Báo cáo khoa học: Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester ppt

Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester Yan Zhou 1 , Barbara I. Chase 1 , Mark Whitmore 2 , Bryan R. G. Williams 2 and Aimin Zhou 1,2 1 Department of Chemistry, Cleveland State University, OH, USA 2 Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, OH, USA The double-stranded (ds) RNA-dependent protein kin- ase (PKR) is a serine ⁄ threonine kinase that is induced in mouse and human cells by interferons (IFN). Upon binding to dsRNA, the activation of PKR by auto- phosphorylation leads to phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF-2a), subsequently resulting in an inhibition of protein syn- thesis. Thus, PKR plays a central role in the antiviral and cell proliferation inhibitory activities of IFN [1]. Overexpression of wild-type PKR greatly enhances the ability of cells to resist viral infection, whereas domin- ant negative PKR suppresses IFN-mediated antiviral activity [2,3]. PKR null mice succumb to encephalo- myocarditis virus (EMCV) infection more rapidly than wild-type mice and display increased susceptibility to the infections of vesicular stomatitis virus and influ- enza virus [4,5]. PKR also exerts an antiproliferative role in cells. Expression of wild-type PKR in yeast inhibits cell proliferation [6,7]. As predicted, NIH3T3 cells expressing various inactive mutants of PKR formed tumours in nude mice. In contrast, overexpres- sion of wild-type PKR in cells does not induce tumour growth in mice [8,9]. PKR can induce apoptosis in cer- tain cell types [10]. HeLa cells overexpressing wild-type PKR undergo apoptosis. However, inactive mutants did not induce apoptosis when similarly expressed [11]. PKR mediates apoptosis in cells induced by different stimuli, including dsRNA, viral infection, endotoxin and cytokines [12–16]. Increased contact hypersensi- tivity responses in PKR null mice compared with Keywords interferon; PKC; PKR; poly I:C; proteasome Correspondence A. Zhou, Clinical Chemistry Program, Department of Chemistry, SI 424, Cleveland State University, Cleveland, OH 44115, USA Fax: +1 216 687 9298 Tel: +1 216 687 2416 E-mail: a.zhou@csuohio.edu (Received 7 October 2004, revised 22 December 2004, accepted 18 January 2005) doi:10.1111/j.1742-4658.2005.04572.x The double-stranded RNA-dependent protein kinase (PKR) is one of the key mediators of interferon (IFN) action against certain viruses. PKR also plays an important role in signal transduction and immunomodulation. Understanding the regulation of PKR activity is important for the use of PKR as a tool to discover and develop novel therapeutics for viral infec- tions, cancer and immune dysfunction. We found that phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), decreased the level of autophosphorylated PKR in a dose- and time-dependent man- ner in IFN-treated mouse fibroblast cells. Polyinosinic–polycytidylic acid (poly I:C) treatment enhanced the activity of PKR induced by IFN, but did not overcome the PMA-induced reduction of PKR autophosphoryla- tion. Western blot analysis with a monoclonal antibody to mouse PKR revealed that the decrease of PKR autophosphorylation in cells by PMA was a result of PKR protein degradation. Selective PKC inhibitors blocked the degradation of PKR stimulated by PMA, indicating that PKC activity was required for the effect. Furthermore, we also found that proteasome inhibitors prevented PMA-induced down regulation of PKR, indicating that an active proteasome is required. Our results identify a novel mechan- ism for the post-translational regulation of PKR. Abbreviations ds, double-stranded; eIF-2a, a-subunit of eukaryotic initiation factor 2; IFN, interferon; PKC, protein kinase C; PKR, RNA-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; poly I:C, polyinosinic–polycytidylic acid. 1568 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS wild-type mice has been suggested by a role for PKR in host immune functions [17]. PKR mediates the activation of signal transduction pathways by a wide range of proinflammatory factors, including lipopolysaccharide, tumour necrosis factor-a, and interleukin-1 [18–21]. Interestingly, the activation of the nuclear factor jB (NFjB) pathway by dsRNA, lipopolysaccharide or tumour necrosis factor-a in cer- tain cells is PKR dependent [22]. PKR is part of the complex of IjB kinase, an upstream effector kinase that phosphorylates critical serine residues in the IjB family of inhibitors [23]. In addition, PKR is also involved in the activation of the stress-activated pro- tein kinases (p38) and c-Jun NH2-terminal kinase [24]. Although PKR is not essential in the most of these pathways, it is required for the maintenance and amplification of cellular responses to proinflammatory and apoptotic stimuli. These results demonstrate that PKR, in addition to its function as a mediator of IFN action, plays broad roles in cell signalling. Although PKR plays an important role in the anti- viral action of IFNs, the control of cell proliferation and the mediation of signal transduction pathways, relat- ively little work has been done on the transcriptional regulation of PKR expression. Furthermore, studies on the regulation of PKR protein levels by agents other than IFN are largely lacking. Here, we report that 4b-phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), inhibits PKR activ- ity with a dose- and time-correlation in IFN-treated mouse fibroblast cells. Western blot analysis revealed that the downregulation of PKR activity is due to the degradation of PKR protein. Pretreatment of cells with selective proteasome inhibitors prevents the PKR degra- dation induced by PMA, suggesting the involvement of an active proteasome. Our results provide the first evidence that PKR is regulated at the post-translational level by a tumour-promoting agent. Results PKR plays an important role in the inhibition of cell proliferation and virus replication. Previously, we have reported that PMA downregulated RNase L, one of the key enzymes in the 2-5 A system that also func- tions in IFN-induced antiviral and antiproliferative activities [25]. To determine if PMA affects PKR activ- ity, lysates from mouse fibroblast L929 cells treated with or without PMA in the presence or absence of 1000 UÆml )1 of IFN-a were analyszed. PKR was isola- ted using the affinity resin, polyinosinic–polycytidylic acid (poly I:C) agarose, and the complex was incuba- ted with a reaction mixture containing [ 32 P]ATP[cP]. As shown in Fig. 1, treatment with 10 ngÆml )1 PMA resulted in a time-dependent decrease in PKR auto- phosphorylation. At 60 min, PMA caused a decrease of the autophosphorylation of PKR to 15% of its induced level. PMA induced downregulation of PKR autophosphorylation was dose dependent (Fig. 2), with maximal reduction observed at 10 ngÆmL )1 , and lesser effects at 20 and 30 ngÆmL )1 , respectively. This result is consistent with the observation in our previous study on the effects of PMA on RNase L [25]. Similar results were also obtained after the treatment of NIH3T3 cells Fig. 1. Double-stranded RNA-dependent protein kinase (PKR) auto- phosphorylation is downregulated by PMA in a time-dependent manner. (A) L929 cells were stimulated with 1000 UÆmL )1 IFN-a for 14 h and then treated with 10 ngÆmL )1 PMA for 5, 30 and 60 min. PKR was isolated using poly I:C–Agarose beads and incubated with [ 32 P]ATP[cP] at 30 °C for 30 min. Phosphorylated PKR was separ- ated by SDS ⁄ PAGE on 10% gels and subjected to autoradiography. (B) PKR autophosphorylation was quantified by PhosphorImager analysis. The results represent data from three separate experi- ments. Y. Zhou et al. Control of protein kinase R degradation FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1569 with PMA in the presence of 1000 UÆml )1 of IFN-a (Fig. 3). These experiments indicate that PMA treat- ment decreases the autophosphorylation of PKR and that this activity is not cell line specific. Poly I:C is a potent inducer of PKR expression and activity in different types of cells. We examined the effect of poly I:C on the downregulation of PKR autophosphorylation by PMA. L929 cells were incuba- ted with 1 lgÆml )1 of poly I:C in the presence of 1000 UÆml )1 of IFN-a for 14 h and treated with or without PMA for 60 min. Poly I:C synergistically induced PKR expression with IFN-a in the cells. How- ever, the relative PMA-induced decrease of autophos- phorylated PKR levels in cells treated with (lane 3 vs. lane 5) or without (lane 2 vs. lane4) poly I:C is almost same (Fig. 4). There was a 3.6-fold decrease in the presence of poly I:C and a 3.4-fold decrease in the absence of poly I:C after PMA treatment. Therefore, this result showed that the downregulation of PKR autophosphorylation by PMA is poly I:C-independent. Following dsRNA binding and autophosphoryla- tion, PKR phosphorylates several cellular substrates. The best characterized one of these is eIF-2a.To determine the effect of PMA-induced PKR downregu- lation on a biologically relevant substrate, we exam- ined the phosphorylation status of eIF-2a in L929 cells. Western blot analysis indicated that while the levels of total eIF-2a and phosphorylated eIF-2a were essentially equivalent in L929 cells treated with or without IFN-a, there was a significant increase of Fig. 2. PKR autophosphorylation is downregulated by PMA in a dose-dependent manner. (A) L929 cells were stimulated with 1000 UÆmL )1 IFN-a for 14 h and then treated with 10, 20 and 30 ngÆmL )1 PMA for 60 min. PKR autophosphorylation was deter- mined as described above. (B) PKR autophosphorylation was quan- tified by PhosphorImager analysis. The results represent data from three separate experiments. Fig. 3. PMA treatment downregulates PKR autophosphorylation in NIH3T3 cells. (A) NIH3T3 cells were stimulated with 1000 UÆmL )1 IFN-a for 14 h, or left unstimulated, and then treated with 10 ngÆmL )1 PMA for 60 min. PKR autophosphorylation was ana- lysed as described above. (B) PKR autophosphorylation was quanti- fied by a densitometry. Control of protein kinase R degradation Y. Zhou et al. 1570 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS phosphorylated eIF-2a, but not eIF-2a protein in the cells treated with dsRNA. Importantly, treatment of dsRNA-induced cells with PMA resulted in a signifi- cant reduction in the level of phosphorylated eIF-2a (Fig. 5). Thus, the PMA mediated downregulation of PKR results in reduced activity towards a key endo- genous substrate. PMA treatment has temporally distinct effects on the activation of PKC. A short-term treatment of cells with PMA results in the activation of PKC, but long- term incubation of cells with PMA depletes PKC protein through the ubiquitin-proteasome pathway [26]. To test if PKC is responsible for the downregula- tion of PKR, L929 cells were treated with 1000 UÆml )1 of IFN-a plus 10 ngÆml )1 PMA for 14 h or IFN-a alone for 14 h, and then 10 ngÆml )1 PMA for 60 min. Short-term treatment of cells with PMA caused a 2.5- fold of PKR autophosphorylation when compared to long-term treatment (Fig. 6). This observation suggests that PKC is involved in the downregulation of PKR autophosphorylation in the cells. To determine further the role of PKC in the downregulation of PKR auto- phosphorylation and investigate the level at which PKR is regulated by PKC, we used GO ¨ 6983, a general PKC inhibitor that can inhibit several PKC isoforms. L929 cells were treated with PMA in the presence of GO ¨ 6983, and PKR levels were determined by western blot analysis probed with a monoclonal antibody to mouse PKR. Interestingly, PMA treatment induced the degradation of PKR protein and the PKC inhib- itor effectively blocked this event, suggesting that PKC-mediated phosphorylation is responsible for the decreased levels of autophosphorylated PKR through the degradation of PKR protein (Fig. 7A). Treatment of cells with the PKC inhibitor alone did not have any Fig. 4. Downregulation of PKR autophosphorylation is independent of PKR activation status. L929 cells were incubated in the presence of 1000 UÆmL )1 IFN-a alone or IFN-a plus 1 lgÆmL )1 poly I:C for 14 h and then treated 10 ngÆmL )1 PMA for 60 min, or left untreated. PKR autophosphorylation was analysed as described above. Fig. 5. PMA treatment disrupts PKR catalytic function. L929 cells were incubated with 1000 UÆmL )1 IFN-a for 14 h before treatment with 10 ngÆmL )1 PMA for 60 min. The cells were then transfected with 1 lgÆmL )1 poly I:C using Lipofectamine reagent. The phos- phorylation of eIF-2a was determined by western blot analysis using monoclonal antibodies to phospho-eIF-2a and total eIF-2a. Fig. 6. Depletion of PKC reduces the downregulation of PKR auto- phosphorylation. L929 cells were untreated (lane 1) or treated with dimethyl sulfoxide (lane2), 1000 UÆ mL )1 IFN-a (lane 3), 1000 UÆmL )1 IFN-a plus 10 ngÆ mL )1 PMA for 14 h (lane 4); 1000 UÆmL )1 IFN-a for 14 h prior to PMA treatment for 60 min (lane 5). PKR autophosphorylation was determined as described above. Y. Zhou et al. Control of protein kinase R degradation FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1571 effect on PKR levels (Fig. 7B). To confirm this result further we treated NIH3T3 cells with two additional PKC inhibitors, GO ¨ 6976 and Rottlerin. As shown in Fig. 7C, GO ¨ 6976 effectively blocked the PMA-induced degradation of PKR, but not Rottlerin, suggesting that PKC-a and b may play a role in this event. The degradation of certain proteins by PMA treat- ment is via a proteasome-dependent mechanism [27]. To test if the downregulation of PKR levels in L929 cells by PMA requires an active proteasome, we used the selective inhibitors of proteasomal proteases, ALLN, MG132 or PSI. The cells were incubated with these inhibitors for 4 h prior to PMA treatment. Pre-incuba- tion of cells with the proteasome inhibitors completely prevented the degradation of PKR caused by PMA (Fig. 8). Proteasome inhibitors induce apoptosis in dif- ferent types of cells [28]. However, these proteasome inhibitors do not induce apoptosis in L929 cells [25]. These experiments suggest that an active proteasome is required for the PMA-induced degradation of PKR. Discussion Studies have revealed that PKR is involved a wide range of biological activities. The structure and function of PKR have been well characterized. However, relatively less work has been done on the regulation of PKR at the transcriptional or post-translational levels. Our results reported here provide the first evidence that PKR protein can be downregulated by PMA, a well-known PKC activator, through proteasome-mediated degrada- tion. As shown in Fig. 7A, GO ¨ 6983, a general inhibitor of PKC completely blocked the degradation of PKR induced by PMA, implicating the involvement of PKC activation. We have further confirmed this observation by using other PKC inhibitors, such as GO ¨ 6976 and Rottlerin. GO ¨ 6976 displays a better inhibitory role in PMA-induced PKR degradation (Fig. 7C), suggesting the involvement of PKC-a and b. Although we have not determined if the PKC-mediated PKR degradation is a direct or indirect effect, our findings raise the possibility that PKR is a substrate of PKC. Katze and colleagues have reported that PKR degradation occurs during poliovirus infection and demonstrated that preincubation of cell extracts with poly I:C, a synthetic dsRNA, largely prevented PKR proteolysis, suggesting that the degradation of PKR during viral infection does not require PKR Fig. 7. Activation of PKC is responsible for the degradation of PKR induced by PMA in L929 cells. (A) L929 cells were incubated in the presence of 1000 UÆmL )1 IFN-a for 14 h and treated with or with- out GO ¨ 6983 (10 n M) for 2 h prior to PMA treatment (10 ngÆmL )1 ) for various times. PKR was detected by western blot analysis probed with a monoclonal antibody to mouse PKR. (B) L929 cells were incubated with 1000 UÆmL )1 IFN-a for 14 h and then treated with GO ¨ 6983 (10 n M) for 30, 60 and 90 min. PKR levels were determined by western blot analysis as described above. (C) NIH3T3 cells were incubated in the presence of 1000 UÆmL )1 IFN-a for 14 h and treated with or without GO ¨ 6976 (1 l M) and Rottlerin (5 l M) for 2 h prior to PMA treatment (10 ngÆmL )1 ) for 30 min. PKR was detected as described above. Fig. 8. PKR degradation induced by PMA is proteasome-dependent L929 cells were incubated with different selective proteasome inhibitors, ALLN (10 l M), MG132 (10 lM) and PSI (1 lM), for 4 h prior to PMA treatment of 60 min. PKR protein levels in the cells were analysed by western blot analysis probed with antibodies to mouse PKR and b-actin. Control of protein kinase R degradation Y. Zhou et al. 1572 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS activation and autophosphorylation [29]. Further- more, cells transfected with poly I:C using the Lipo- fectamine plus reagent for 4 h displayed a markedly high level of autophosphorylated PKR, suggesting that autophosphorylated PKR is not the target of the proteasome and is relatively stable [5]. Viral infection results in the activation of PKC in cells [30–32]. Entry of influenza viruses into cells is inhib- ited by a highly specific PKC inhibitor [33]. Other viruses including vesicular stomatitis virus, herpes simplex I and vaccinia virus, are also inhibited by H7, a broader PKC inhibitor [34], indicating that the activation of PKC in cells by virus is important for virus to infect host cells. Taken together, these observations and our results suggest a possibility that virus may counteract the antiviral activity of PKR in cells through the activation of PKC, resulting in the degradation of PKR protein. Therefore, investigating the regulation of PKR by PKC may provide useful information for designing therapeutic methods for viral infectious diseases. Autophosphorylation is the first step in converting PKR to its active form. Several critical autophospho- rylation sites in human PKR have been identified, including S242, S448, T255, T258, T446 and T451. Autophosphorylated PKR in cells infected by virus or transfected by dsRNA is relatively stable [5,35]. There- fore, if PKR is indeed a PKC substrate, it is unlikely that PKC-mediated phosphorylation sites are the same as the autophosphorylation sites on PKR. Mouse PKR has 74 serine ⁄ threonine amino acids and human PKR has 87 serine ⁄ threonine amino acids. Should PKR prove to be a direct target of PKC, identifying the location of PKC-phosphorylated serine ⁄ threonine residues on PKR and mutating these amino acids will be very important for studying the role of PKR in growth suppression, apoptosis and antiviral infection. It is possible that the effect of PMA on the downregu- lation of PKR is indirect. For example, a PKR interact- ing protein (several have been identified [1]), may be phosphorylated in response to PMA and, in turn, recruit PKR to the proteasome. Phosphorylation of target pro- teins promotes ubiquitylation and accelerates protea- some mediated proteolysis. PKC is involved in inducing a wide range of proteasome-mediated protein degrada- tion, including p21(WAF1 ⁄ CIP1), STAT3, IjB, estro- gen receptor and beta-amyloid precursor [26,27,36–39]. Interestingly, we have been unable to observe the higher molecular weight bands of ubiquitylated PKR in the presence of proteasome inhibitors after the cells were treated with PMA. This observation is similar to the result obtained by Halvorsen and colleagues for the transcription factor STAT3 [27]. Thus, PMA-induced PKR degradation is likely to involve proteasome- dependent degradation. However, whether or not PKR is directly ubiquitylated requires further investigation. PMA treatment causes a degradation of STAT2 and STAT3 in neuroblastoma cells through the PKC- dependent phosphorylation and the proteasome path- way, suggesting a role of PMA in the regulation of cytokine signalling transduction [27]. Furthermore, Larner and colleagues have reported that the inhibi- tory effect of PMA on the IFN signalling pathway results in suppression of the expression of IFN-stimu- lated genes [40]. Previously, we reported that the deg- radation of RNase L (an IFN inducible gene) induced by PMA in L929 cells is PKC dependent [25]. RNase L and PKR are important enzymes in the molecular mechanism of IFN functions against viral infection and cellular proliferation. RNase L displays its antiproliferative effects in cells through regulating the expression of genes at the post-transcripational levels, whereas PKR works at the translational levels. It is possible that the tumour promoting activity of PMA may be mediated, in part, through downregulating the products of tumour suppressor genes, such as RNase L and PKR, providing a preferable environment for tumour growth. Further study of the disruption of IFN-stimulated gene products by PMA in cells may shed new light on the molecular mechanism by which this agent promotes tumorigenesis. Experimental procedures Cell culture and treatments Murine L929 and NIH3T3 cells (ATCC, Manassas, VA, USA) were grown in DMEM (Cleveland Clinic Foundation Core Facility) supplemented with 10% fetal bovine serum (Biosource, Camarillo, CA, USA) and antibiotics in a humidified atmosphere of 5% CO 2 at 37 °C. The cells were grown to 90% confluence and incubated with 1000 UÆmL )1 murine IFN-a (R & D Systems, Minneapolis, MN, USA) for 14 h. Cells then were treated with or without 10 ngÆmL )1 PMA (Sigma, St. Louis, MO, USA) for various times. Cells were incubated with selective proteasome inhibitors, ALLN, MG132, and PSI (Calbiochem, La Jolla, CA, USA) for 4 h prior to PMA treatment as indicated. The PKC inhibitor, GO ¨ 6983, GO ¨ 6976 and Rottlerin were purchased from Calbiochem. Poly I:C was purchased from Sigma. Preparation of cellular extracts Cells were harvested by washing twice with ice-cold phos- phate-buffered saline (NaCl ⁄ P i ) and collected with a scraper. Cytoplasmic extracts were prepared by suspension of cell Y. Zhou et al. Control of protein kinase R degradation FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS 1573 pellets in NP-40 lysis buffer [10 mm Tris ⁄ HCl pH 8.0, 5 mm Mg(OAc) 2 ,90mm KCl, 0.2 mm phenylmethylsulfonyl fluor- ide, 100 unitsÆmL )1 aprotinin, 10 lgÆmL )1 leupeptin and 2% NP-40). After centrifugation at 10 000 g in a microcentrifuge at 4 °C for 10 min, the supernatant was removed and stored at )80 °C. Autophosphorylation assay Cell extracts were incubated with poly I:C–Agarose (Roche) prewashed with 1 · DBG buffer [20 mm Tris ⁄ HCl pH 7.5, 50 mm KCl, 5 mm Mg(OAc) 2 , 0.2 mm phenylmethylsulfo- nyl fluoride, 100 unitsÆ mL )1 aprotinin, 10 lgÆmL )1 leupep- tin, 7 mm mercaptoethanol, and 10% glycerol] on ice for 1 h. After centrifugation, the beads were washed three times with NP-40 lysis buffer as described above. The washed beads were incubated in NP-40 buffer containing 2 mm MnCl 2 ,1lm cold ATP and 1 lm [ 32 P]ATP[cP] (50 CiÆm- mol )1 , Amersham, Piscataway, NJ, USA) at 30 °C for 30 min. Proteins were separated by SDS ⁄ PAGE on 10% gels and the autphosphorylated PKR was detected by auto- radiography. Western blot analysis Cellular extracts (150 lgÆsample )1 ) were fractionated by SDS ⁄ PAGE on 10% gels and transferred to PVDF mem- branes (Millipore, Bedford, MA, USA). Western blot analysis was performed using a monoclonal antibody to mouse PKR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1 : 2000 and PKR was detected by a chemiluminescence method according to the manufac- turer’s specification (Amersham). The phosphorylated eIF-2a antibody was from Cell Signaling Technology, Beverly, MA, USA, and the total eIF-2a antibody was from Santa Cruz Biotechnology. 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Mol Cell Biol 16, 1419–1424. Control of protein kinase R degradation Y. Zhou et al. 1576 FEBS Journal 272 (2005) 1568–1576 ª 2005 FEBS . Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester Yan Zhou 1 , Barbara I. Chase 1 ,. treatment of NIH3T3 cells Fig. 1. Double-stranded RNA-dependent protein kinase (PKR) auto- phosphorylation is downregulated by PMA in a time-dependent manner.