Double-stranded RNA-activated protein kinase interacts with apoptosis signal-regulating kinase 1 Implications for apoptosis signaling pathways Takenori Takizawa 1 , Chizuru Tatematsu 1 and Yoshinobu Nakanishi 2 1 Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan; 2 Graduate school of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan Double-stranded RNA-activated protein kinase (PKR), a serine/threonine kinase, is activated in virus-infected cells and acts as an antiviral machinery of type I interferons. PKR controls several stress response pathways induced by double-stranded RNA, tumor necrosis factor-a or lipo- polysaccharide, which result in the activation of stress-acti- vated protein kinase/c-Jun NH 2 -terminal kinase and p38 of the mitogen-activated protein kinase family. Here we showed a novel interaction between PKR and apoptosis signal-regulating kinase 1 (ASK1), one of the members of the mitogen-activated protein kinase kinase kinase family, which is activated in response to a variety of apoptosis- inducing stimuli. PKR and ASK1 showed predominant cytoplasmic localization in COS-1 cells transfected with both cDNAs, and coimmunoprecipitated from the cell extracts. A dominant negative mutant of PKR (PKR-KR) inhibited both the apoptosis and p38 activation induced by ASK1 in vivo. Consistently, PKR-KR inhibited the auto- phosphorylation of ASK1 in vitro, and exposure to poly(I)– poly(C) increased the phosphorylation of ASK1 in vivo. These results indicate the existence of a link between PKR and ASK1, which modifies downstream MAPK. Keywords: ASK1; apoptosis; MAPK; PKR; signal trans- duction. The interferon-inducible, double-stranded RNA (dsRNA)- activated protein kinase (PKR) is a serine/threonine kinase ubiquitously expressed in mammalian cells [1,2]. PKR is activated by a variety of dsRNA molecules generated during viral infection [3]. Upon its activation, PKR autophosphorylates and then phosphorylates eukaryotic translational initiation factor 2 (eIF-2a) [4], thereby inhi- biting cell growth or viral replication [5,6]. Thus PKR mediates the antiviral and antiproliferative actions of type I interferons [6]. On the other hand, catalytically inactive mutants of PKR transform NIH-3T3 cells [7,8], while overexpression of wild-type PKR induces apoptosis of HeLa cells [9,10]. PKR appears to up-regulate expression of the apoptotic receptor Fas induced by viral infection [11,12]. Moreover, mouse embryonic fibroblasts deleted of the PKR gene have been shown to resist apoptosis in response to dsRNA, tumor necrosis factor-a (TNF-a) or lipopolysac- charide (LPS) [13]. PKR has been shown to play some role in the activation of p38 mitogen activated protein kinases (MAPKs) and the stress-activated protein kinase (SAPK)/ c-Jun amino-terminal kinases (JNKs) that are strongly activated in response to TNF-a,dsRNAorLPS[13]. However, the precise pathway linking PKR and the MAPK family remains to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAPKKK) that acts upstream of JNK and p38 MAPKs [14,15]. ASK1 phosphorylates SEK1/MKK4 or MKK3/MKK6, one of the members of the MAPK kinase family, which in turn activates JNK or p38 MAPK, respectively [15]. A wide variety of stress-related stimuli activate ASK1, including serum withdrawal, TNF-a, react- ive oxygen species, microtubule-interfering agents, genoto- xic stress, and possibly Fas ligand [16]. Overexpression of the wild type or constitutively active form of ASK1 induces cell death through signals involving the mitochondrial cell death pathway [17]. ASK1 binds proteins associated with death receptors as TNF-receptor-associated proteins (TRAFs) or Daxx, which also results in MAPK activation [18,19]. In the present study, we show that PKR interacts with ASK1 and modifies the ASK1 signaling pathway both in vivo and in vitro. These results suggest that PKR acts as a signal transducer by interacting with MAPKKKs, which then modifies the downstream MAPK cascade. MATERIALS AND METHODS Plasmids and DNA transfection Human PKR cDNA was kindly provided by A. Hovanes- sian (Institut Pasteur, France). A mutant PKR cDNA Correspondence to T. Takizawa, Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan. Fax: + 81 568 88 0829, Tel.: + 81 568 88 0829, E-mail: takizawa@inst-hsc.pref.aichi.jp Abbreviations: ASK1, apoptosis signal-regulating kinase 1; dsRNA, double-stranded RNA; eIF-2a, eukaryotic translational initiation factor 2a; EGFP, enhanced green fluorescence protein; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; MAPK, mitogen activated protein kinase; PKR, double-stranded RNA-acti- vated protein kinase; RITC, tetaramethyl rhodamine isothiocyanate; DMEM, Dulbecco’s modified Eagle’s medium. (Received 29 June 2002, revised 4 September 2002, accepted 22 October 2002) Eur. J. Biochem. 269, 6126–6132 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03325.x carrying a point mutation of K to R at position 296 (PKR-KR) was constructed as described [20]. cDNAs for human ASK1 and dominant negative mutant of ASK1 carrying a point mutation of K to M at position 709 (ASK- KM) were kindly provided by H. Ichijo (Laboratory of Cell Signaling, Tokyo Medical and Dental University). The plasmid encoding PKR or PKR-KR fused to enhanced green fluorescent protein (EGFP) (Clontech Laboratories, Inc., Palo Alto, CA, USA) was described previously [21]. Human embryonic kidney 293 (HEK293), COS-1 and NIH-3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and maintained under 5% CO 2 at 37 °C. Cells were transfected with 2 lg of plasmid DNA using 6 lLof Lipofectamine-plus and 4 lL of Lipofectamine (Gib- coBRL) according to the manufacturer’s instructions. To establish permanent transfectants, HEK293 cells were diluted about 10-fold and replated in medium containing 750 lgÆmL )1 of G418 two days after transfection, and then drug-resistant colonies were isolated. For microscopic observation, cells were seeded on a cover glass and DNAs were transfected as described above. For in vivo labeling of ASK1 or PKR, cells were washed with phosphate-free DMEM, and then incubated in phosphate-free DMEM in the presence of [ 32 P]-orthophosphate (400 lCiÆmL )1 )(ICN Biomedicals, CA, USA) for the indicated period as described in the figure legend. The phosphorylation reaction was resolved by SDS/PAGE and visualized by autoradio- graphy. Autoradiogram was scanned and densitometric analysis was performed with Kodak DIGITAL SCIENCE 1 D software (Eastman Kodak). Indirect immunofluorescence The day after transfection, cells were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 in NaCl/P i for 30 min and washed with NaCl/P i . Subse- quently, cells were incubated with anti-HA mAb (12CA5, Boehringer Mannheim, Germany) or anti-PKR polyclonal antibody (N-18, Santa Cruz, CA, USA) at a dilution of 200 or 100, respectively, for 60 min. Cells were then stained with anti-(mouse IgG) conjugated with fluorescein isothiocya- nate (FITC) (MBL, Nagoya, Japan) or anti-(rabbit IgG) conjugated with tetaramethyl rhodamine isothiocyanate (RITC) (Jackson Immunoresearch Laboratory, West Grove, PA, USA) at a dilution of 200 for 60 min, and observed under a fluorescence microscope at a magnifica- tion of 270 (Olympus BX-60, Tokyo, Japan). For the expression of EGFP, cells were fixed with 4% paraformal- dehyde, and observed under a fluorescence microscope as described above. In vitro kinase assay Cell extracts were prepared with PKR buffer I (20 m M Tris/ HClpH7.6,50m M KCl, 400 m M NaCl, 1 m M EDTA, 5m M 2-mercaptoethanol, 1% Triton X-100, 0.2 m M phe- nylmethane sulfonyl fluoride, 100 UÆmL )1 aprotinin, and 20% glycerol) and cleared by centrifugation. Then ASK1 in the cell extract was precipitated by incubation with anti-HA mAb (5 lL) for 1 h at 4 °C followed by 50 lLofa1:1 slurry of protein–G Sepharose 4FF (Pharmacia, Piscata- way, NJ, USA). The immune complex on the beads was washed four times with PKR buffer I and then once with PKR buffer III (20 m M Tris/HCl pH 7.6, 80 m M KCl, 5m M b-mercaptoethanol, 2 m M MgCl 2 ,2m M MnCl 2 ,and 20% glycerol). The beads were then resuspended in PKR buffer III containing 2 l M [c- 32 P]ATP (5 lCi) (ICN Biomedicals) in the presence or absence of poly(I)–poly(C) at the concentration indicated in the legends for 15 min at 30 °C. The phosphorylation reaction was resolved by SDS/ PAGE and visualized by autoradiography. PKR activity was measured as described [11]. Immunoprecipitation and Immunoblot analyses Cell extracts were prepared with PKR buffer I and incubated with anti-PKR mAb (2 lL) or anti-HA antibody (5 lL) for 1 h at 4 °C followed by 50 lL of a 1 : 1 slurry of protein-G Sepharose 4FF for another 1 h at 4 °C. The immune complex on the beads was washed four times with PKR buffer I. The beads were then boiled in Laemmli’s sample buffer [22] and resolved by SDS/PAGE. Proteins were transferred onto nitrocellulose filters (Bio-Rad Labor- atory, Hercules, CA, USA) and were incubated with polyclonal anti-PKR Ig or anti-HA Ig followed by anti- (rabbit IgG) or anti-(mouse IgG) conjugated with peroxidase. Signals were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham, Boston, MA, USA). Protein was measured by Bradford reagent (Bio-Rad Laboratory). RESULTS PKR interacts with ASK1 To investigate whether PKR and ASK1 interact with each other, the localization of PKR and ASK1 was first examined by indirect immunofluorescence. As the wild type of PKR is hardly expressed at all by transfection due to translational inhibition [23], we used the kinase negative mutant of PKR (PKR-KR), which shows the same localization pattern as the wild type as previously reported [24]. The signal for PKR-KR was predominantly localized in the cytoplasm, whereas that for ASK1 distributed diffusely with relatively intense staining at the periphery of the cells (Fig. 1A, a and b). When PKR-KR and ASK1 were cotransfected into COS-1, both proteins showed predominant cytoplasmic localization (Fig. 1A, c and d), indicating colocalization of PKR and ASK1. We next used a coimmunoprecipitation assay to define the interaction between PKR and ASK1. They were transfected into COS-1 cells, and immunoprecipitated with antibody against either PKR or ASK1. When the immune- complexes were precipitated with anti-PKR Ig and analyzed by Western blotting with anti-HA Ig, the signal of ASK1 was detected only in the cell extracts transfected with both cDNAs (lane 2 in Fig. 1B). PKR-KR was also coimmuno- precipitated with anti-HA Ig (lane 4 in Fig. 1B). Expression of these proteins was verified by Western blotting (Fig. 1B, right panel). To examine whether endogenous PKR is coimmunoprecipitated with ASK1, we established two HEK 293 cells permanently expressing ASK1 (ASK-4 and ASK-8 cells) and control cells containing empty plasmid (pcDNA). The expressions of ASK1 and endogenous PKR in these cells were confirmed by Western blotting (Fig. 1C, Ó FEBS 2002 PKR interacts with ASK1 (Eur. J. Biochem. 269) 6127 right panel). ASK1 or endogenous PKR was immunopre- cipitated with anti-PKR or anti-HA Ig, respectively (Fig. 1C, left panel). All these results indicate that PKR directly interacts with ASK1. Alternatively, the interaction might be bridged by RNA. However, as all these coimmu- noprecipitation assays were conducted in the presence of high salt (0.45 M ), and immunecomplexes were resistant to RNase treatment (data not shown), direct protein–protein interaction seems to be likely. Dominant negative mutant of PKR inhibits ASK1 activity To explore the potential influence of PKR on ASK1 in vivo, the effect of PKR-KR on the ASK1-induced apoptosis was investigated. We used constructs of PKR fused with EGFP to directly visualize cell morphology. We have shown that EGFP-PKR induced apoptosis without poly(I)–poly(C), whereas EGFP-PKR-KR inhibited Fas-induced apoptosis [21]. NIH-3T3 cells were transfected with ASK1 and either pEGFP-PKR-KR or pEGFP. Serum was removed from the medium 24 h after transfection, and the cells were incubated for another 24 h. The cells were then fixed and ASK1 was stained with anti-HA Ig followed by RITC- labeled secondary antibody. ASK1 and EGFP-expressing cells exhibited a round shrunken morphology indicating an induction of apoptosis (Fig. 2B, arrow heads in upper panel), whereas the cells expressing both ASK1 and pEGFP-PKR-KR exhibited a flat spread shape (Fig. 2B, arrow heads in lower panel). EGFP-expressing cells without ASK1 expression were a flat shape as well (Fig. 2B, upper panel). The expression of these proteins was verified by Western blotting (Fig. 2A). The number of cells exhibiting a shrunken morphology was counted in several fields and summarized (Fig. 2C). Transfection of ASK1 and subse- quent serum deprivation caused about 60% of cells to die, whereas cotransfection of pEGFP-PKR-KR suppressed the cell death to almost the control level (Fig. 2C). Transfection of either empty vector or PKR-KR alone did not cause significant cell death. As PKR-KR inhibited the ASK1-induced apoptosis, it seems reasonable to speculate that PKR-KR inhibited ASK1 signaling. As ASK1 has been shown to activate stress-activated MAPKs, the effect of PKR-KR on the activation of p38 by ASK1 was examined. COS-1 cells were transfected with ASK1 and/or PKR-KR, and p38 activation was examined by Western blotting using antibody against the phosphorylated form of p38 (Fig. 3). ASK1 increased p38 phosphorylation 24 h after transfection (lane 6 in Fig. 3) (an average of 5.4-fold increase in the intensity from three independent experi- ments compared with that of pcDNA at 24 h), whereas cotransfection of PKR-KR inhibited its increase to about 60% level of ASK1 (lane 8 in Fig. 3) (an average of 3.4- fold increase). Fig. 1. PKR interacts with ASK1. (A) Cytoplasmic localization of ASK1 with PKR. COS-1 cells were transfected with pcDNA-PKR- KR (a), pcDNA-ASK1-HA (b), or both (c and d). Cells were fixed 18 h after transfection and then incubated with anti-PKR Ig (a) or anti-HA Ig (b) followed by FITC-labeled anti-rabbit or anti-mouse immunoglobulin, respectively. For double staining of PKR-KR (c) and ASK1 (d), cells were incubated with both antibodies, followed by RITC-labeled and FITC-labeled secondary antibodies. Cells were observed under a fluorescent microscope at a magnification of 270. (B) PKR and ASK1 were coimmunoprecipitated. COS-1 cells (approxi- mately 4 · 10 5 cells) were transfected with the plasmid DNAs indi- cated above the lanes, and lysed in a lysis buffer 48 h after transfection. Lysates (200 lg of total protein) were incubated with anti-PKR (lanes 1 and 2) or anti-HA (lanes 3 and 4) antibody, followed by protein G–Sepharose. Immunecomplexes were then analyzed by Western blotting with anti-HA (lanes 1 and 2) or anti-PKR (lanes 3 and 4) Ig. Expression of ASK1 and PKR-KR in a total lysate (100 lgoftotal protein) was examined by Western blotting using anti-HA (ASK1) or anti-PKR Ig (PKR) (right panel). Signals were visualized by ECL. Ig denotes immunoglobulin. (C) ASK1 and endogenous PKR were coimmunoprecipitated. HEK293 cell lines permanently transfected with pcDNA (pcDNA) or ASK1 (ASK-4, ASK-8) were established. Approximately 2 · 10 7 celles were lysed in a lysis buffer, and ASK1 or PKR in a lysate (12 mg of total protein) was immunoprecipitated with anti-HA or anti-PKR Ig (immppt) as described in (B), and Immune- complexes were analyzed by Western blotting with anti-HA or anti- PKR Ig (WB). Expression of ASK1 or endogenous PKR in a total lysate (100 lg of total protein) was examined by Western blotting (right panel). Signals were visualized by ECL. 6128 T. Takizawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Dominant negative mutant of PKR inhibits ASK1 activity in vitro The above results suggested that PKR modifies ASK1 activity. We therefore examined by means of an in vitro kinase assay whether PKR directly affects ASK1 activity. ASK1 was immunoprecipitated with anti-HA Ig from the extract of HEK293 cells permanently expressing ASK1, and an autophosphorylation reaction was induced in the presence or absence of poly(I)–poly(C). Poly(I)–poly(C), however, did not affect ASK1 activity at all (Fig. 4A). This might be due to the amount of PKR coimmunoprecipitated, which was so small that its effect could not be detected. Therefore, PKR-KR was further transfected into ASK-8 cells and ASK1 activity was examined. An increase in the intensity of the PKR signal was observed in PKR-KR- transfected cells by Western blotting (lanes 2 and 4 in Fig. 2. Effect of dominant negative mutant of PKR (PKR-KR) on ASK1-induced apoptosis. (A) Western blotting of PKR-KR or ASK1. NIH-3T3 cells were transfected with empty vector of pEGFP (GFP), pEGFP-PKR-KR (PKR-KR), pEGFP and ASK1 (ASK + GFP) or pEGFP-PKR-KR and ASK1 (ASK + PKR-KR) as indicated above the lanes. Cell lysates were prepared 48 h after transfection and then examined by Western blotting using anti-GFP, anti-PKR, or anti-HA Ig. Signals were visualized by ECL. (B) Effect of PKR-KR on the ASK1-induced apoptosis. NIH-3T3 cells were transfected with 1.5 lg of pcDNA-ASK1-HA with either 0.5 lg of pEGFP or pEGFP-PKR- KR. Serum was removed from the medium 24 h after transfection, and the cells were incubated for another 24 h. They were then fixed with 4% PFA and stained with anti-HA Ig followed by anti-(mouse IgG) conjugated with RITC. Arrowheads indicate the cells expressing ASK1 and either EGFP or PKR-KR fusion protein. (C) Rounded and shrunken cells were counted as apoptotic. The percentages of apop- totic cells are shown as an average of three independent experiments ±S.D. Fig. 3. Effect of dominant negative mutant of PKR (PKR-KR) on activation of p38 induced by ASK1. COS-1 cells (approximately 4 · 10 5 cells) were transfected with the plasmid DNAs described above the lanes, and cells were harvested 12 h or 24 h after transfection. Cell lysates (100 lg of total protein) were resolved by SDS/PAGE. Expression of the phosphorylated form of p38, total p38, ASK1 and PKR was examined by Western blotting using anti-(p38-P), anti-(p38- total), anti-HA, and anti-PKR Ig, respectively. Signals were visualized by ECL. The representative data of three independent experiments with similar results were shown. Ó FEBS 2002 PKR interacts with ASK1 (Eur. J. Biochem. 269) 6129 Fig. 4B), while the amount of ASK1 in ASK-8 cells did not change (lanes 3 and 4 in Fig. 4B). Transfection of PKR-KR revealed a 35% decrease (an average from three independ- ent experiments) in the autophosphorylation activity of ASK1 (lanes 7 and 8 in Fig. 4B), suggesting that PKR affects the activity. Effect of PKR on the activity of ASK1 in vivo To examine in vivo the effect of PKR on ASK1 activity, HEK293 cells permanently transfected with wild type or dominant negative mutant of ASK1 were exposed to either poly(I)–poly(C) or H 2 O 2 in the presence of [ 32 P] orthophosphate, and cell lysates were prepared. ASK1 was immunoprecipitated with anti-HA Ig. Immunecomplex was then resolved by SDS/PAGE, and phosphorylation reaction was visualized by autoradiography. Exposure to H 2 O 2 markedly increased the intensity of ASK1 about 2.7-fold (an average of two independent experiments) compared with that without H 2 O 2 (AR in Fig. 5B), indicating H 2 O 2 activates ASK1 as described [25]. Expo- sure to poly(I)–poly(C) also increased the signal for ASK1 about 2.1-fold (an average of two independent experi- ments) compared with that without poly(I)–poly(C) (AR in Fig. 5A), suggesting that PKR could activate ASK1. The amount of ASK1 in the immunecomplexes was not changed by these exposures, which was verified by Western blotting (WBs in Fig. 5A and B). On the other hand exposure to poly(I)–poly(C) did not markedly Fig. 5. Effect of poly(I)–poly(C) on ASK1 activity in vivo. (A) HEK293 cells (approximately 4 · 10 5 cells) permanently transfected with empty vector (pcDNA), wild type (ASK-8) or dominant negative mutant of ASK1 (ASK-KM-2) were incubated with or without poly(I)–poly(C) (polyI–C) (100 lgÆmL )1 ) in the presence of [ 32 P]-orthophosphate (400 lCiÆmL )1 ) for 2 h and harvested. ASK1 was immunoprecipitated with anti-HA mAb, and resolved by SDS/PAGE. Phosphorylation reactions were visualized by autoradiography (AR). Expression of ASK1, ASK-KM was verified by Western blotting (WB). (B) Cells were incubated with or without H 2 O 2 (1 m M ) in the presence of [ 32 P]- orthophosphate for 1 h and harvested. Phosphorylation reactions (AR) and expression of ASK1, ASK-KM (WB) were examined as described in (A). The representative data of two independent experi- ments with similar results were shown. Fig. 4. Effect of PKR-KR on ASK1 activity in vitro . (A) ASK1 activity in ASK1-expressing HEK293 cells (ASK-4, ASK-8). Cells were lysed in a lysis buffer, and ASK1 in a lysate (400 lg of total protein) was immunoprecipitated with anti-HA monoclonal Ig followed by protein G-sepharose. Protein G-sepharose was then suspended in PKR III buffer with [c- 32 P]ATP in the absence or presence of poly(I)–poly(C) (polyI–C) (1.0 lgÆmL )1 ) for 15 min at 30 °C. Reactions were resolved by SDS/PAGE and visualized by autoradiography. (B) Effect of PKR- KR on ASK1 activity. Control (pcDNA) or ASK-8 cells were trans- fected with PKR-KR (+) or empty vector (–), and cell lysates were prepared 48 h after transfection. Expression of ASK1 and PKR-KR was verified by Western blotting (lanes 1–4). ASK1 activity in the absence or presence of PKR-KR was examined as described in (A) and visualized by autoradiography (lanes 5–8). The representative data of three independent experiments with similar results were shown. 6130 T. Takizawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 increase the phosphorylation state of ASK-KM, a kinase negative mutant of ASK1 [15,19]. This may indicate that PKR does not directly phosphorylate ASK1 but rather supports autophosphorylation activity of ASK1. All these results suggest that PKR could activate ASK1, although it remains possible that poly(I)–poly(C) directly activates ASK1. However, the latter might be unlikely, as poly(I)– poly(C) did not activate ASK1 in vitro (Fig. 4A). DISCUSSION Besides having the antiviral activity of type I interferons, PKR has been shown to transduce signals such as dsRNA, LPS, platelet-derived growth factor, Fas, and TNF-a [13,26,27]. As most of these signals are capable of inducing apoptosis, PKR seems to transduce apoptotic signals, especially receptor-mediated stimuli. As these stimuli also have been shown to activate protein kinases of the MAPK family [28], cross talk between PKR and the MAPK cascade has been proposed. However, the pathway linking PKR and the MAPK family remains to be clarified. In the present study, we showed that PKR colocalized and was coimmunoprecipitated with ASK1 when cotrans- fected into COS-1 cells. The interaction of PKR with ASK1 does not require kinase activity of PKR, as a kinase negative mutant as well as endogenous PKR were coimmunoprecipitated with ASK1. This seems to be consistent with recent reports that the interaction of PKR with IjBkinaseb or the signal transducer and activator of transcription1 does not require kinase activity of PKR [29,30]. However, PKR-KR decreased the autophosphorylation activity of ASK1 in vitro,and inhibited both the activation of p38 MAPK and apoptosis induced by ASK1 in vivo. Moreover, exposure to poly(I)– poly(C) increased ASK1 phosphorylation. All these results indicate that PKR dose not play only a structural role but rather modulates a signaling pathway of ASK1. Therefore the binding of PKR to ASK1 might cause a conforma- tional change in ASK1 for activation, of which is dependent on PKR activity, or an additional factor(s) might be requited to activate ASK1. It has been shown that the activation p38 by poly(I)– poly(C) or LPS treatment was abrogated in PKR-null fibroblasts, while generally acting stimuli such as osmotic shock or H 2 O 2 did not require PKR to activate MAPKs [13]. By contrast, a variety of stimuli such as TNF-a, IL-1, Fas, ceramide, H 2 O 2 , osmotic shock, heat shock, anticancer drugs, protein synthesis inhibitors and so on activate ASK1 [16]. 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Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAPKKK) that acts upstream of JNK and p38 MAPKs [14 ,15 ]. ASK1 phosphorylates SEK1/MKK4 or