perk-ire1
Divergent Effects of PERK and IRE1Signaling on Cell Viability
Jonathan H.Lin1,2*,Han Li1,2,Yuhong Zhang3,David Ron3,Peter Walter1,2
1Howard Hughes Medical Institute,University of California San Francisco,San Francisco,California,United States of America,2Department of Biochemistry and Biophysics,University of California San Francisco,San Francisco,California,United States of America,3Departments of Cell Biology and Medicine,Kimmel Center for Biology and Medicine,Skirball Institute,New York University School of Medicine,New York,New York,United States of America
Abstract
Protein misfolding in the endoplasmic reticulum(ER)activates a set of intracellular signaling pathways,collectively termed the Unfolded Protein Response(UPR).UPR signaling promotes cell survival by reducing misfolded protein levels.If homeostasis cannot be restored,UPR signaling promotes cell death.The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood.The ER-resident proteins,PERK and IRE1,control two key UPR signaling pathways.Protein misfolding concomitantly activates PERK and IRE1and has clouded insight into their contributions toward life or death cell fates.Here,we employed chemical-genetic strategies to activate individually PERK or IRE1uncoupled from protein misfolding.We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis.By contrast,equivalent durations of IRE1signaling enhanced cell proliferation without promoting cell death.
These results demonstrate that extended PERK and IRE1signaling have opposite effects on cell viability.Differential activation of PERK and IRE1may determine life or death decisions after ER protein misfolding.
Citation:Lin JH,Li H,Zhang Y,Ron D,Walter P(2009)Divergent Effects of PERK and IRE1Signaling on Cell Viability.PLoS ONE4(1):e4170.doi:10.1371/ journal.pone.0004170
Editor:Andreas Bergmann,UT MD Anderson Cancer Center,United States of America
Received October16,2008;Accepted December6,2008;Published January12,2009
Copyright:?2009Lin et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.
Funding:This work was supported by NIH grants DK047119,ES08681,EY018313,and GM032384,and the Cystic Fibrosis Foundation.Peter Walter is an Investigator of the Howard Hughes Medical Institute.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist.
*E-mail:JLin@https://www.wendangku.net/doc/4317126462.html,
Introduction
Physiologic or pathologic processes that disturb protein folding in the endoplasmic reticulum(ER)activate a set of signaling pathways termed the Unfolded Protein Response(UPR).The molecular gatekeepers of the UPR are ER-resident transmembrane proteins that monitor the quality of protein folding in the ER and relay that information to the rest of the cell.In mammalian cells,PERK and IRE1independently govern two key UPR signal transduction pathways[1].PERK is a transmembrane kinase that phosphorylates translation initiation factor eIF2a,thereby reducing cellular protein synthesis and with it the load of proteins entering into the ER[2]. eIF2a phosphorylation also allows the translation of select mRNAs that contain small open reading frames in their59untranslated regions,leading to the production of transcription activators,such as ATF4and ATF5[3,4].IRE1is a bifunctional transmembrane kinase/endoribonuclease that induces the non-conventional splicing of Xbp1mRNA to produce another b-ZIP transcription activator, XBP1[5].In addition to splicing Xbp1mRNA,IRE1’s kinase can also activate the c-Jun N-terminal kinase(JNK)signaling pathway through the MAP3K cascade[6,7].The transcription factors produced by PERK,IRE1,and other UPR signaling pathways collaborate to control behavior,metabolism,and ultimately cell fate in response to ER stress by inducing a wide array of targets that include protein folding chaperones such as ERdj4[8]and additional transcriptional activators such as Chop[3].
Genetic and pharmacological experiments have demonstrated that PERK signaling can confer both protective and proapoptotic effects in the face of ER stress.For instance,genetic deletion of Perk or impairment of eIF2a activity impaired cell survival[9,10]. Conversely,transient artificial PERK activation or pharmacolog-ical eIF2a activation enhanced cell survival in response to ER protein misfolding[11,12].Deletion of downstream components of PERK signaling,Atf4and Chop,impaired or enhanced cell survival in response to protein misfolding depending on the cell type studied[3,13,14,15,16,17].
Like PERK,IRE1signaling has also been implicated in enhancing or impairing cell survival.Artificial extension of IRE1’s RNAse function enhanced cell survival in the face of ER stress [18,19].RNAi knockdown of Xbp1,IRE1’s RNAse target, impaired cell survival after protein misfolding in vitro and was required for the survival of multiple secretory cell types in vivo [20,21].Genetic deletion of Ask1,the MAP3K proposed to link IRE1signaling to JNK,conferred resistance to ER stress-induced cell death[7,22].JNK can prevent or promote cell death depending on the specific stimuli,intensity,and/or duration of activation[23,24,25].
These findings demonstrate that PERK and IRE1signaling can regulate cell survival after protein misfolding.How do cells modulate PERK and IRE1activities to arrive at either cell fate? Previous studies demonstrated that the duration of PERK and IRE1signaling varied markedly after the imposition of protein misfolding[18].In particular,chronic ER stress led to IRE1 branch inactivation while PERK signaling was unaffected.These observations suggested that the progression toward cell death from unmitigated protein misfolding involved attenuation of IRE1
signaling coupled with persistent PERK activity.Previously,we employed chemical-genetic tools to artificially activate IRE1and demonstrated a cytoprotective effect for its RNAse function in isogenic human cells[18].Here,we employed a similar strategy to selectively activate PERK.We observed that sustained PERK signaling was detrimental to cell viability whereas the equivalent duration of IRE1signaling was not,suggesting that extended PERK activity contributes to the cell death that occurs with chronic ER stress.
Results
Chemical-Genetic Control of PERK and IRE1Signaling in Human Cells
We previously used recombinase-directed site-specific DNA integration to introduce alleles into the genome of human embryonic kidney293(HEK293)cells[18].This technique minimized perturbation of the native UPR as well as differences arising from position insertion variegation effects.We extended this strategy to create additional isogenic cell lines stably expressing an artificial PERK allele,Fv2E-Perk,which had previously been demonstrated to activate wild-type PERK signaling in hippocampal neurons and CHO cells upon addition of the dimerizing molecule,AP20187[11,26].We observed stable Fv2E-Perk mRNA and protein expression at all times examined in our cells(Fig.1A and Fig.S1).To determine how effectively we could recapitulate PERK branch signaling in HEK293cells expressing Fv2E-Perk,we monitored multiple specific parameters of PERK activity after addition of the dimerizing agent,AP20187. After application of drug,we observed production of phosphor-ylated eIF2a and a downstream translational target ATF4that approached levels seen with exposure to thapsigargin,an ER toxin that strongly induces all branches of the UPR(Fig.1A and Fig. S1).Consistent with activation of these proximal parameters of PERK branch activity,we also observed increased mRNA levels of downstream PERK signaling transcriptional targets,Chop and Gadd34,after AP20187application(Fig.1A and Fig.S1).The GADD34phosphatase has been demonstrated to target phos-phorylated eIF2a and thereby deactivate PERK branch signaling [27].Interestingly,we observed no diminution in phosphorylated eIF2a levels in the presence of AP20187,even though Gadd34was induced,suggesting that drug-activated Fv2E-PERK overcame the negative feedback effects of GADD34on eIF2a(Fig.1A).Lastly, to determine if AP20187’s effects were confined to PERK or had non-specifically triggered ER stress,we examined a specific marker of IRE1activation,splicing of Xbp1mRNA.Cells expressing Fv2E-Perk spliced Xbp-1mRNA in response thapsi-
gargin,but no Xbp1mRNA splicing was observed at all concentrations and durations of AP20187exposure that activated Fv2E-PERK(Fig.1B and Fig.S1).Hence,these cells provide a system to examine the effects of selective PERK branch signaling. To study the effects of selective IRE1branch activity on cell viability,we used transgenic HEK293cells expressing an artificial Ire1[I642G]allele which we had previously shown could be regulated by addition of the ATP analogue,1NM-PP1[18,19].As a control for the specificity of IRE1[I642G]’s effects,we created additional cells expressing an allele of IRE1,Ir e1[I642G/K599A], that bore a second missense mutation at amino acid position599, which converted an essential lysine residue to alanine in the catalytic kinase domain of IRE1[28].We observed stable expression of IRE1[I642G]or IRE1[I642G/K599A]protein at all times examined in transgenic HEK293cells(Fig.2A). However,1NM-PP1application triggered Xbp1mRNA splicing and ERdj4mRNA induction,two parameters of IRE1branch signaling,only in cells expressing IRE1[I642G],indicating that the additional K599A point mutation in IRE1[I642G/K599A] abolished its activity(Fig.2A).To ascertain that1NM-PP1’s effects were confined to activation of IRE1[I642G],we examined a marker of PERK branch signaling,production of ATF4protein. Parental and transgenic cells produced ATF4in response to thapsigargin treatment,but no ATF4protein was observed at all durations of1NM-PP1treatment that activated IRE1signaling (Fig.2B).Hence,these cells provided a system to examine the effects of selective IRE1branch signaling on cell viability. Divergent Effects of Extended PERK and IRE1Signaling on Cell Proliferation and Apoptosis
We used these isogenic cell lines bearing Fv2E-Perk,Ire1[I642G], or Ire1[I642G/K599A]to address how selective IRE1or PERK signaling affected cell viability with respect to proliferation and Figure1.Selective and specific activation of PERK signaling.(A) Parental wild-type and transgenic HEK293cells expressing the AP20187-sensitized Fv2E-Perk allele were treated for the indicated times with AP20187(2nM).Fv2E-Perk,Gadd34,and Chop mRNA levels were measured by quantitative PCR,normalized to levels of a housekeeping gene,Rpl19,and are shown relative to levels in untreated cells.Fv2E-PERK,phospho-eIF2a,and ATF4proteins were detected by immuno-blotting.Total eIF2a protein was measured as a loading control.(B) Parental wild-type and transgenic HEK293cells expressing the AP20187-sensitized Fv2E-Perk allele were treated for the indicated times with AP20187(2nM).Xbp1mRNA splicing was assesed by RT-PCR.The unspliced(u)and spliced(s)Xbp1mRNA products are indicated as labeled.The asterisk indicates the position of a hybrid amplicon. doi:10.1371/journal.pone.0004170.g001
apoptosis.Chronic protein misfolding induced by multi-day exposure to tunicamycin or thapsigargin severely impaired cell proliferation and triggered apoptosis in wild-type cells (Video S1,Fig.3A,Fig.4).When we selectively activated PERK signaling in cells bearing Fv2E-Perk by application of AP20187for up to 48hours,we observed a pronounced reduction in cell numbers compared to mock treated or parental cells exposed to AP20187(Video S2,Fig.3A,Fig.3B).By contrast,when we selectively activated IRE1signaling in transgenic cells bearing Ire1[I642G]by application of 1NM-PP1,we observed increased cell numbers compared to mock treated or parental cells exposed to the drug (Video S3,Fig.3A,Fig.3C).The advantage in proliferation specifically required functional IRE1branch activity,since 1NM-PP1exposure did not enhance survival in cells bearing the doubly-mutated Ire1[I642G/K599A]allele (Fig.3A,Fig.3B).In sum,these studies clearly demonstrated that sustained PERK signaling impairs cell proliferation while IRE1signaling promotes cell growth.
We also observed striking morphologic changes in cells,in which PERK signaling was selectively activated,including retraction of cellular extensions,loss of refractiveness under phase-contrast microscopy,and detachment from the underlying
matrix (Video S2,Fig.3A).These physical changes resembled those seen in cells undergoing cell death after exposure to lethal concentrations of ER stress-inducing agents,such as tunicamycin or thapsigargin (Video S1,Fig.3A).By contrast,none of these morphologic changes were seen in cells in which IRE1branch signaling had been selectively activated (Video S3,Fig.3A).These morphologic changes suggested that sustained PERK activity triggered apoptosis in addition to impairing cell proliferation.To determine if molecular markers of apoptosis occurred in these cells,we next examined cleavage of poly(ADP-ribose)polymerase (PARP),a nuclear DNA repair enzyme that undergoes proteolysis in response to many apoptotic stimuli [29].Robust production of cleaved PARP was observed after 48to 72hours of exposure to tunicamycin or thapsigargin (Fig.4and Fig.S2).Minimal PARP cleavage was seen in wild-type cells exposed to 1NM-PP1or AP20187,indicating that these small molecules did not trigger cell death at bio-efficacious concentrations (Fig.S2).When AP20187was applied to cells expressing Fv2E-PERK,we saw strong production of cleaved PARP (Fig.4).By contrast,when 1NM-PP1was added to cells expressing IRE1[I642G],PARP was not cleaved (Fig.4).Taken together with the cytomorphologic changes,these findings indicate that sustained PERK signaling triggers apoptosis,whereas IRE1signaling does not when activated for equivalent duration.Consistent with the incompat-ibility of extended PERK signaling with viability,loss of the Fv2E-PERK transgene was observed in all cells that were able to proliferate in the presence of AP20187(Fig.S3).
Discussion
The UPR detects and responds to ER protein misfolding acutely by enhancing the protein folding capacity of the ER,but,if protein misfolding persists,the UPR promotes cell death.The molecular basis for this switch between protective and proapopto-tic UPR function is poorly understood.Prior studies from our group had delineated distinct molecular phases of UPR signaling in which acute ER stress activated both PERK and IRE1,but persistent chronic ER stress activated only the PERK pathway and attenuated IRE1signaling [18].These observations led to the hypothesis that the switch in IRE1signaling coupled with unabated PERK activity contributed to the transition from protective to proapoptotic UPR function.To examine this model,we used chemical-genetic approaches to activate PERK or IRE1in isolation in isogenic ‘‘sister’’human cell lines and observed that chronic PERK signaling promotes cell death.By contrast,IRE1activity enhances cell survival.Coupled with our previous studies,these findings provide compelling evidence that the time course of PERK and IRE1signaling plays a critical role in determining how the UPR selects between life and death cell fates.
Our current finding that chronic PERK activity impairs cell viability is consistent with our prior study showing that selective activation of PERK triggered cell death in other cell types [11]as well numerous reports demonstrating that the CHOP transcrip-tion factor,produced by PERK signaling,actively promotes apoptosis in vitro and in vivo [3,13,15,16].How can this proapoptotic capacity of PERK signaling be reconciled with its ability to enhance cell survival in the face of protein misfolding [9,10,11,12]?Our findings suggest that the duration and/or strength of PERK signaling may determine whether cytoprotective or proapoptotic outcomes predominate.In our model,transient PERK signaling protects cells by temporarily dampening cellular protein synthesis and thus reducing misfolded protein levels in the ER.Transient PERK signaling may also be insufficient to induce CHOP levels to proapoptotic threshholds,given Chop’s
inherent
Figure 2.Selective and specific activation of IRE1signaling.(A)Parental wild-type and transgenic HEK293cells expressing the Ire1[I642G],or Ire1[I642G/K599A]allele were treated for the indicated times with 1NM-PP1(1m M),and wild-type cells were treated for 4hours with thapsigargin (tg)(300nM).IRE1[I642G]and IRE1[I642G/K599A]protein was detected by immunoblotting for the FLAG epitope.GAPDH levels were assessed as a protein loading control.Xbp1mRNA splicing was determined by RT-PCR.The unspliced (u )and spliced (s )Xbp1mRNA products are indicated as labeled.ERdj4mRNA levels were measured by quantitative PCR,normalized to Rpl19mRNA levels,and are shown relative to levels in untreated cells.(B)Parental wild-type and transgenic HEK293cells expressing the Ire1[I642G]or Ire1[I642G/K599A]alleles were treated for the indicated times with 1NM-PP1(1m M);wild-type cells were also treated for 4hours with thapsigargin (tg)(300nM).ATF4protein was detected by immunoblotting.GAPDH levels were assessed as a protein loading control.doi:10.1371/journal.pone.0004170.g002
mRNA and protein instability [30].However,persistent PERK signaling could ultimately impair cell viability if extended translational inhibition interrupted the generation of proteins vital for cellular homeostasis.Persistent PERK signaling could also lead to the accumulation of sufficient CHOP to drive cell death.Intriguingly,in some cell types,CHOP directly induces the transcription of Bim ,a proapoptotic member of the BCL2protein family that directly elicits cell death by permeabilizing the mitochondrial outer membrane [31].A PERK-CHOP-BIM signaling axis could link chronic protein misfolding in the ER to
activation of the intrinsic apoptosis machinery in the mitochon-dria.Additional parallel proapoptotic signaling pathways must also exist given the continued sensitivity of Perk and Chop null cells to ER protein misfolding [9,13].
Can IRE1also transmit apoptotic signals from the ER?While we demonstrate a cytoprotective function for IRE1signaling through its RNAse activity,in mammalian cells,IRE1has acquired additional properties independent of splicing that include activation of the JNK signaling pathway and selective biochemical interactions with the BAK and BAX proteins of the BCL2family of apoptotic regulators [6,32].The JNK signaling pathway and BCL2proteins are key regulators of cell survival and apoptosis in response to numerous stimuli [23,33].Although the consequences of their interactions with the IRE1signaling pathway on cell survival are unknown,they raise the possibility that IRE1employs multiple downstream modules besides XBP1generation to regulate cell fate after activation by protein misfolding.IRE1’s oligomerization status has recently been shown to regulate its RNAse activity [34].Investigating the effect of IRE1polymerization status on JNK and BAX/BAK activity may shed additional insight into IRE1’s effects on cell survival.
Divergent effects of persistent PERK and IRE1signaling on cell proliferation and survival may also underlie the phenotypes observed in several pathologic and physiological situations in vivo.Mice on high-fat diets developed hepatocyte steatosis,accompa-nied by inflammation and PERK activation,suggesting a link between PERK signaling and cellular dysfunction [35,36].
By
Figure 3.Sustained Perk signaling impairs cell proliferation.(A)Parental wild-type and isogenic HEK293cells expressing Fv2E-Perk ,Ire1[I642G],or Ire1[I642G/K599A]alleles were treated with tunicamycin (5m g/ml),1NM-PP1(1m M),or AP20187(2nM),videographed for 48hours,and frames from indicated time points are shown.Magnification bar,125m m.(B)Parental wild-type and transgenic HEK293cells expressing the Fv2E-Perk allele were treated with AP20187(2nM),counted,and are shown relative to numbers of mock-treated cells at the indicated times.(C)Parental wild-type and transgenic HEK293cells expressing Ire1[I642G]or Ire1[I642G/K599A]alleles were treated with 1NM-PP1(1m M),counted,and are shown relative to numbers of mock-treated cells at the indicated times.
doi:10.1371/journal.pone.0004170.g003
Figure 4.Sustained Perk signaling promotes apoptosis.Parental wild-type and isogenic HEK293cells expressing Ire1[I642G]or Fv2E-Perk were treated with thapsigargin (300nM);1NM-PP1(1m M);or AP20187(2nM)for the indicated times.Cleaved PARP protein was assessed by immunoblot.GAPDH protein levels served as a loading control.doi:10.1371/journal.pone.0004170.g004
contrast,selective expression of spliced XBP1protein in B-cells dramatically enhanced cell numbers,leading to a multiple myeloma-like phenotype[37],consistent with the ability of IRE1’s RNAse function to promote cell proliferation and survival. Pharmacological modulation of PERK or IRE1signaling could provide new approaches to treat diseases associated with ER stress. Materials and Methods
Molecular Biology
Generation of the AP20187dimerizable Fv2E-Perk allele and 1NM-PP1sensitized Ire1[I642G]allele has been previously described (Lu et al.,2000;Lin et al.,2007).To construct the Ire1[I642G/ K599A]allele,QuikChange site-directed mutagenesis(Stratagene, San Diego,CA)was used to insert a lysine to alanine missense mutation in the Ire1[I642G]allele at amino acid position599.
RT-PCR analysis of Xbp1mRNA splicing was performed as previously described(Lin et al.,2007).Primers used for quantita-tive PCR analysis included:Fv2E-Perk mRNA,59-TGAGTGT-GGGTCAGAGAGCCAAAC-39and59-ACGGAGTCGTATT-TACTTTCAGTC-39;human Rpl19mRNA,59-ATGTATCA-CAGCCTGTACCTG–39and59-TTCTTGGTCTCTTCCTC-CTTG-39;human Chop mRNA,59-ACCAAGGGAGAACCAG-GAAACG-39and59-TCACCATTCGGTCAATCAGAGC-39; human Gadd34mRNA,59-CCTCTACTTCTGCCTTGTCTC-CAG-39and59-TTTTCCTCCTTCTCCTCGGACG-39;and human ERdj4mRNA,59-TGGTGGTTCCAGTAGACAA-AGG-39and59-CTTCGTTGAGTGACAGTCCTGC-39. Quantitative PCR was performed using a MJ Opticon2DNA Engine(Bio-Rad,Hercules,CA)as previously described(Lin et al., 2007).
Protein Analysis
The following antibodies and dilutions were used for Western analyses:anti-FKBP at1:1000(Affinity BioReagents,Golden,CO); anti-eIF2a at1:2000(Cell Signaling,Natick MA);anti-phospho-eIF2a at1:500(Cell Signaling,Natick,MA);anti-ATF4at1:2000 (Santa Cruz Biotechnologies,Santa Cruz,CA);anti-GAPDH at 1:10000000(AbCAM,Cambridge,MA);anti-FLAG at1:5000 (Sigma,St.Louis,MO);and anti-PARP at1:2000(Cell Signaling, Natick,MA).
Cell Culture
HEK293cell lines were maintained at37u C,5%CO2in DMEM media supplemented with fetal calf serum,glutamine,and antibiotics (Invitrogen,San Diego,CA).Tunicamycin and thapsigargin were obtained from Calbiochem EMD Bioscience Inc.(Darmstadt, Germany).AP20187was provided by Ariad Pharmaceuticals (Cambridge,MA)and used as directed.1NM-PP1was used as previously described[18].The Fv2E-Perk,Ire1[I642G],and Ire1[I642G/K599A]alleles were integrated into HEK293cells bearing frt sites as previously described(Lin et al.,2007).Multiple independent isogenic clones were analyzed with identical findings. The CHO cell line bearing Fv2E-Perk has been previously described[26].To obtain resistant cells,CHO cells bearing Fv2E-Perk were plated at clonal density and grown for10days in 100nM AP20187(and3m g/ml puromycin to enforce expression of the Fv2E-Perk retroviral transgene).Multiple resistant clones were identified under such conditions and individually expanded for Fv2E-PERK protein expression analysis.
Cell Microscopy,Image Acquisition,and Cell Counts Wild-type or isogenic HEK293cells bearing Fv2E-Perk,Ir-e1[I642G],or Ire1[I642G/K599A]alleles were plated at densities of 75000cells/ml and live-cell imaging was performed using an inverted microscope(Nikon TE2002E2)with a1060.3NA objective and a cooled charge-coupled device camera(Coolsnap HQ2,Photometrics)in a sealed humidified5%CO2,37C chamber.Images were acquired at5-minute intervals for48hours after application of tunicamycin(5m g/ml),1NM-PP1(1m M), AP20187(2nM),or dimethylformamide solvent using Nikon Imaging Systems Elements2.3software.Images were exported as TIFF files into ImageJ software to compile into video files and to capture frames for cell counts.Three to six independent imaging experiments were conducted for each condition,and representative videos are shown.
Supporting Information
Figure S1Titration of AP20187on HEK293cells expressing Fv2E-Perk.(A)Transgenic HEK293cells were treated for4hours with thapsigargin(300nM)or the indicated concentrations of AP20187.Fv2E-PERK and ATF4proteins were detected by immunoblotting.Chop mRNA levels were measured by quanti-tative PCR,normalized to Rpl19mRNA levels,and shown relative to levels in mock-treated cells.(B)Transgenic HEK293 cells were treated for4hours with thapsigargin(300nM)or the indicated concentrations of AP20187.Xbp1mRNA splicing was assesed by RT-PCR.The unspliced(u)and spliced(s)Xbp1 mRNA products are indicated as labeled.
Found at:doi:10.1371/journal.pone.0004170.s001(1.67MB EPS) Figure S2Effect of tunicamycin,1NM-PP1,or AP20187on PARP processing in HEK293cells.Parental wild-type HEK293 cells were treated for the indicated times with tunicamycin(tu)(5 I?J g/ml),1NM-PP1(1I?J M),or AP20187(2nM).Cleaved PARP protein was assessed by immunoblot.GAPDH protein levels served as a loading control.
Found at:doi:10.1371/journal.pone.0004170.s002(1.94MB EPS) Figure S3Loss of Fv2E-PERK restores cell viability in CHO cells.Fv2E-PERK protein(+/2phosphorylation)was examined by immunoblotting in parental CHO cells expressing stably-integrated Fv2E-Perk and6clonal derivatives that grew in the presence of AP20187(100nM).Ponceau S staining of the immunoblot revealed equivalent protein levels and served as a loading control(data not shown).Where indicated,cells were exposed to AP20187(100nM)for30minutes.
Found at:doi:10.1371/journal.pone.0004170.s003(0.63MB EPS) Video S1HEK293cells treated with mock solvent(left frame)or tunicamycin(right frame)for48hours.
Found at:doi:10.1371/journal.pone.0004170.s004(7.81MB MOV)
Video S2HEK293cells expressing Fv2E-PERK treated with mock solvent(left frame)or AP20187(right frame)for48hours. Found at:doi:10.1371/journal.pone.0004170.s005(10.37MB MPG)
Video S3HEK293cells expressing IRE1[I642G]treated with mock solvent(left frame)or1NM-PP1(right frame)for48hours. Found at:doi:10.1371/journal.pone.0004170.s006(10.15MB MPG)
Acknowledgments
We thank B.Farese,M.Karin,and B.Yen for helpful comments.We thank K.Thorn.F.Sanchez,and the Nikon Imaging Center for technical assistance.We thank C.Zhang and K.Shokat for generous provision of 1NM-PP1.
Author Contributions
Conceived and designed the experiments:JHL HL YZ DR PW.Performed the experiments:JHL HL YZ.Analyzed the data:JHL HL YZ DR PW.Contributed reagents/materials/analysis tools:JHL YZ DR PW.Wrote the paper:JHL PW.
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Fv2E-PERK
ATF-4
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C h o p m R N A r e l a t i v e f o l d i n c r e a s
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Figure S 1
cleaved PARP GAPDH 4872
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Figure S 2
100
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AP20187:
P a r e n t a l
R e s i s t a n t C l o n e #1
R e s i s t a n t C l o n e #2
R e s i s t a n t C l o n e #3
R e s i s t a n t C l o n e #4
R e s i s t a n t C l o n e #5
R e s i s t a n t C l o n e #6
Figure S 3