs. 1 and 5 at lower concentrations oftunicamycin. The apoptotic defect observed inPKR−/− and PACT−/− cells was also most pro-nounced below the tunicamycin concentrations of0.1 μg/ml. Thus, PACT and PKR are essential foreIF2α phosphorylation and apoptosis in response tolower concentrations of tunicamycin. At lowerlevels of ER stress induced by tunicamycin, PACT-mediated PKR activation seems to be the primarymechanism utilized by the cells to bring abouteIF2α phosphorylation. At higher concentrations oftunicamycin, an additional, alternate cellular path-way may bring about the eIF2α phosphorylationand consequent apoptosis.

Discussion

The ER is the primary site of synthesis, posttrans-lational modifications, folding, and oligomerizationof secreted, membrane-bound, and some organelle-resident proteins.43 The ER also functions as anefficient quality-control system and prevents incom-pletely folded molecules from moving along secre-tory pathways. Some forms of cellular stress (knowncollectively as ER stress), such as depletion of cal-cium ions from the ER lumen, inhibition of proteinglycosylation, reduction of disulfide bonds, expres-sion of mutant proteins, and ischemic insults, lead tothe accumulation and aggregation of unfolded pro-teins in the ER.44,45 Since protein aggregation andmisfolding is toxic to cells, several pathophysiolo-gical conditions are associated with ER stress,including diabetes, ischemia, and neurodegenera-tive diseases.46,47 The attenuation of general proteinsynthesis to cope with ER stress is mainly achievedby phosphorylation of the initiation factor eIF2α onserine 51.37 PKR-like ER resident kinase (PERK) hasbeen shown to be activated in response to misfoldedproteins and to phosphorylate eIF2α.43,48,49 Phos-phorylation of serine 51 of eIF2α by PERK in res-ponse to ER stress is a protective response since itattenuates the new protein synthesis and the cellsgain time to manage correct folding or degrade theaccumulated misfolded proteins. In accordance withthis, homozygous mutations in PERK cause pan-creatic beta-cell death and infancy-onset diabetes inhuman Wollcot–Rallison syndrome and in geneti-cally engineered mice.50–54 Thus, lack of PERKkinase activity and lack of eIF2α phosphorylationmakes the cells more vulnerable to apoptosis inresponse to ER stress.55–58 In contrast to this, PKRactivation by ER stressors such as thapsigargin(which causes Ca2+ release from the ER) leads toactivation of apoptotic pathways.59–62 Although theprimary result of PKR activation by ER stress is alsophosphorylation of eIF2α on serine 51, the outcome

of this event is opposite of PERK activation andleads to cell death by apoptosis. In accordance withthis, inhibition of PKR activation in response to ERstress leads to a reduction in apoptosis.60,63–66In this study, we investigated the mechanism of

PKR activation in response to ER stressor tunicamy-cin. Our results demonstrate that PACT mediatesPKR activation in response to tunicamycin. Usingmetabolic labeling with [32P]orthophosphate, weestablished that PACT is phosphorylated aftertunicamycin treatment and phosphorylated PACTassociates with PKR with higher affinity, therebyleading to its activation. Consequently, PACT−/−

cells show no PKR activation in response to tuni-camycin. Furthermore, MEFs isolated from PKR−/−

and PACT−/− mice are resistant to tunicamycin-induced apoptosis. This defect in null cells specifi-cally results from lack of PKR and PACT activity,since a reconstitution of PKR and PACTexpression inthe null cells rescues the defect in apoptosis. BotheIF2α phosphorylation and the consequent CHOPinduction was absent in PACT−/− MEFs in responseto tunicamycin. Furthermore, the results of tunica-mycin dose response (Fig. 8) clearly indicated thatPACT-induced PKR activation seems to be theprimary mediator of eIF2α phosphorylation at tuni-camycin concentrations of 0.1 μg/ml and lower. Ourresults indicate that the MEFs use two differentmechanisms to phosphorylate eIF2α depending onthe level of stress they are experiencing. PERKactivation, which is known to occur in response toER stress, may require higher levels of tunicamycin(ER stress). On the contrary, PACT-induced PKRactivation occurs at lower concentrations of tunica-mycin. A cross talk between the PERK and PKRpathways has been noted before for eIF2α phospho-rylation in response to viral infections.67 Thus, ourresults for the first time establish PACT phosphory-lation in response to tunicamycin as the mechanismof PKR activation during ER stress. In addition, ourresults also establish that PACT-dependent PKRactivation is essential for induction of apoptosis inresponse to tunicamycin at lower concentrations.However, it is also worth noting that the PACT andPKR null MEFs showed consistently lower amountof apoptosis even at higher tunicamycin concentra-tions (Figs. 2 and 5).Recently, PKR was identified as one of the media-

tors of tunicamycin-induced cell death in neuronalcells.63 Interestingly, phosphorylated PKR is presentin brain tissues in patients with Alzheimer's disease,Parkinson's disease, Huntington's disease, andamyotrophic lateral sclerosis.68–71 Thus, PKR maynot only be involved in apoptosis induced by viralinfections, but may also be involved in inducingapoptosis in response to ER stress. Inhibition of PKRactivity by overexpression of its trans-dominantmutant or by using a chemical inhibitor confersresistance to apoptosis in response to a variety ofstress signals including ER stressors.59,63,72,73 How-ever, the exact mechanism of PKR activation in thispathway had remained elusive thus far. Our resultsnow establish PACT as the activator of PKR in


response to ER stressor tunicamycin. Using a smallinterfering RNA-mediated downregulation of PKRactivity in HeLa and HEK293 cells, it was reportedrecently that PKR plays a significant role in ERstress-mediated apoptosis.73 In the same report, itwas shown that thapsigargin treatment of HEK293cells increased the PACT mRNA and protein levels,which are essential for PKR activation. We did notobserve any induction of PACT protein levels inresponse to tunicamycin in MEFs or in SK-N-SHcells. Although the reasons for these differences areunknown at the present, it is possible that they mayresult from the different cell types or from differentinducers of ER stress. On the other hand, phospho-rylation of PACT in response to cellular stressresulting in its increased affinity for PKR and conse-quent PKR activation has been established.26,29,32 Itis known that PACT is phosphorylated on serine 287in response to stress signals and that constitutivephosphorylation of serine 246 is a prerequisite forthe stress-induced phosphorylation.32 Both theseresidues reside in the PKR activation domain ofPACT, which has been mapped to its third carboxy-terminal copy of dsRBM.27,28 Considering thatphosphorylation of PACT in response to tunicamy-cin resulted in an increased association with PKR, itis possible that the phosphorylation observed inresponse to ER stress occurs at these sites.There are four eIF2α kinases in mammalian cells,

heme-regulated inhibitor, mammalian homologueof the Saccharomyces cerevisiae protein kinase generalcontrol nonderepressible-2 (GCN2), PERK, andPKR.74–77 Functional characterization of these kina-ses has indicated that each is involved in phospho-rylation of eIF2α in response to a distinct stimulus.Heme-regulated inhibitor is known to regulateeIF2α phosphorylation and protein synthesis in res-ponse to heme availability in erythroid cells. GCN2is involved in regulating translation in response toamino acid deprivation in mammalian cells. Ingeneral, it is thought that the ER stress-activatedkinase PERK phosphorylates the eIF2α in responseto ER stressors. Our results establish that PKR alsoplays a major functional role in tunicamycin-induced eIF2α phosphorylation and induction ofapoptosis. We also observed that the apoptotic res-ponse of PKR and PACT null cells was identical tothat of their wt counterparts in response to thap-sigargin and DTT, which induce ER stress by Ca2+mobilization and by inhibition of disulfide bondformation (data not shown). In this regard, it wasreported recently that PACT null MEFS were notresistant to thapsigargin-induced apoptosis.78 Thus,it is possible that, although seemingly similar, thedifferent inducers of ER stress may utilize separatekinases as effectors to bring about an inhibition ofprotein synthesis by phosphorylation of eIF2α. Theexistence of several related eIF2α-specific kinaseslikely provides the cells some degree of redundancyof function. Our work presented here neverthelessestablishes that PACT-induced PKR activation playsan important functional role in tunicamycin-induced apoptosis.

Materials and Methods

Reagents, cells, and antibodies

Tunicamycin was obtained from Sigma. PACT+/+ andisogenic PACT−/− MEFs were a generous gift from Dr.Ganes Sen (Lerner Research Institute, Cleveland ClinicFoundation) and PKR+/+ and isogenic PKR−/− MEFs werea kind gift from Dr. Bryan Williams (Monash Institute ofMedical Research, Australia). MEFs, SK-N-SH, andHT1080 cells were cultured in Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal bovine serum andpenicillin/streptomycin. The following antibodies wereused: anti-Flag monoclonal M2 (Sigma), anti-PKR(human) monoclonal (71/10, R & D systems), anti-PKR(murine) monoclonal (Transduction Laboratories), anti-phospho-PKR (Thr451) (Cell Signaling), anti-phospho-eIF2α (Ser51) (Invitrogen), anti-eIF2α, and anti-his tag(Santa Cruz), anti-CHOP (Santa Cruz). PACT polyclonalantibody has been described.26

Flow cytometry analysis to monitor apoptosis

MEFs were treated with 0.1 μg/ml of tunicamycin. Thecells were permeabilized 24, 48, and 72 h after thetreatment with Vindelov's solution,79 stained with propi-dium iodide, and analyzed by flow cytometry using aCoulter cell sorter.

DNA fragmentation analysis

The MEFs were treated with different concentrations oftunicamycin for 48 h. Low molecular weight fragmentedDNAwas prepared and analyzed as described.29

Western blot analysis

Western blot analysis of the total proteins from cellextracts or of the immunoprecipitates was performed asdescribed before.25 For the analysis of eIF2α phosphoryla-tion and CHOP induction from MEFs, 10 μg of the totalprotein was analyzed for eIF2α phosphorylation and 50 μgof the total protein was analyzed for CHOP induction.

PKR activity assay

The SK-N-SH cells were treated with 1 μg/ml tunica-mycin for indicated time intervals and PKR activity assayswere performed as described before25 without addition ofany exogenous activator using anti-PKR monoclonalantibody (R & D Systems).

PACT–PKR interactions

SK-N-SH cells grown to 70% confluency in 100-mmplates were treated with 1 μg/ml of tunicamycin forindicated times. The cell extracts were prepared in 100 μllow-salt buffer (20 mM Tris–HCl, pH 7.5, 100 mMKCl, 100U/ml aprotinin, 0.2 mM PMSF, 20% glycerol, 0.1% TritonX-100, and phosphatase inhibitor cocktail, Sigma). Cellextract (500 μg) was bound to 1 μg of recombinant, hexa-histidine-tagged PKR protein immobilized on Ni-agaroseresin in 100 μl of low-salt buffer at 4 °C for 1 h. The beadswere washed in 500 μl of low-salt buffer four times and


PACT bound to beads was analyzed by SDS-PAGEfollowed by Western blot analysis using anti-PACT poly-clonal antibody. Aliquots of whole-cell lysate were exa-mined without immunoprecipitation by Western blotanalyses with anti-PACT antibody. To ascertain thatequal amounts of pure PKR protein was analyzed in theassays, the same blots were stripped and reprobed withhexahistidine tag-specific antibody.

In vivo phosphate labeling

For analyzing phosphorylation of PACT, SK-N-SH cellswere transfected with Flag-PACT/pcDNA3.1− constructin 100-mm dishes. Twenty-four hours after transfection,the transfected cells were kept in phosphate-free DMEMcontaining 10% serum for 1 h before the tunicamycintreatment. The tunicamycin treatment was done in 3 mlmedium in the presence of 500 μCi/ml of [32P]orthophos-phoric acid (Perkin Elmer). Four hours after the tunica-mycin treatment, cell extracts were prepared in 100 μl oflow-salt buffer (20 mM Tris–HCl, pH 7.5, 100 mM KCl,100 U/ml aprotinin, 0.2 mM PMSF, 20% glycerol, 0.1%Triton X-100, and phosphatase inhibitor cocktail, Sigma).Flag-PACT was immunoprecipitated from 500 μg totalprotein extract using the Flag M2 antibody-agarose(Sigma) in low-salt buffer at 4 °C for 2 h. The beadswere washed in 500 μl low-salt buffer four times andPACT bound to beads was analyzed by SDS-PAGEfollowed by phosphorimager analysis. Another samplesimilarly immunoprecipitated with Flag M2 antibody-agarose was used for Western blot analysis with anti-PKRmonoclonal antibody (Transduction Laboratories).

Reconstitution of PKR and PACT expression in MEFsand apoptosis assays

The PKR−/− and PACT−/− cells grown on coverslipswere transfected with 600 ng total of the indicatedplasmids using the Effectene (Qiagen) reagent. The cellswere observed for GFP fluorescence 24 h after transfectionwith an inverted fluorescence microscope and weretreated with 0.1 μg/ml tunicamycin. The morphology ofcells was monitored at regular time intervals every 12 h.At 72 h after treatment, the cells were washed twice withphosphate-buffered saline and fixed in 1:1 acetone/methanol for 1 min, and the cover slips were mountedin Vectashield (Vector Laboratories) mounting mediumcontaining DAPI. At least 300 GFP-positive cells werecounted as live or dead based on their morphology. Thecells showing normal flat morphology were scored as liveand the cells showing cell shrinkage, membrane blebbing,rounded morphology, partial detachment from the plate,and nuclear condensation with intense DAPI fluorescencewere counted as dead cells. The percentage of apoptoticcells was calculated with the formula, % apoptosis=(fluorescent dead cells/total fluorescent cells)×100.

Acknowledgements

We are grateful to Ganes Sen and Bryan Williamsfor PACT null and PKR null MEFs, respectively. Thiswork was supported by an American Heart Asso-ciation grant-in-aid (0555503U to R.C.P.).

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essential role of pact-mediated pkr activation in tunicamycin-induced apoptosis

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