P58IPK, a Novel Endoplasmic Reticulum Stress-inducible Protein and Potential Negative Regulator of eIF2α Signaling
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m212074200
ISSN1083-351X
AutoresRika van Huizen, Jennifer L. Martindale, Myriam Gorospe, Nikki J. Holbrook,
Tópico(s)Autophagy in Disease and Therapy
ResumoThe unfolded protein response, which is activated in response to the loss of endoplasmic reticulum (ER) Ca2+ homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen, involves both transcriptional and translational regulation. In the current studies we sought to identify novel ER stress-induced genes by conducting microarray analysis on tunicamycin-treated cells. We identified P58IPK, an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase, as induced during ER stress. Additional studies suggested that p58 IPK induction was mediated via ATF6 and that P58IPK played a role in down-regulating the activity of the pancreatic eIF2 kinase/eukaryotic initiation factor 2α (eIF2α)-like ER kinase/activation transcription factor (ATF) 4 pathway. Modulation of P58IPK levels altered the phosphorylation status of eIF2α, and thereby affected expression of its downstream targets, ATF4 and Gadd153. Overexpression of P58IPK inhibited eIF2α phosphorylation and reduced ATF4 and Gadd153 protein accumulation, whereas silencing of P58IPK expression enhanced pancreatic eIF2α-like ER kinase and eIF2α phosphorylation and increased ATF4 and Gadd153 accumulation. These findings implicate P58IPK as an important component of a negative feedback loop used by the cell to inhibit eIF2α signaling, and thus attenuate the unfolded protein response. The unfolded protein response, which is activated in response to the loss of endoplasmic reticulum (ER) Ca2+ homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen, involves both transcriptional and translational regulation. In the current studies we sought to identify novel ER stress-induced genes by conducting microarray analysis on tunicamycin-treated cells. We identified P58IPK, an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase, as induced during ER stress. Additional studies suggested that p58 IPK induction was mediated via ATF6 and that P58IPK played a role in down-regulating the activity of the pancreatic eIF2 kinase/eukaryotic initiation factor 2α (eIF2α)-like ER kinase/activation transcription factor (ATF) 4 pathway. Modulation of P58IPK levels altered the phosphorylation status of eIF2α, and thereby affected expression of its downstream targets, ATF4 and Gadd153. Overexpression of P58IPK inhibited eIF2α phosphorylation and reduced ATF4 and Gadd153 protein accumulation, whereas silencing of P58IPK expression enhanced pancreatic eIF2α-like ER kinase and eIF2α phosphorylation and increased ATF4 and Gadd153 accumulation. These findings implicate P58IPK as an important component of a negative feedback loop used by the cell to inhibit eIF2α signaling, and thus attenuate the unfolded protein response. endoplasmic reticulum activation transcription factor CCAAT/enhancer-binding protein homologous protein α subunit of eukaryotic initiation factor-2 glucose-regulated protein 78 pancreatic eIF2 kinase PKR-like ER kinase double stranded RNA double-stranded RNA-dependent protein kinase unfolded protein response X-box binding protein-1 mouse embryo fibroblasts reverse transcriptase 4,6-diamidino-2-phenylindole small interference RNA glyceraldehyde-3-phosphate dehydrogenase poly(ADP-ribose) polymerase The endoplasmic reticulum (ER)1 is an important organelle in which newly synthesized secretory and membrane-associated proteins are correctly folded and assembled. Perturbations in the ER environment result in a condition known as ER stress, which can threaten cell survival. ER stress can be induced in cells by a variety of treatments including agents known to affect calcium homeostasis, inhibitors of glycosylation, and overloading of the cell with mutant proteins that cannot be properly folded. Such stress triggers the activation of a complex response termed the unfolded protein response (UPR), which in mammalian cells is characterized by coordinate transcriptional up-regulation of a number of proteins including molecular chaperones and folding enzymes, global inhibition of protein synthesis, and activation of apoptotic pathways (1Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar). The first two components serve to reduce the load of client proteins and alleviate the stress, whereas the third functions to eliminate severely damaged cells. At least three separate mechanisms contribute to the transcriptional response to ER stress in mammalian cells. The first involves IRE1α and IRE1β, transmembrane protein kinases with endoribonuclease function, that sense the presence of unfolded proteins in the ER, leading to their activation (2Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2133) Google Scholar, 3Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (751) Google Scholar, 4Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (661) Google Scholar). In turn, activation of IRE1 results in the splicing of mRNA encoding the transcription factor XBP-1, increasing its efficiency of translation, thereby enhancing its expression (5Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2161) Google Scholar, 6Shen X. Ellis R.E. Lee K. Liu C.Y. Yang K. Solomon A. Yoshida H. Morimoto R. Kurnit D.M. 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J. 1999; 339: 135-141Crossref PubMed Scopus (371) Google Scholar, 18Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). Repression of protein synthesis in response to ER stress is mediated through the increased phosphorylation of eukaryotic initiation factor 2 (eIF2α), a modification that interferes with the formation of an active 43 S translation-initiation complex (19Brostrom C.O. Brostrom M.A. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 79-125Crossref PubMed Scopus (250) Google Scholar). Phosphorylation of eIF2α during ER stress is carried out by the pancreatic eIF2α kinase (PEK or PERK), which is activated as part of the unfolded protein response (20Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2558) Google Scholar, 21Sood R. Porter A.C. Ma K. Quilliam L.A. Wek R.C. Biochem. 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Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar, 2Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2133) Google Scholar, 30Liu C.Y. Schroder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Although PERK-mediated eIF2α phosphorylation leads to a general suppression of translation, it promotes the preferential translation of certain mRNAs. Most notable among these is the transcription factor ATF4 (18Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). Genome-wide expression analysis using DNA microarrays has revealed that activation of the UPR in yeast results in the up-regulation of more than 350 genes. These include genes involved in various aspects of the secretory pathway, such as protein folding, ER to Golgi vesicular transport, and ER-associated protein degradation (22Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar). Although many of the molecular details of the UPR have been conserved in yeast and mammalian systems, the scope of UPR outputs in the mammalian cell is more complex and diverse. Accordingly, it is likely to involve a greater number of proteins and gene expression changes than seen in yeast. The present study utilized DNA microarray analysis to search for novel genes induced by ER stress in mouse embryo fibroblasts (MEFs). We report here the identification of P58IPK, an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR), as a gene whose expression is up-regulated in response to ER stress. Additional studies provide evidence that ATF6 contributes to the induction of P58IPK and P58IPK plays a role in regulating the activity of the PERK/eIF2α/ATF4 pathway. MEFs, human embryo kidney fibroblasts (HEK-293), and human cervical carcinoma HeLa cells were cultured in Dulbecco's modified essential medium (Invitrogen). All media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units of penicillin/ml, and 100 μg of streptomycin (Invitrogen) per ml, and were maintained in a humidified atmosphere containing 5% CO2. Recombinant DNA techniques were performed by standard procedures. Plasmid pCGN-atf6 was kindly provided by Dr. R. Prywes (Columbia University) and used to constructpCGN-p50atf6 by inserting a PCR fragment spanning the NH2-terminal 1–373 amino acids of atf6, followed by a stop codon into the XbaI and BamHI sites of the pCGN vector. HEK-293 and HeLa cells were grown to 60% confluency in 100-mm plates and transfected using Polyfect reagent (Qiagen, Valencia, CA). Six μg ofpCGN-p50atf6,pCDNA3-atf4 (kindly provided by Dr. J. Leiden, University of Chicago),pCDNA1-p58 IPK (kindly provided by Dr. M. G. Katze, University of Washington), pcDNA-perk(kindly provided by Dr. D. Ron, New York University School of Medicine), or an empty vector were transfected per plate. Tunicamycin and thapsigargin were from Sigma. Whole cell lysate protein aliquots (20–30 μg) were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Schleicher & Schuell) by standard techniques. Blots were hybridized with the following antibodies: monoclonal anti-P58IPK (gift from Dr. M. G. Katze, University of Washington), polyclonal anti-Gadd153 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), polyclonal anti-ATF4 (Santa Cruz Biotechnology Inc.), monoclonal anti-KDEL (Stressgen, Victoria, BC, Canada), polyclonal anti-eIF2α (Cell Signaling Technology, Beverly, MA), polyclonal anti-phospho-eIF2α (Cell Signaling Technology, Beverly, MA), polyclonal anti-phospho-PERK (Cell Signaling Technology), polyclonal anti-PKR (Cell Signaling Technology), polyclonal anti-phospho-PKR (Cell Signaling Technology), monoclonal anti-cleaved PARP (Cell Signaling Technology), polyclonal anti-cleaved caspase-3 (Cell Signaling Technology), monoclonal anti-myc (9E10) (Santa Cruz Biotechnology Inc.), and monoclonal anti-GAPDH (Abcam Ltd., Cambridge, UK). Secondary horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were from Amersham Biosciences. Proteins signals were detected by using Western Lightning Chemiluminescence ReagentPlus (PerkinElmer Life Sciences). To analyze RNA expression by reverse transcription-PCR (RT-PCR), total RNA from each sample was treated with DNase I and used for RT-PCR with SuperScript One-Step RT-PCR with the Platinum Taq system (Invitrogen). The primers for gene-specific RT-PCR analysis were as follows: for P58IPK, GAGGTTTGTGTTGGGATGCAG (5′) and GCTCTTCAGCTGACTCAATCAG (3′); for ATF4, AGGAGTTCGCCTTGGATGCCCTG (5′) and AGTGATATCCACTTCACTGCCCAG (3′); for ATF6, ATCAGTTTACAACCTGCACCCAC (5′) and CTGTCTCCTTAGCACAGCAATATC (3′); for Gadd153, CTGAGTCATTGCCTTTCTCTTCG (5′) and CTCTGACTGGAATCTGGAGAGTG (3′); GAPDH, ACATCAAGAAGGTGGTGAAGCAGG (5′) and CTCTTGCTCTCAGATCCTTGCTGG (3′). Equal aliquots of the PCR products were electrophoresed through 2% agarose gels. For Northern blot analysis, 4-μg aliquots of total RNA (harvested using Nucleo Spin RNAII kit (Clontech, Palo Alto, CA)) were run on agarose-formaldehyde gels and transferred onto GeneScreen Plus membranes (PerkinElmer Life Sciences). cDNAs corresponding toatf4, gadd153, and grp78 (a generous gift from Amy S. Lee), were labeled by the random primer method and used to detect corresponding mRNAs on Northern blots. An end-labeled 24-bp oligonucleotide complementary to 18 S rRNA (ACGGTATCTGATCGTCTTCGAACC) was used as a probe to verify RNA integrity and loading differences. Total RNA was extracted from all samples using a NucleoSpin RNAII kit (Clontech, Palo Alto, CA). Atlas Human 1.2 K filters (www.Clontech) each containing 1174 genes were used. Total RNA (5 μg) was reverse transcribed and labeled with [α-32P]dATP using the Clontech cDNA array labeling kit. Hybridizations and washes were performed as recommended by the manufacturer. The cDNA array membranes were visualized for analysis by using a PhosphorImager (Amersham Biosciences), and were quantitated as described (31Mayne M. Cheadle C. Soldan S.S. Cermelli C. Yamano Y. Akhyani N. Nagel J.E. Taub D.D. Becker K.G. Jacobson S. J. Virol. 2001; 75: 11641-11650Crossref PubMed Scopus (69) Google Scholar). DAPI staining was performed as described previously (24Han A.P. Yu C. Lu L. Fujiwara Y. Browne C. Chin G. Fleming M. Leboulch P. Orkin S.H. Chen J.J. EMBO J. 2001; 20: 6909-6918Crossref PubMed Scopus (283) Google Scholar). In brief, prior to staining, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed with phosphate-buffered saline. DAPI was added to the fixed cells for 30 min, after which they were examined by fluorescence microscopy. Apoptotic cells were identified by condensation and fragmentation of nuclei. Percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted ×100. A minimum of 400 cells were counted for each treatment. Twenty-one nucleotide double-stranded RNAs were transcribed in vitro using the SilencerTM siRNA construction kit according to the manufacturer instructions (Ambion Inc.). The targeting sequence of human P58IPK (accession number U28424), corresponding to nucleotide positions 137–157 (coding region), was AATTACTTGCAGCTGGACAGC. An siRNA targeting the luciferase mRNA (accession number X65324) served as a control. Cells were seeded in 6-well plates on the day before transfection at a concentration of 105 cells per well. Cells were transfected with OligofectAMINE reagent according to the manufacturer's instructions (Invitrogen). Briefly, Opti-MEM (165 μl) was mixed with 20 μl of 1 μm siRNA duplex. In a separate tube, 12 μl of Opti-MEM I was incubated with 3 μl of OligofectAMINE for 5 min at room temperature. The two mixtures were combined, gently mixed, and incubated for another 20 min at room temperature. The entire mixture was added to the cells in 0.8 ml of 10% fetal bovine serum-containing Dulbecco's modified Eagle's medium without antibiotics. Cells were assayed at different time intervals after transfection. Cells were seeded into 6-well plates at a density of 105 cells per well. Twenty-four h following P58IPK siRNA transfection, cells were placed for 30 min in methionine-free minimal essential medium (BIOSOURCE, Camarillo, CA), and labeled by addition of [35S]methionine (20 μCi/ml; 1,000 Ci/mmol;Amersham Biosciences) to the culture medium for 2 h. Cells were washed twice with ice-cold phosphate-buffered saline and collected in lysis buffer (20 mm Hepes, pH 7.4, 2 mm EGTA, 50 mm β-glycerophosphate, 1 mmNa3VO4, 5 mm NaF, 1% Triton X-100, 10% glycerol, 1 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin). The protein concentration was measured using the Bio-Rad protein DC assay kit. The [35S]methionine incorporation was measured by cold trichloroacetic acid precipitation and analyzed by SDS-PAGE. For trichloroacetic acid precipitation, equal amounts of protein were added to 0.5 ml of 0.1 mg/ml bovine serum albumin containing 0.02% sodium azide and placed on ice. Ice-cold 20% trichloroacetic acid (0.5 ml) was added and samples were vortexed vigorously and incubated for 30 min on ice. Cell suspensions were filtered through glass microfiber discs (Whatman). Discs were washed three times with ice-cold 10% trichloroacetic acid, and twice with 100% ethanol, after which they were air-dried, and the radioactivity was measured by scintillation counting. We sought to investigate the UPR stress response in mammalian cells by assessing changes in gene expression profiles after ER stress using the Atlas cDNA Gene Array (Clontech). This array contains cDNAs for 1174 genes involved in apoptosis, cell cycle control, stress responses, transcription, and signaling. Total RNA was isolated from MEFs that were either left untreated or treated for 6 h with 2 μg/ml tunicamycin, an agent that causes ER stress by inhibiting proteinN-glycosylation. Analysis of the resulting signals on cDNA arrays (carried out as described under "Materials and Methods") revealed 19 candidate genes whose expression was increased in response to tunicamycin treatment. Among these were a number of genes previously shown to play a role in the UPR response, includinggrp78, gadd153, and erp72. One novel gene whose expression was significantly elevated in MEFs following ER stress was p58 IPK, an established inhibitor of the eIF2α kinase PKR, a kinase related to the ER-specific kinase, PERK (Fig. 1 A). Hence, we further investigated the regulation of p58 IPK by ER stress and examined its potential role during the UPR response. To validate the findings obtained by microarray analysis,p58 IPK expression levels in MEFs and HEK-293 cells were examined using RT-PCR after treatment with either tunicamycin or thapsigargin (another ER stress agent that inhibits ER Ca2+-ATPase, thereby causing ER stress through a different mechanism). As shown, p58 IPK mRNA levels increased in both cell lines in response to each ER stress agent in a time-dependent manner (Fig. 1 B). Further characterization of the response was carried out through kinetic analysis of P58IPK protein expression by Western blot analysis. As depicted in Fig. 1 C, P58IPK protein levels similarly increased following treatment with tunicamycin in both cell lines (Fig. 1 C). Because ATF4 and ATF6 are known to play a role in the transcriptional activation of ER stress-inducible genes, we examined their contribution to the induction of p58 IPK expression. To investigate the potential role of ATF6, we transfected HEK-293 cells with a plasmid that expresses p50 ATF6 (the soluble form of ATF6, capable of translocating into the nucleus). Cells were harvested 48 h after transfection and p58 IPK mRNA was examined using RT-PCR (Fig. 2). Overexpression of p50 ATF6 alone in HEK-293 cells resulted in up-regulation of the p58 IPK mRNA, even in the absence of ER stress. In contrast, overexpression of full-length ATF4 alone did not alter p58 IPK mRNA expression. To ensure that the ATF4 construct was functional in this assay, we also analyzed expression of gadd153, an established transcriptional target of both ATF4 and ATF6. gadd153mRNA was induced similarly by ectopically overexpressed p50 ATF6 and ATF4, indicating that the ATF4 construct was functional. These observations indicate that p58 IPK is a novel target of the ATF6, but not ATF4 pathway. To gain a better understanding of the biological role of P58IPK in the UPR, we tried to generate stable cell lines overexpressing P58IPK using both HEK-293 and HeLa cells. Despite our best efforts, no stable P58IPK-overexpressing clones could be obtained (data not shown). As an alternative strategy, we transiently transfected a plasmid expressing P58IPK into HeLa cells, where high transfection efficiencies could be achieved. ER stress is known to induce translational repression, which is mediated by phosphorylation of eIF2α. Phosphorylation of eIF2α leads to down-regulation of translation initiation through a well characterized mechanism involving inhibition of eIF2β activity (32Rowlands A.G. Panniers R. Henshaw E.C. J. Biol. Chem. 1988; 263: 5526-5533Abstract Full Text PDF PubMed Google Scholar). P58IPK is a known inhibitor of the eIF2α kinase PKR, and it has been shown that overexpression of P58IPK can inhibit dsRNA-induced phosphorylation of eIF2α by PKR. Because ER stress leads to elevated P58IPK expression, we hypothesized that P58IPK might affect the eIF2α phosphorylation during the UPR. To address this possibility HeLa cells were transiently transfected with a P58IPK expression vector or empty vector (Fig. 3 A). Forty-eight hours post-transfection, cells were treated with tunicamycin, and protein lysates were analyzed for both eIF2α phosphorylation and total eIF2α protein levels by Western blotting. As shown in Fig.3 B, overexpression of P58IPK significantly attenuated tunicamycin-induced eIF2α phosphorylation. Given that elevated P58IPK expression decreased the phosphorylation levels of eIF2α, we sought to analyze the levels of ATF4, a downstream target of this pathway, and Gadd153, whose expression is in turn regulated (at least in part) by ATF4. P58IPK overexpression profoundly inhibited ATF4 protein accumulation in tunicamycin-treated HeLa cells (Fig. 3 C), consistent with previous reports showing that the production of ATF4 protein requires eIF2α phosphorylation (18Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). Gadd153 protein induction in the P58IPK overexpressed cells was ∼70% of that seen in control cells after 4–6 h treatment with tunicamycin (Fig. 3 C). Previous reports have shown that P58IPK can protect cells from dsRNA or tumor necrosis factor-α-induced apoptosis. We were therefore interested in determining whether P58IPK could protect cells from ER stress-induced apoptosis. HeLa cells transiently transfected with either a p58 IPK -containing expression vector or an empty vector were treated with tunicamycin for different time periods and then analyzed for apoptosis. A hallmark of apoptosis is cleavage of the nuclear 116-kDa PARP (poly(ADP-ribose) polymerase) protein to an 85-kDa inactive polypeptide. Inactivation of PARP through proteolytic cleavage facilitates chromosomal DNA fragmentation as part of the cellular apoptotic program (33Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2351) Google Scholar). Our results show that the tunicamycin-induced PARP proteolysis was similar in vector and P58IPK-transfected HeLa cells (Fig.3 D). DAPI staining revealed a similar pattern of condensed and fragmented nuclei for both the control and P58IPK-transfected cells (Fig. 3 E). Taken together, these results suggest that under the conditions utilized here, overexpression of P58IPK does not alter the apoptotic response to ER stress. Whereas elevated P58IPK expression failed to alter the cellular outcome following ER stress, it remained possible that a reduction in P58IPK could influence the response. We decided to employ the RNA interference technique to address this possibility. A small inhibitory double-stranded RNA homologous to a 21-nucleotide sequence unique to the human P58IPK was used to reduce P58IPK expression. As shown in Fig.4 A, transfection of HeLa or HEK-293 cells with P58IPK siRNA resulted in a reduction of P58IPK protein levels, although control transfections with siRNA specific for luciferase, carried out in parallel, showed no effect on P58IPK expression. These observations indicate that the P58IPK siRNA treatment specifically reduced the abundance of P58IPK protein. Approximately 28 h after transfection with the P58IPKsiRNA duplex, we observed that many cells showed reduced ability to adhere to the plate and floated in the medium. To determine whether silencing P58IPK causes cell death, we examined the cell growth and viability of P58IPK siRNA-transfected cells. Cells transfected with the control siRNA grew very well. By contrast, cells transfected with P58IPK siRNA showed a marked reduction in viability (Fig. 4 B). Caspase-3 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many key proteins such as the nuclear enzyme PARP. Activation of caspase-3 requires the proteolytic processing of its inactive zymogen into activated p17 and p12 subunits. Cleavage of caspase-3 and PARP can be detected by Western blot analysis and was apparent 20 h after transfection of P58IPKsiRNA in HeLa cells, whereas no caspase-3 or PARP cleavage were detected in the control cells (Fig. 4 C). These results indicate that silencing P58IPK decreases viability by causing apoptotic cell death. Because overexpression of P58IPK can inhibit eIF2α phosphorylation, we postulated that silencing of P58IPKwould induce eIF2α phosphorylation and inhibit protein translation. To determine the effect of P58IPK siRNA on eIF2α phosphorylation, HeLa and HEK-293 cells were transfected with the control siRNA or P58IPK siRNA and 20 h post-transfection, protein lysates were prepared and analyzed by Western blotting (Fig. 5 A). The results show that P58IPK siRNA treatment markedly increased the level of eIF2α phosphorylation compared with control transfected cells. To further establish th
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