Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

pTRS1 inhibits PKR activation by dsRNA.

We began by examining the ability of pTRS1 to inhibit PKR activation in transfected cells. Increasing amounts of a synthetic dsRNA [poly(I·C)] were added to lysates of cells transfected with either a GFP or pTRS1 expression plasmid, and PKR activation was measured by Western blotting. PKR is activated by autophosphorylation on Thr446 (T446) and Thr451 (T451), although phosphorylation of T451 is dependent upon T446 phosphorylation (23, 50). We therefore used PKR T446 phosphorylation as a marker of PKR activation. Low levels of phosphorylated PKR were present in the absence of poly(I·C) in cells expressing GFP, likely due to stress from transfection. Poly(I·C) activated PKR in cells expressing GFP (Fig. 1A). In contrast, the presence of pTRS1 completely suppressed PKR activation, even at the highest doses of poly(I·C). Thus, pTRS1 is sufficient to prevent PKR activation by dsRNA in cell culture.

We next examined the regions of pTRS1 necessary for PKR antagonism. In these experiments, we used a series of plasmids expressing amino- or carboxyl-terminal truncations of pTRS1, as they have previously been characterized in vitro (37, 39) (Fig. 1B and Table 1) and measured PKR T446 phosphorylation after poly(I·C) treatment (Fig. 1C). It is important to note that truncation mutants may unintentionally affect protein folding, and thus negative results using these mutants should be interpreted with caution. As before, full-length pTRS1 blocked activation of PKR by poly(I·C) treatment. However, pTRS1 lacking its amino-terminal 240 or 390 amino acids (pTRS1 240-795 or pTRS1 390-795, respectively), which contain the dsRNA binding domain, did not inhibit PKR activation. Similarly, a pTRS1 mutant lacking the final 116 amino acids (pTRS1 1-679), containing the PKR binding domain, also failed to antagonize PKR. Curiously, the removal of an additional 180 amino acids from the C terminus (pTRS1 1-550) restored PKR inhibition, but further deletion (pTRS1 1-370) again led to an inability to inhibit PKR activation.

Purified pTRS1 and PKR directly interact in vitro, and deletion or mutation of the PKR binding domain ablates the ability of pTRS1 to inhibit PKR during HCMV infection (6, 7, 37). These results suggest that pTRS1 interaction with PKR is critical for PKR antagonism. We therefore tested the ability of each pTRS1 mutant to bind PKR in transfected cells (Fig. 1D). Cells were transfected with vectors expressing pTRS1 mutants together with kinase-dead PKR (K296R), and the presence of pTRS1 in PKR-specific immune complexes was determined by Western blotting. Only full-length pTRS1 bound to PKR. None of the pTRS1 truncation mutants copurified with PKR, even pTRS1 240-795 and pTRS1 390-795, which both contain the previously described PKR binding domain. Interestingly, pTRS1 1-550 inhibited PKR activation (Fig. 1C) but did not bind PKR. These data suggest that pTRS1 binding to PKR is sufficient, but not necessary, to prevent PKR activation by dsRNA. However, as noted above, negative results in this assay should be interpreted cautiously due to the potential effect of the truncations on protein folding.

Relative role of dsRNA and PKR binding by pTRS1 in PKR antagonism.

Amino acids 84 to 246 of pTRS1 contain a noncanonical double-stranded RNA binding domain (RBD) that allows purified pTRS1 to competitively inhibit the binding of purified PKR to dsRNA (39). This function suggests that in addition to inhibiting PKR via a direct interaction, pTRS1 may also inhibit PKR by preventing recognition of its dsRNA ligands (38, 39). Consistent with this idea, the pTRS1 1-550 mutant, which lacks the PKR binding domain, did not copurify with PKR but could still prevent PKR activation in response to dsRNA treatment. To determine if pTRS1 1-550 inhibits PKR by binding to dsRNA, we introduced three amino acid changes in the dsRBD of pTRS1 1-550 (pTRS1 1-550triple) that prevent binding of purified pTRS1 to dsRNA (38). We then measured the ability of this mutant to prevent poly(I·C)-induced PKR activation (Fig. 2A). pTRS1 1-550triple could not prevent PKR activation in response to poly(I·C) treatment. Thus, in the context of the truncated pTRS1 protein (pTRS1 1-550), RNA binding is necessary for PKR antagonism.

In contrast, we found that pTRS1 could still inhibit PKR activation when the same dsRBD mutations were introduced into full-length pTRS1 (pTRS1triple) (Fig. 2A). We reasoned this could be due to pTRS1 binding to PKR through the previously defined PKR binding domain (PBD). We therefore mutated amino acids in pTRS1 necessary for binding to PKR (7) either alone (pTRS1mut1) or in combination with the dsRBD mutations (pTRS1mut1.triple). Mutation of the pTRS1 PBD ablated both binding to PKR (Fig. 2C) and the ability to inhibit poly(I·C)-induced PKR activation, even when the dsRBD was intact (Fig. 2B), suggesting that the primary mechanism of PKR antagonism by full-length pTRS1 is mediated through interactions with PKR. To further explore this hypothesis, we tested if pTRS1 associated with PKR via an RNA intermediate (Fig. 2D). Micrococcal nuclease treatment had no effect on pTRS1 binding to PKR. We also demonstrated that pTRS1 interacts with PKR in HCMV-infected cells, which had not been previously demonstrated (Fig. 2E). Together, these data suggest that pTRS1 has two distinct mechanisms to inhibit PKR activation. In some circumstances, pTRS1 may prevent PKR activation by sequestering dsRNA from PKR, independent of binding to PKR. However, at least in this system, the primary mechanism of PKR inhibition by full-length pTRS1 requires an RNA-independent interaction with PKR.

pTRS1 blocks PKR activation at a step after PKR dimerization.

Binding to dsRNA induces PKR dimerization, which is necessary for PKR activation (3, 51). We thus hypothesized that pTRS1 might block PKR activation by preventing PKR dimerization. To test this hypothesis, cells expressing pTRS1 were cotransfected with plasmids encoding Myc- and Flag-tagged PKR K296R. Catalytically inactive PKR was used in these experiments to limit toxicity. Poly(I·C) was added to cell lysates to induce PKR dimerization, and the ability of Flag-tagged PKR to be copurified with Myc-tagged PKR was measured by immunoprecipitation followed by Western blotting (Fig. 3). As expected, Flag-tagged PKR was copurified with Myc-tagged PKR in the absence of pTRS1. Similar amounts of Flag-PKR associated with Myc-PKR in the presence of pTRS1, suggesting that pTRS1 does not affect PKR homodimer formation. In addition, we detected pTRS1 in the Myc-PKR immune complexes, suggesting that pTRS1 might bind to PKR dimers. These data show that pTRS1 does not inhibit PKR activation by preventing PKR dimerization and suggest that pTRS1 may in fact bind to PKR dimers.

To further test if pTRS1 prevents PKR activation at a step after dimerization, we determined if pTRS1 could bind and inhibit a constitutively dimerized, active PKR. The glutathione S-transferase (GST) protein constitutively forms dimers; replacing the amino-terminal dsRBD of PKR with GST results in a constitutively dimerized, and thus constitutively active, PKR kinase domain (GST-KD) (52). We asked if pTRS1 copurified with GST-KD to determine if pTRS1 could bind the dimerized PKR kinase domain (Fig. 4A). As long-term expression of GST-KD is toxic, a doxycycline-inducible promoter was used to regulate GST-KD expression. Wild-type pTRS1 was copurified with glutathione resin from cells expressing GST-KD, while the PKR binding domain mutant (pTRS1mut1) was not. We next determined if pTRS1 could inhibit the activity of a constitutively dimerized PKR KD by measuring the effect of pTRS1 expression on GST-KD autophosphorylation (Fig. 4B). pTRS1 expression decreased GST-KD autophosphorylation compared to those of GFP and pTRS1mut1 controls. Together, these results suggest that pTRS1 binds and inhibits dimerized PKR and that pTRS1 binds PKR through an interaction with the PKR KD.

Amino acids in the PKR eIF2α contact site are important for pTRS1 binding.

Previous studies identified key amino acids in the αG helix of the PKR kinase domain that are necessary for binding of PKR to its substrate eIF2α (Fig. 5A) (3, 4). To determine if pTRS1 interacted with the PKR eIF2α binding site, we examined the ability of pTRS1 to bind and inhibit previously characterized PKR mutants with single amino acid changes in the αG helix (Table 1). Mutation of PKR residues Thr487 and Phe495 to alanine and arginine (T487A and F495R, respectively) ablate PKR phosphorylation of eIF2α but do not affect PKR catalytic activity or autophosphorylation (3). Two amino acids within the PKR αG helix, T487 and Glu490 (E490), are conserved in all eIF2α kinases (4). Therefore, we generated plasmids expressing PKR T487A, F495R, or E490A mutants and measured their binding to pTRS1 (Fig. 5B). While pTRS1 was efficiently copurified with wild-type PKR, very low levels of pTRS1 were recovered with the T487A and E490A PKR mutants, which were visible only after overexposure of the blot. pTRS1 was copurified with the PKR F495R mutant, but at reduced levels. pTRS1 also maintained the ability to bind a PKR mutant in which the activating phosphorylation site had been mutated to alanine (T446A) (Fig. 5B). These data suggest that amino acids in the PKR eIF2α contact site are important for pTRS1 binding and that PKR activation is not necessary for its interaction with pTRS1.

We next tested the ability of pTRS1 to prevent activation of each of the above-mentioned PKR mutants. pTRS1 and the PKR mutants were expressed in PKR-deficient cells, and PKR autophosphorylation was measured after poly(I·C) transfection (Fig. 5C). Consistent with previous results, poly(I·C) induced the autophosphorylation of each PKR mutant in control cells expressing GFP, demonstrating that each PKR mutant was catalytically active (3, 4, 27). Though the PKR F495 mutant was activated to lower levels than wild-type PKR in control cells, pTRS1 prevented autophosphorylation of the PKR F495R mutant. In contrast, the PKR T487A and E490A mutants were resistant to inhibition by pTRS1, consistent with the inability of pTRS1 to bind these mutants. From these data, we conclude that binding of pTRS1 to the PKR αG helix is necessary for inhibition of PKR activation.

The vaccinia virus PKR antagonist K3L also binds PKR at an eIF2α contact residue within the αG helix, Asp486 (D486). Recently, PKR mutants resistant to K3L binding and inhibition were identified (Fig. 5A) (27). To determine if pTRS1 bound PKR in a manner structurally similar to that of K3L, we tested the ability of pTRS1 to bind and inhibit three PKR mutants (the E375V, A473T, and D486V mutants) that escape inhibition by K3L (Fig. 5D). We found that pTRS1 efficiently bound both the PKR E375V and A473T mutants but showed decreased binding to the PKR D486V mutant. pTRS1 prevented autophosphorylation of the PKR E375V and A473T mutants and reduced autophosphorylation of the PKR D486V mutant (Fig. 5E). These data suggest that while pTRS1 and K3L interact with the same region of PKR, these interactions occur in a structurally distinct manner.

pTRS1 inhibits PKR kinase activity.

The inhibition of PKR autophosphorylation by pTRS1 at a step after PKR dimerization suggested that pTRS1 might inhibit PKR kinase activity. To test this hypothesis, we measured PKR kinase activity in the presence of pTRS1 (Fig. 6). Cells were transfected with a Myc-PKR expression plasmid together with plasmids expressing the control protein GFP, wild-type pTRS1, or pTRS1mut1, which cannot bind PKR. We then measured PKR kinase activity in Myc-specific immune complexes using a peptide from histone H2A as a substrate. The histone H2A peptide has previously been shown to be a substrate for PKR in vitro (3). PKR kinase activity was significantly lower in pTRS1-expressing cells than in control cells expressing GFP or cells expressing kinase-dead PKR K296R. The pTRS1mut1 protein did not inhibit PKR kinase activity, consistent with its inability to bind PKR and prevent PKR autophosphorylation. In fact, in some experiments slightly elevated PKR kinase activity was found in cells expressing pTRS1mut1, though the difference was not statistically significant. Together with the above-described data, these results suggest that pTRS1 binding to the PKR kinase domain inhibits PKR kinase activity, preventing activating autophosphorylation of PKR and subsequent phosphorylation of its substrate eIF2α.

pTRS1 binds and inhibits an additional eIF2α kinase.

Our results show that pTRS1 binds and inhibits PKR through interactions with amino acids in the PKR eIF2α contact site. Interestingly, two of the amino acids in the PKR αG helix important for pTRS1 binding are conserved in other eIF2α kinases. To further test the hypothesis that interaction with the PKR eIF2α contact site is critical for pTRS1 binding, we determined if pTRS1 could also bind an additional eIF2α kinase, HRI. We found that HRI was copurified with pTRS1 in transfected 293T cells (Fig. 7A) and in HCMV-infected fibroblasts (Fig. 7B). Treating cells with arsenite leads to an accumulation of reactive oxygen species (ROS), which activates the HRI kinase, resulting in eIF2α phosphorylation (53). To determine if pTRS1 could prevent HRI-dependent eIF2α phosphorylation, we measured eIF2α phosphorylation after arsenite treatment in PKR-deficient cells (Fig. 7C). Arsenite induced a dose-dependent increase in eIF2α phosphorylation, which was decreased in cells expressing pTRS1. This result is consistent with our previous study showing that pTRS1 inhibits stress granule formation in PKR-deficient cells treated with arsenite (6). As HRI activation does not require an interaction with dsRNA, these data further confirm that pTRS1 can inhibit eIF2α kinases independent of its ability to bind RNA. In addition, these data further support the conclusion that residues in the eIF2α contact site are critical for pTRS1 interaction with PKR and may mediate interactions with additional eIF2α kinases.

Cells and viruses.

HEK293T cells and MRC-5 fibroblasts were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 100 U/ml of penicillin-streptomycin (Sigma) at 37°C and 5% CO₂. PKR-deficient HeLa cells (PKR KO), HEK293T cells containing an inducible TRS1 expression cassette (293T-pTRS1i), and PKR-deficient cells containing an inducible TRS1 expression cassette (PKR KO-pTRS1i) were maintained in 1 μg/ml of puromycin in normal growth media. PKR-deficient HeLa cells have been previously described (35). The HCMV AD169inGFP strain was used for all infections. This strain is derived from a bacterial artificial chromosome (BAC) and contains a green fluorescent protein (GFP) reporter driven by the simian virus 40 (SV40) promoter.

Generation of recombinant plasmids.

Vectors expressing full-length TRS1 or TRS1 truncation mutants fused to a carboxyl-terminal 6×His epitope were a kind gift from Adam Geballe (University of Washington [39]). To generate full-length TRS1 containing arginine-to-alanine mutations that disrupt dsRNA binding (R121A, R124A, and R125A [38]), the full-length TRS1 expression plasmid was amplified with primers Triple F and Triple R (Table 2), and recircularized using Gibson cloning (New England BioLabs [NEB]). The R121A, R124A, and R125A point mutations were introduced into the 1-550 TRS1 expression vector, as described above, using the pcDNATRS1 1-550 plasmid as a template. Point mutations within the pTRS1 PKR binding domain that disrupt PKR binding (R658A, D660A D,661A, and E662A [7]) were incorporated into full-length TRS1 and TRS1triple using primers Mut1 F and Mut1 R (Table 1) followed by recircularization using a Gibson reaction (New England BioLabs), in which a mixture of DNA exonuclease, DNA polymerase, and DNA ligase anneal overlapping cDNA ends into a contiguous double-stranded DNA molecule. The portion of each clone containing the TRS1 open reading frame (ORF) was sequenced to ensure the absence of unintended mutations.

The human protein kinase R (PKR) ORF containing homology arms to the pcDNA3.1 V5 His expression vector was ordered as a gBlock gene fragment (IDT; Table 2). The pcDNA3.1 vector was digested with BamHI and XhoI, and the full-length PKR ORF was inserted using a Gibson cloning reaction according to the manufacturer's directions. The kinase-dead PKR (K296R) ORF containing homology arms to the pcDNA3.1 expression vector was also ordered as a gBlock gene fragment (Table 2) and introduced into pcDNA3.1 by Gibson cloning as described above. A Flag epitope was fused to the N terminus of PKR K296R by amplification of the PKR ORF with primers PKRflag F and PKR R (Table 2) followed by Gibson cloning into the pcDNA3.1 vector digested with BamHI and XhoI as described above. An N-terminal Myc tag was added to both wild-type PKR and PKR K296R as described above using the primers PKRmyc F and PKR R. The following point mutations were introduced into PKR: E375V, T446A, A473T, D486V, T487A, E490A, and F495R using primer pairs PKR_E375V F and PKR_E375V R, PKR_A446T F and PKR_A446T R, PKR_T473A F and PKR_T473A R, PKR_D486V F and PKR_D486V R, PKR_T487A F and PKR_T487A R, PKR_E490A F and PKR_E490A R, and PKR_F495R F and PKR_F495R R, respectively. All primer sequences are listed in Table 2. The full-length PKR expression plasmid was amplified from the Myc-tagged wild-type PKR and the Myc-tagged PKR K296R expression plasmids with the above-listed primer pairs. The PCR products were then gel extracted and circularized using Gibson cloning. For each PKR mutant, the portion of each vector containing the PKR ORF was sequenced to confirm the absence of additional mutations.

The PKR kinase domain (KD) fused to glutathione S-transferase (GST) was created by first amplifying GST from pET-41 b(+) using primers GST-pCW F and GST R (Table 2). The PKR KD (amino acids 259 to 551) was then amplified from the full-length PKR expression vector using primers KDGST F and KDGSTpCW R (Table 2). The plasmid pCW-Cas9 (Addgene; number 50661) was digested with NheI and BamHI to remove the Cas9 open reading frame. The GST ORF and PKR KD were then cloned into the digested pCW vector using a three-part Gibson reaction. Full-length TRS1 was introduced into the pCW plasmid by amplifying the TRS1 ORF using primers TRS1ind F and TRS1ind R (Table 2), followed by Gibson assembly into pCW-Cas9 previously digested with NheI and BamHI. Sanger sequencing was used to confirm the absence of unintended mutations.

The heme-regulated inhibitor (HRI) ORF was amplified from the pJP1563 vector (DNASU; number 38827) using primers HRImyc F and HRImyc R (Table 2). The HRI ORF was then cloned into the pcDNA3.1 vector digested with BamHI and XhoI via Gibson reaction. The complete HRI ORF was sequenced to ensure the absence of unintended mutations.

Production of lentivirus stocks and pTRS1-inducible cell lines.

Lentivirus stocks were generated by transfecting HEK293T cells with the pCW TRS1 expression plasmid (described above) and lentivirus packaging mix (Sigma) using polyethylenimine (PEI; Polysciences). The medium was collected at 48 h and 72 h after transfection and clarified by passage through a 0.22-μm filter. The virus was then stored at −80°C until use. 293T-pTRS1i and PKR KO-pTRS1i cells were generated by transduction with the pCW TRS1 lentivirus in the presence of 8 μg/ml of Polybrene. After bulk selection in 1 μg/ml of puromycin for 48 h, clonal cell lines were isolated by limiting dilution in media containing puromycin. Clonal cell lines were assayed for minimal basal expression of pTRS1 and maximal induction of pTRS1 expression after treatment with 1 μg/ml of doxycycline by Western blotting.

PKR and HRI activation assays.

The ability of pTRS1 or pTRS1 mutants to inhibit PKR was measured in HEK293T cells. Cells were transfected with 1 μg of either a GFP or TRS1 expression plasmid using PEI and harvested at 36 h after transfection. Cells were washed with phosphate-buffered saline (PBS), pelleted, and lysed in 120 μl of PKR lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 10% glycerol, and 1 mM EDTA with protease and phosphatase inhibitors [Roche]) on ice for 15 min. Insoluble debris was removed by centrifugation at 21,000 × g for 10 min at 4°C. Unless otherwise indicated, 1 mg/ml of poly(I·C) (LMW; Invivogen) was added to 100 μl of supernatant for 30 min at 30°C to activate PKR. To examine PKR KD activation, HEK293T cells were transfected with 500 ng of GST-KD expression plasmid together with 500 ng of either GFP, TRS1 or TRS1mut1 expression plasmid for 36 h. Doxycycline (1 μg/ml) was added to cells overnight prior to harvest to induce GST-KD expression. Cells were washed once with PBS, pelleted, and stored at −80°C until processed. To measure activation of PKR mutants in PKR KO cells, PKR KO-pTRS1i cells were cotransfected with 500 ng of PKR expression plasmid and 500 ng of either GFP or TRS1 expression plasmid. Twenty hours after DNA transfection, cells were transfected with 20 μg of poly(I·C) using Lipofectamine 2000 (Thermo Fisher) for 4 h. Cells were washed once in PBS, pelleted, and stored at −80°C until processed.

PKR KO-pTRS1i cells were used to measure the effect of pTRS1 on arsenite-induced HRI activation. Cells were treated overnight with doxycycline (1 μg/ml) to induce pTRS1 expression. The next day, cells were treated with 0.05, 0.25, or 0.5 mM sodium arsenite (Sigma) for 2 h to activate HRI (53). Cells were then washed once with PBS, pelleted, and stored at −80°C until processed.

Analysis of protein expression.

Unless otherwise stated, cells were lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, and 1 mM EDTA containing protease and phosphatase inhibitors; Roche). Protein concentration was determined using a Bradford assay. Equal amounts of protein were resolved on 10% SDS-PAGE gels, transferred to nitrocellulose membranes (Amersham), and blocked in 5% nonfat milk in TBS-T (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature (RT) or overnight at 4°C. For mouse monoclonal antibodies, membranes were incubated with primary antibodies for either 1 h at RT or overnight at 4°C in 1% bovine serum albumin (BSA) in TBS-T. For rabbit polyclonal antibodies, membranes were blocked as described above and incubated at 4°C overnight in 5% BSA in TBS-T, with the exception of the anti-His antibody, which was diluted in 1% BSA in TBST-T. Membranes were incubated in appropriate secondary antibodies (KPL) at a 1:10,000 dilution in 1% BSA in TBS-T for 1 h at RT. A modified protocol was used for total and phosphorylated eIF2α blots. Membranes probed for total eIF2α were blocked and incubated in primary antibody as described above and then incubated in secondary antibody (1:10,000) in 5% milk in TBS-T for 1 h at RT before being developed. Membranes probed for phospho-eIF2α (Ser51) were blocked in 5% BSA in TBS-T overnight at 4°C prior to an overnight incubation in primary antibody at 4°C. Membranes were then incubated in secondary antibody (1:10,000) in 1% BSA in TBS-T for 1 h at RT. Antibodies against the following were used: pTRS1 (1:100 [43]), His (1:1,000; CST; number 2365S), total PKR (1:1,000; Santa Cruz; sc-707), phospho-PKR (Thr446) (1:1,000; Abcam; ab32036), Myc (1:1,000; CST; number 2276S), Flag (1:1,000; Sigma; F3165), eIF2α (1:1,000; CST; number 9722S), phospho-eIF2α (Ser51) (1:1,000; CST; number 3398S), HRI (1:1,000; Abcam; ab28530), and tubulin (1:20,000; Sigma; T6199).

Immunoprecipitation.

HEK293T cells were transfected with 5 μg of the desired plasmid for a total of 36 h. Where indicated, doxycycline (1 μg/ml) was added 18 h prior to harvest to induce pTRS1 expression. Cells were lysed in 1 ml of PKR lysis buffer for 15 min on ice. Insoluble debris was removed by centrifugation for 10 min at 21,000 × g at 4°C. One hundred microliters of protein A/G Sepharose beads (Santa Cruz) in PKR lysis buffer (20-μl packed-bead volume) was added to each sample and nutated at 4°C for 30 min to preclear the lysate. Where indicated, 2,000 U of micrococcal nuclease (NEB) and 5 μl of 0.5 M CaCl₂ were added to each sample prior to the preclearing step and incubated at 37°C for 30 min to digest nucleic acid species. The beads were then removed and the lysate was transferred to a new tube containing 1 μg of the desired antibody, or 1 μg of either mouse IgG or tubulin antibody as a negative control. Samples were nutated with antibody at 4°C for 4 h, after which 100 μl of protein A/G beads in PKR lysis buffer (20-μl packed-bead volume) was added to each sample. For the anti-TRS1 immunoprecipitation, antibodies were preconjugated to protein A/G beads overnight before addition to sample lysates. Samples were nutated for 90 min after the addition of the beads. The beads were collected by brief centrifugation and washed three times with 1 ml of PKR lysis buffer before addition of 30 μl of 1× sample buffer (0.1 M Tris-HCl [pH 6.8], 6% glycerol, 2% SDS, 0.1 M dithiothreitol [DTT], 0.002% bromophenol blue). The samples were boiled at 95°C for 10 min, briefly centrifuged at 21,000 × g, and stored at −20°C prior to analysis by Western blotting.

For the glutathione Sepharose pulldown, HEK293T cells were cotransfected with 2.5 μg of inducible GST-PKR kinase domain (GST-KD) expression plasmid and 2.5 μg of either TRS1 or TRS1mut1 expression plasmid for 36 h. GST-KD expression was induced by addition of 1 μg/ml of doxycycline 18 h before harvest. Upon harvest, cells were washed once with PBS, pelleted, and lysed in 500 μl of wash buffer (20 mM HEPES [pH 7.0], 100 mM NaCl, 1 mM DTT, 1% glycerol) for 15 min on ice. Insoluble debris was removed by centrifugation at 21,000 × g for 10 min at 4°C. One hundred microliters of washed glutathione Sepharose 4B (GE) (15-μl packed-Sepharose volume) was added to each sample. Samples were nutated at RT for 1 h and then washed three times with 1 ml of wash buffer. Thirty microliters of 1× sample buffer was added to the beads, after which samples were boiled, briefly centrifuged, and stored at −20°C until analysis by Western blotting.

PKR kinase assay.

HEK293T cells were transfected with 5 μg of the desired plasmids for 36 h. For cotransfections, 2.5 μg of each plasmid was transfected. Immunoprecipitation using anti-Myc or mouse IgG was performed as described above. After a washing in PKR lysis buffer, beads were resuspended in 25 μl of 1× kinase reaction buffer (40 mM Tris [pH 7.5], 20 mM MgCl₂, 0.1 mg/ml of BSA, 500 μM ATP [Promega]) containing 7.5 μg of histone H2A peptide (NEB) as a substrate. The kinase reaction was run for 30 min at RT. The ADP-Glo kinase assay (Promega) was then used to determine PKR kinase activity. Briefly, 25 μl of ADP-Glo reagent was added to each kinase reaction and incubated at RT for 40 min. Fifty microliters of kinase detection reagent was added to each sample and incubated at RT for an additional 40 min, after which the luminescence of each sample was read using a luminometer (Molecular Devices). Fold change in luminescence compared to the IgG control immunoprecipitation was calculated, and statistical significance was determined using an unpaired Student t test.