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• W1978340521 abstract "Article3 August 1998free access PACT, a protein activator of the interferon-induced protein kinase, PKR Rekha C. Patel Rekha C. Patel Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Ganes C. Sen Corresponding Author Ganes C. Sen Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Rekha C. Patel Rekha C. Patel Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Ganes C. Sen Corresponding Author Ganes C. Sen Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Author Information Rekha C. Patel1 and Ganes C. Sen 1 1Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4379-4390https://doi.org/10.1093/emboj/17.15.4379 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PKR, a latent protein kinase, mediates the antiviral actions of interferon. It is also involved in cellular signal transduction, apoptosis, growth regulation and differentiation. Although in virus-infected cells, viral double-stranded (ds) RNA can serve as a PKR activator, cellular activators have remained obscure. Here, we report the cloning of PACT, a cellular protein activator of PKR. PACT heterodimerized with PKR and activated it in vitro in the absence of dsRNA. In mammalian cells, overexpression of PACT caused PKR activation and, in yeast, co-expression of PACT enhanced the anti-growth effect of PKR. Thus, PACT has the hallmarks of a direct activator of PKR. Introduction The interferon (IFN)-induced, double-stranded (ds) RNA-activated protein kinase (PKR), is a key mediator of the antiviral and antiproliferative effects of IFN (Hovanessian, 1989; Sen and Ransohoff, 1993; Williams, 1995; Clemens and Elia, 1997). PKR is present at low constitutive levels in cells and its expression can be induced by treatment with IFN. The most well known activator of PKR is dsRNA, but other polyanionic agents such as heparin have also been shown to activate it in vitro (Galabru and Hovanessian, 1987; Hovanessian and Galabru, 1987). The only known physiological substrate of PKR activity is the α subunit of the eukaryotic initiation factor eIF-2 (Lengyel, 1993; Samuel, 1993). Phosphorylation of eIF-2α on Ser51 by PKR leads to inhibition of protein synthesis (Colthurst et al., 1987; Hershey, 1991). eIF-2α phosphorylation leads to its increased affinity for eIF-2B, thus sequestering eIF-2B in an inactive complex with phosphorylated eIF-2 and GDP (Clemens et al., 1994; De Haro et al., 1996). As a result, eIF-2B is not available to catalyze nucleotide exchange on non-phosphorylated eIF-2. Because eIF-2B is present in cells at a lower molar concentration than eIF-2, phosphorylation of a small fraction of cellular eIF-2α can lead to a severe block in protein synthesis. Upon viral infection of IFN-treated cells, PKR is activated by viral dsRNA which leads to an inhibition of viral and cellular protein synthesis (Samuel et al., 1984; Rice et al., 1985). Thus, PKR plays a major role in the antiviral activity of IFN. The importance of antiviral effects of PKR is manifested by the fact that many viruses employ a variety of mechanisms to counteract the actions of PKR (Sonenberg, 1990; Katze, 1992). Although the majority of research on PKR has focused on its participation in the inhibitory action of IFN on viral infection, there is now substantial evidence for PKR's role in uninfected cells as well (Proud, 1995). PKR has been implicated in several diverse cellular functions such as growth regulation, apoptosis, differentiation and signaling pathways (Proud, 1995; Williams, 1997). A potential role for PKR in cell growth regulation has been suggested by the fact that overexpression of human PKR inhibits cell proliferation in yeast (Chong et al., 1992), insect (Barber et al., 1992) and mammalian cells (Koromilas et al., 1992). On the other hand, expression of catalytically inactive mutants of PKR in NIH 3T3 cells results in tumorigenicity in nude mice (Koromilas et al., 1992; Meurs et al., 1993). This result has been ascribed to a transdominant inhibitory effect of mutant enzyme on the endogenous wild-type PKR, resulting in derepression of growth. It is relevant to the proposed role of PKR in growth regulation that oncogenic Ras protein has been reported to induce an inhibitor of PKR activation (Mundschau and Faller, 1992, 1994). Surprisingly, mice devoid of functional PKR develop normally and are free of any tumors (Yang et al., 1995), suggesting that a cellular system, absent in NIH 3T3 cells, may be able to replace PKR functionally in the PKR null mice. Several studies have shown the involvement of PKR in cellular apoptosis (Yeung et al., 1996; Kibler et al., 1997). Overexpression of PKR in HeLa cells leads to apoptosis (Lee and Esteban, 1994), and recent studies with mouse embryonic fibroblasts derived from the PKR null mice have also shown its involvement in dsRNA- and lipopolysaccharide (LPS)-induced apoptosis (Der et al., 1997). PKR has also been implicated in the onset of differentiation. Activation of PKR has been shown to be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts and subsequent differentiation to adipocytes (Judware and Petryshyn, 1991, 1992; Petryshyn et al., 1997). However, the identity of an activator of PKR under differentiation permissive conditions has remained unknown. A more recent observation also correlates the levels of active PKR with the degree of cellular differentiation. It was observed that in the myogenic line L8, transforming growth factor-β (TGF-β) and EGTA reduced both the PKR activity and the level of muscle-specific proteins (Salzberg et al., 1995). PKR participates in transcriptional signaling pathways used by dsRNA and IFN-γ: in PKR null cells, dsRNA fails to activate NFκB and IRF-1, and the IFN-γ responses of these cells are also impaired (Maran et al., 1994; Yang et al., 1995; Kumar et al., 1997; Wong et al., 1997). Treatment of cells with IFN-γ has been shown to result in phosphorylation and activation of PKR (Kumar et al., 1997). However, no cellular activator of PKR in response to IFN-γ in the absence of dsRNA has been identified thus far. In addition, PKR has also been implicated in platelet-derived growth factor (PDGF) (Mundschau and Faller, 1995) and interleukin-3 (IL-3) (Ito et al., 1994) signaling. The dsRNA-binding domain (DRBD) of PKR lies near its N-terminus (Katze et al., 1991; Feng et al., 1992; Green and Mathews, 1992; McCormack et al., 1992; Patel and Sen, 1992) and it contains two dsRNA-binding motifs conserved in a large family of dsRNA-binding proteins (St. Johnston et al., 1992). Deletion or specific substitution mutations in this region result in the loss of the dsRNA binding and activation (McCormack et al., 1994; Patel et al., 1994, 1996; Green et al., 1995; McMillan et al., 1995). These mutants can, however, be activated in vitro by heparin (Patel et al., 1994, 1996) and may be functionally active in yeast (McMillan et al., 1995; Romano et al., 1995) and mammalian cells (Lee et al., 1994), suggesting the existence of additional cellular activators of PKR. The N-terminal domain of PKR also mediates its dsRNA-independent dimerization in vitro and in vivo (Cosentino et al., 1995; Patel et al., 1995; Romano et al., 1995; Wu and Kaufman, 1997). Similar dimerization domains are present in other dsRNA-binding proteins, and these proteins can heterodimerize with PKR through these domains (Cosentino et al., 1995; Benkirane et al., 1997). Since dsRNA is thought to be present only in virally infected cells, it is unclear what activates PKR in the absence of virus infection, and potential protein activators of PKR are likely to exist in cells. Here we report the identification of a new protein, PACT, which interacts with PKR through dimerization domains similar to PKR's own and can activate PKR in vitro and in vivo in the absence of dsRNA. Results Cloning of PACT, a new dsRNA-binding protein that interacts with PKR PACT was cloned by virtue of its interaction with PKR. K296R, an enzymatically inactive mutant of PKR (Katze et al., 1991), was used as the bait in a yeast two-hybrid screen of a human cDNA library, and a novel 1.8 kb PKR-interacting cDNA containing an open reading frame encoding a protein of 313 residues was isolated (Figure 1A). PACT was represented in the database only as several expressed sequence tags, and its sequence contains three motifs (Figure 1A, underlined residues) similar to the dimerization motifs present in PKR and other dsRNA-binding proteins. A comparison of the first two motifs of PACT with the first motif of PKR and two motifs of TAR RNA-binding protein (TRBP) (St. Johnston et al., 1992), another human protein of this family, revealed strong sequence conservation (Figure 1B). Primer extension analysis of cellular mRNA confirmed that the clone represented a full-length cDNA (data not shown), and Northern analysis showed a corresponding mRNA of 2.0 kb. PACT mRNA was expressed at varying levels in all of the cell lines tested (Figure 1C). Since PKR is an IFN-inducible protein, it was of interest to investigate if either IFN or dsRNA treatment had any effect on PACT mRNA levels. To test this, we have used the human glioblastoma cell line, GRE, in which the IFN genes have been deleted. This makes it suitable for studying the dsRNA-mediated transcriptional induction because these cells do not induce IFN production upon treatment with dsRNA. The transcriptional induction of PACT was assessed by an RNase protection assay. As represented in Figure 1C, PACT mRNA levels were the same in untreated cells as in cells treated with IFN-β, -γ or dsRNA. As expected, the 561 mRNA was induced both by IFN-β and dsRNA treatments, and the IRF-1 mRNA was induced by treatment with IFN-γ (Leonard and Sen, 1997). Figure 1.(A) Protein sequence of PACT. The deduced amino acid sequence of PACT cDNA is represented. The conserved dsRNA-binding motifs are underlined. (B) Alignment of the three human protein sequences that contain the dsRNA-binding domain. Each of the three proteins has the full-length domain and C-terminal short domains. The shaded residues indicate amino acids that are identical in at least two motifs. The consensus sequence derived from St. Johnston et al. (1992) is shown below. Upper case letters indicate residues that occur in at least three motifs, and lower case letters indicate residues shared by two motifs or conservative substitutions. The sequences are as follows: PACT-1, PACT 35–99; TRBP-1, human TAR-binding protein 10–74; PKR-1, human dsRNA-activated kinase 10–75; PACT-2, PACT 127–192; TRBP-2, 139–204. The similarities between PACT, TRBP and PKR were identified initially in searches of the GenBank and EMBL databases. The multiple motifs in these sequences subsequently were aligned using the MegAlign v3.05 (DNASTAR Inc.) program. The following nucleic acid sequences were used to obtain protein sequences: human TRBP, M60801; human PKR, M35663. (C) Northern blot analysis. A multiple human cancer cell line blot (Clontech) was hybridized to random primer-labeled PACT cDNA insert. Each lane contains ∼2 μg of poly(A)+ RNA isolated from the following cell lines; lane 1, melanoma G361; lane 2, lung carcinoma A549; lane 3, colorectal adenocarcinoma SW 480; lane 4, Burkit's lymphoma Raji; lane 5, lymphoblastic leukemia MOLT-4; lane 6, chronic myelogenous leukemia K- 562; lane 7, HeLa S3; and lane 8, promyelocytic leukemia HL-60. (D) Ribonuclease protection assay. Total RNA isolated from untreated GRE cells, or GRE cells treated for 6 h with IFN β (500 U/ml), IFN-γ (500 U/ml) or 100μg/ml of poly(I)·poly(C) was analyzed by RNase protection assay. Thirty μg of total RNA was hybridized with 32P-labeled PACT (296 bases), IRF-1 (220 bases), 561 (195 bases) and γ-actin (140 bases) antisense RNA probes. Following RNase digestion, the protected RNA probes (PACT, 196 bases; IRF-1, 175 bases; 561, 142 bases; and γ-actin, 135 bases) were resolved in a 6% polyacrylamide, 8 M urea gel and visualized by autoradiography. The positions of the undigested probes are indicated on the left and the positions of the protected fragments are indicated on the right. The RNA samples are as indicated on the top of the lanes. Download figure Download PowerPoint PACT interacts with PKR through its dsRNA-binding motifs The interaction of PACT with PKR was confirmed by co-immunoprecipitation of the in vitro translated products. 35S-Labeled, flag epitope-tagged PACT protein was synthesized by in vitro translation (Figure 2A, lane 1). This protein could be immunoprecipitated using anti-flag monoclonal antibody (mAb)–agarose (Figure 2A, lane 2), but not by agarose alone (lane 3). The specificity of this immunoprecipitation is shown by the fact that PKR protein, similarly synthesized, was not immunoprecipitated either with the anti-flag mAb–agarose or with agarose alone (lanes 5 and 6). Since flag-PACT could be immunopecipitated specifically, we then assayed for PKR's interaction with PACT by co-immunoprecipitation and mapped the PACT-interacting domain within PKR. PKR could be co-immunoprecipitated with flag epitope-tagged PACT (Figure 2B, lane 5). A similar interaction was also observed between DRBD, the dimerization domain of PKR, and PACT (lane 6). Δ170, the C-terminal half of PKR (residues 171–551), did not co-immunoprecipitate with PACT (lane 7), thereby indicating that PACT interacts with PKR through its DRBD. The specificity of these co-immunoprecipitations was confirmed by the fact that another dsRNA-binding protein, the 69 kDa 2′–5′ oligoadenylate synthetase (Marie et al., 1997), did not co-immunoprecipitate with PKR. We further checked if when a fixed amount of PACT protein was mixed with increasing amounts of PKR protein, the same stoichiometric ratio was maintained in the co-immunoprecipitations. Since we were using flag-PACT to pull down untagged PKR, we varied the amount of PKR in the mixture while keeping the amount of flag-PACT constant. As seen in Figure 2C (lanes 6–10), a fixed amount of PACT was able to co-immunoprecipitate increasing quantities of PKR from the mixtures containing increasing amounts of PKR. About 30% of the total PKR protein could be co-immunoprecipitated from the mixture at all ratios. The amount of PKR co-immunoprecipitated with PACT followed the same ratio at which these two proteins were mixed. The ratios at which these proteins were mixed (lanes 1–5) were 1, 2, 5, 10 and 30, and the ratios of the immunoprecipitated PKR (lanes 6–10) were 1, 1.8, 4.25, 9.4 and 21.3. Figure 2.(A) In vitro translation and immunoprecipitation of flag-tagged PACT. In vitro translated, 35S-labeled wild-type PKR and flag-PACT were synthesized using the TNT T7 coupled rabbit reticulocyte lysate system from Promega. Three μl of the reticulocyte lysate proteins were immunoprecipitated using anti-flag mAb–agarose (IBI). Total lanes represent proteins from 1 μl of the lysate, and the anti-flag mAb and agarose lanes represent the proteins immunoprecipitated from 3 μl of the lysates. The positions of the molecular weight markers are indicated on the left. The positions of the two proteins are as indicated on the right. (B) PACT interacts with PKR through its DRBD. In vitro translated, 35S-labeled proteins were synthesized using the TNT T7 coupled rabbit reticulocyte lysate system from Promega. Flag-tagged PACT, wild-type PKR, Δ170, 2′–5′ oligoadenylate synthetase and DRBD were translated independently. Three μl of the reticulocyte lysate containing the flag-PACT were mixed with 3 μl of the lysates containing either wild-type PKR, Δ170, 2′–5′ oligoadenylate synthetase or DRBD. Flag-tagged PACT was immunoprecipitated from the reticulocyte lysate using the anti-flag mAb–agarose (IBI), and the proteins co-immunoprecipitating with it were analyzed by SDS–PAGE analysis followed by fluorography. Lanes 1–4 show all proteins in the mixture before immunoprecipitation, and lanes 5–8 represent immunoprecipitated proteins. Lanes 1 and 5, wild-type PKR and flag-PACT; lanes 2 and 6, flag-PACT and DRBD; lanes 3 and 7, flag-PACT and Δ170; lanes 4 and 8, flag-PACT and 2′–5′ oligoadenylate synthetase. The ‘total’ lanes contain 1 μl of the reticulocyte lysates each and the ‘CO-IP’ lanes contain 3 μl of the lysates each. The positions of the molecular weight markers are indicated on the right. The positions of different proteins are as indicated on the left. (C) Dose-dependent association between PKR and PACT. In vitro translated, 35S-labeled wild-type PKR and flag-PACT proteins were mixed in different proportions. Two μl of the reticulocyte lysate containing the flag-PACT was mixed with 0.1–3 μl of the lysate containing wild-type PKR. Flag-tagged PACT was immunoprecipitated from the reticulocyte lysate using the anti-flag mAb–agarose (IBI), and the wild-type PKR co-immunoprecipitating with it was analyzed by SDS–PAGE analysis followed by fluorography. The relevant bands were quantitated by phosphorimager analysis. Lane 1, 3 μl of flag-PACT + 0.1 μl of wild-type PKR; lane 2, 3 μl of flag-PACT + 0.2 μl of wild-type PKR; lane 3, 3 μl of flag-PACT + 0.5 μl of wild-type PKR; lane 4, 3 μl of flag PACT + 1 μl of wild-type PKR; lane 5, 3 μl of flag-PACT + 3 μl of wild-type PKR. The positions of PKR and flag-PACT are as indicated on the right. Download figure Download PowerPoint PACT binds dsRNA Since the same motifs of PKR that mediate interaction with PACT also mediate dsRNA binding by PKR, we examined the ability of PACT to bind dsRNA (Figure 3). This was assayed by a poly(I)·poly(C)–agarose binding assay previously described for assaying the binding of PKR to dsRNA (Patel and Sen, 1992). The 35S-labeled in vitro synthesized PACT and PKR proteins were assayed for binding to poly(I)·poly(C)–agarose at 50, 300 and 500 mM salt. As represented in lanes 2–4, PKR bound strongly, showing 54, 50 and 26% binding at 50, 300 and 500 mM salt concentrations, respectively. PACT showed similar binding characteristics, with 40, 31 and 6% binding at these salt concentrations (lanes 6–8). To ascertain the specificity of dsRNA binding by PACT, various nucleic acids were added in excess during the binding step as competitors (lanes 9–12). The binding was not affected by single-stranded RNA and DNA or DNA–RNA hybrid but was competed out by dsRNA. Thus, PACT was identified as a new dsRNA-binding protein that could heterodimerize directly with PKR. Figure 3.PACT binds dsRNA. The 35S-labeled PKR and PACT protein were synthesized using the TNT T7 coupled reticulocyte lysate system from Promega. The dsRNA-binding activity was measured by poly(I)·poly(C)–agarose binding assay, using 4 μl of translation products at 50, 300 or 500 mM NaCl concentrations. The proteins bound to beads after washing were analyzed by SDS–PAGE followed by fluorography. The relevant bands were quantitated by phosphorimager analysis. Competition by different nucleic acids for binding of PACT to poly(I)·poly(C)–agarose is shown in the extreme right panel. The competitor nucleic acid was added during the binding reaction at 10 μg/ml concentration. The lanes are labeled on top with the competitor used. Lanes 1–4, poly(I)·poly(C)–agarose binding activity of PKR; lanes 5–8, poly(I)·poly(C)–agarose binding activity of PACT; and lanes 9–12, competition of PACT's poly(I)·poly(C)–agarose binding activity. The positions of the molecular weight markers are indicated on the right. Download figure Download PowerPoint PACT activates PKR in a dsRNA-independent manner To determine if PACT could affect the activity of PKR, polyhistidine-tagged PACT was expressed in Escherichia coli and purified under denaturing conditions in the presence of 6 M urea, to prevent RNA binding to PACT. The validity of this procedure was confirmed by adding 32P-labeled dsRNA to the bacterial lysate before purification. Under native conditions, 90% of the added dsRNA co-purified with PACT, but no detectable radioactivity was associated with PACT that had been purified in the presence of 6 M urea. From the specific activity of the labeled dsRNA that was added to the bacterial lysate, it was calculated that 10−13 g of dsRNA may have been associated with PACT after purification under denaturing conditions. This amount of dsRNA is far less (∼104-fold less) than that required for activation of PKR. Denatured PACT, thus purified, was renatured by stepwise dialysis against decreasing concentrations of urea. The purified renatured PACT was homogeneous as confirmed by silver staining after SDS–PAGE analysis (data not shown). Purified PACT, devoid of any associated RNA, was incubated with immunopurified PKR, eIF-2 and [γ-32P]- ATP. In the absence of activators, neither PKR nor eIF-2 was phosphorylated (Figure 4A, lane 1), whereas the addition of two known activators, dsRNA and heparin, caused phosphorylation of both proteins (lanes 8 and 9). PACT could also activate PKR, causing the phosphorylation of both PKR and eIF-2. PKR activation by PACT was dose-dependent and biphasic: the maximum activation was observed between 400 pg and 4 ng of PACT, which was similar to that achieved by 1 μg/ml of dsRNA (lane 8). These results clearly demonstrated that PACT could activate PKR directly. To confirm further that PACT activates PKR without the involvement of any dsRNA, we assayed the ability of heat-inactivated and micrococcal nuclease-treated PACT to activate PKR (Figure 4B, lanes 3 and 4). Heat inactivation is expected to result in loss of activity due to protein denaturation, but the dsRNA-dependent activation will be unaffected by this treatment. As expected, the heat inactivation resulted in a loss of PACT's ability to activate PKR (lane 3), while micrococcal nuclease treatment did not (lane 4). To rule out the possibility of any dsRNA remaining protected because it is bound by PACT, we assayed the effect of artificially added dsRNA during the micrococcal nuclease treatment. However, since PACT treated in such a way would activate PKR in the absence of dsRNA, we needed to inactivate PACT by heat after the nuclease treatment. Any protected dsRNA during this treatment could now be assayed for its ability to activate PKR. As seen in lane 5, the artificially added dsRNA was not protected during the nuclease treatment. As seen in lane 6, dsRNA's ability to activate PKR is not destroyed by heat inactivation. When an activating amount of PACT was incubated in the kinase assay buffer with purified K296R, an inactive mutant of PKR, no phosphorylation of K296R protein was detected (data not shown). This result rules out the possibility of a bacterial kinase co-purifying with PACT and phosphorylating PKR. Figure 4.(A) Purified PACT activates PKR. PACT was expressed as a hexahistidine-tagged protein in E.coli, purified by Ni affinity chromatography under denaturing conditions and renatured by stepwise dialysis. A 100 μg aliquot of total protein from HeLa M cells was immunoprecipitated using PKR monoclonal antibody (Ribogene) and PKR assay was performed in activity buffer containing 500 ng of purified eIF-2, 0.1 mM ATP and 10 μCi of [γ-32P]ATP at 30°C for 10 min. Poly(I)·poly(C) (100 ng/ml; lane 8) or heparin (10 U/ml; lane 9) was used as the standard activator for the enzyme. Purified PACT in amounts of 4 pg (lane 2), 40 pg (lane 3), 400 pg (lane 4), 4 ng (lane 5), 40 ng (lane 6) and 400 ng (lane 7) was added to PKR prior to addition of eIF-2 and [γ-32P]ATP to test its effect on PKR activity. PKR activity without any added activator is in lane 1. (B) Heat inactivation of PACT's PKR activating capacity. The PKR activity assay was done as described in (A). The additions to the assay mixture were as follows: lane 1, no activator; lane 2, 4 ng of native PACT; lane 3, 4 ng of heat-inactivated (90°C for 15 min) PACT; lane 4, 4 ng of PACT treated with micrococcal nuclease; lane 5, 4 ng of PACT with 1 ng of poly(I)·poly(C), treated with micrococcal nuclease and then heat inactivated; and lane 6, 1 ng of dsRNA heated at 90°C for 15 min. Download figure Download PowerPoint We previously have identified two mutants of PKR, K150A and A158D, which can neither bind to nor be activated by dsRNA (Patel et al., 1996). These mutants retained their ability to interact with PACT in a co-immunoprecipitation assay (Figure 5A), thereby enabling us to test if they could be activated by PACT. As shown in Figure 5B, these mutants could be activated by PACT, confirming that activation of PKR by PACT can be achieved in the absence of dsRNA binding. Although these mutants are unable to bind dsRNA, it is conceivable that their interaction with PACT may complement this defect, thereby enabling them to bind dsRNA that may be associated with either protein. Since our preparation of purified PACT was devoid of any dsRNA, we needed to ascertain that no dsRNA was associated with the in vitro translated mutant PKR molecules. For this purpose, we added labeled dsRNA to the reticulocyte lysate containing the in vitro translated PKR mutant proteins and analyzed its association with these proteins after the immunoprecipitation. No significant counts were found to be associated with immunoprecipitates. Since neither of the protein partners (PACT and PKR mutants) in the kinase reaction mix had any dsRNA bound to them, it can be concluded that PACT activates K150A and A158D mutants by direct interaction. Figure 5.(A) PACT interacts with the dsRNA binding-defective mutants of PKR. In vitro translated, 35S-labeled proteins were synthesized using the TNT T7 coupled rabbit reticulocyte lysate system from Promega. Flag-tagged PACT was co-translated either with wild-type PKR, K150A or A158D. Three μl of the reticulocyte lysate containing the synthesized proteins were used for immunoprecipitation. The flag-PACT was immunoprecipitated using the anti-flag mAb–agarose (IBI), and the proteins co-immunoprecipitating with it were analyzed by SDS–PAGE analysis followed by fluorography. ‘Total’ lanes show all proteins in the mixture before immunoprecipitation, and CO-IP lanes represent immunoprecipitated proteins. The positions of PKR and flag-PACT proteins are as indicated on the left. (B) PACT can activate dsRNA-unresponsive PKR mutants. Wild-type PKR, K150A mutant and A158D mutant were translated in vitro. Five μl of the reticulocyte lysates were immunoprecipitated with the anti-PKR monoclonal antibody and the immunoprecipitates were assayed for PKR in activity buffer containing 500 ng of purified eIF-2, 0.1 mM ATP and 10 μCi of [γ-32P]ATP at 30°C for 10 min in the presence of [γ-32P]ATP without any added activator or in the presence of either 1 μg/ml of poly(I)·poly(C) or 4 ng of purified PACT. Download figure Download PowerPoint Overexpression of PACT in mammalian cells leads to activation of PKR, enhanced phosphorylation of eIF-2α and inhibition of translation To determine if PACT could activate PKR in vivo, an expression construct of flag-tagged PACT was transfected into human HT1080 cells and co-immunoprecipitation experiments performed. The endogenous cellular PKR could be co-immunoprecipitated with transfected PACT protein (Figure 6A). PKR precipitated from PACT-expressing cells was more active, as judged by autophosphorylation and eIF-2α phosphorylation (Figure 6B), than PKR from the vector control. As expected, PKR from the K296R-transfected cells was even less active than the vector control, since K296R inhibits PKR activity. Western blotting confirmed that all immunoprecipitates contained similar amounts of PKR. Figure 6.(A) PKR can be co-immunoprecipitated with PACT from mammalian cells. HT 1080 cells were transfected in 100 mm culture dishes with 5 μg of pCB6+ vector and flag-PACT/pCB6+ DNAs. At 24 h post-transfection, cells were harvested and cell extracts were prepared. A 100 μg aliquot of total cell extracts was used to immunoprecipitate flag-PACT with anti-flag mAb–agarose as described in Materials and methods. The immunoprecipitates were then analyzed by a Western blot analysis with the anti-PKR and anti-flag polyclonal antibodies (Santa Cruz Biotech. Inc.). (B) Expression of PACT in mammalian cells leads to PKR activation. HT 1080 cells were transfected in 100 mm culture dishes with 5 μg of pCB6+ vector, flag-PACT/pCB6+ or K296R/pCB6+ DNAs. At 24 h post-transfection, cells were harvested and 100 μg of total cell extracts were used to immunoprecipitate PKR with monoclonal antibody. The immunoprecipitates were assayed for kinase activity in the presence of [γ-32P]ATP and purified eIF-2. No activator of PKR was added to these kinase assays. Western blot analysis performed with anti-PKR monoclonal antibody with the extracts is shown in the lower strip. The order of samples is the same in all three strips. Download figure Download PowerPoint The above results suggested that PACT could activate PKR in vivo. This was confirmed by examining the level of phosphorylation of PKR and eIF-2 in vivo in the transfected cells. The cells t" @default.
• W1978340521 created "2016-06-24" @default.
• W1978340521 creator A5028524295 @default.
• W1978340521 creator A5084601919 @default.
• W1978340521 date "1998-08-03" @default.
• W1978340521 modified "2023-10-16" @default.
• W1978340521 title "PACT, a protein activator of the interferon-induced protein kinase, PKR" @default.
• W1978340521 cites W116205901 @default.
• W1978340521 cites W1485999498 @default.
• W1978340521 cites W1537683572 @default.
• W1978340521 cites W1545795848 @default.
• W1978340521 cites W1548937543 @default.
• W1978340521 cites W1549105044 @default.
• W1978340521 cites W1567253068 @default.
• W1978340521 cites W1569857352 @default.
• W1978340521 cites W1575516617 @default.
• W1978340521 cites W1579430004 @default.
• W1978340521 cites W1582095946 @default.
• W1978340521 cites W1596444914 @default.
• W1978340521 cites W179070074 @default.
• W1978340521 cites W1792328490 @default.
• W1978340521 cites W1845690770 @default.
• W1978340521 cites W1878018656 @default.
• W1978340521 cites W1895900311 @default.
• W1978340521 cites W1915631065 @default.
• W1978340521 cites W1970812281 @default.
• W1978340521 cites W1974129290 @default.
• W1978340521 cites W1975967072 @default.
• W1978340521 cites W1977824741 @default.
• W1978340521 cites W1981348971 @default.
• W1978340521 cites W1985703111 @default.
• W1978340521 cites W1989136432 @default.
• W1978340521 cites W1992583586 @default.
• W1978340521 cites W1996826162 @default.
• W1978340521 cites W2001068917 @default.
• W1978340521 cites W2001464207 @default.
• W1978340521 cites W2005968120 @default.
• W1978340521 cites W2010623923 @default.
• W1978340521 cites W2012690458 @default.
• W1978340521 cites W2014851246 @default.
• W1978340521 cites W2015686762 @default.
• W1978340521 cites W2021174259 @default.
• W1978340521 cites W2022846624 @default.
• W1978340521 cites W2023530592 @default.
• W1978340521 cites W2024437842 @default.
• W1978340521 cites W2027565750 @default.
• W1978340521 cites W2040500676 @default.
• W1978340521 cites W2042159800 @default.
• W1978340521 cites W2043442231 @default.
• W1978340521 cites W2044884879 @default.
• W1978340521 cites W2050469761 @default.
• W1978340521 cites W2058982642 @default.
• W1978340521 cites W2068576524 @default.
• W1978340521 cites W2074168438 @default.
• W1978340521 cites W2076767893 @default.
• W1978340521 cites W2076941373 @default.
• W1978340521 cites W2077808289 @default.
• W1978340521 cites W2080028459 @default.
• W1978340521 cites W2088959549 @default.
• W1978340521 cites W2089033636 @default.
• W1978340521 cites W2089232394 @default.
• W1978340521 cites W2089402352 @default.
• W1978340521 cites W2099674663 @default.
• W1978340521 cites W2105746094 @default.
• W1978340521 cites W2118635464 @default.
• W1978340521 cites W2128674562 @default.
• W1978340521 cites W2130104095 @default.
• W1978340521 cites W2150344629 @default.
• W1978340521 cites W2159873784 @default.
• W1978340521 cites W2160414774 @default.
• W1978340521 cites W2165055107 @default.
• W1978340521 cites W2166497605 @default.
• W1978340521 cites W2168409709 @default.
• W1978340521 cites W2169333620 @default.
• W1978340521 cites W2183562487 @default.
• W1978340521 cites W2252225016 @default.
• W1978340521 cites W2411153978 @default.
• W1978340521 cites W2419602391 @default.
• W1978340521 cites W2576734941 @default.
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