INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

PKR, the double-stranded RNA (dsRNA)-activated protein kinase (also known as DAI), is transcriptionally induced by interferon and activated in virus-infected mammalian cells, where it plays an important role in cellular antiviral defense mechanisms. PKR interferes with virus replication by phosphorylating eukaryotic initiation factor 2 (eIF2) subunit  (eIF2), converting eIF2 from a substrate to an inhibitor of its guanine nucleotide exchange factor, eIF2B. This reduction in the recycling of eIF2 by eIF2B leads to a general inhibition of translation that limits viral protein synthesis (30). PKR may also have an important role in controlling cellular proliferation, as the expression of catalytically defective PKR alleles transforms mammalian cells in culture and leads to tumor formation in mice (25, 31).

The yeast Saccharomyces cerevisiae also contains an eIF2 kinase, known as GCN2, that is activated by uncharged tRNA when cells are starved for one or more amino acids. Limited phosphorylation of eIF2 under these conditions leads to increased translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes. This induction of GCN4 translation in response to diminished eIF2 recycling occurs because ribosomes are prevented from initiating at short upstream open reading frames in the GCN4 mRNA leader, enabling them to initiate at the GCN4 start codon instead. The newly synthesized GCN4 protein leads to increased expression of amino acid biosynthetic genes, reversing the amino acid limitation which triggered the activation of GCN2 (reviewed in reference 20). Low-level expression of PKR in yeast mutants lacking GCN2 leads to eIF2 phosphorylation at a level sufficient to induce GCN4 expression without inhibiting general translation initiation (13). When expressed at higher levels, PKR phosphorylates eIF2 to an extent that inhibits general protein synthesis and prevents yeast cell growth (9, 13).

PKR kinase activity is stimulated in vitro by dsRNA, and the N-terminal 171 amino acids of the protein contain two copies of a dsRNA-binding motif (dsRBM) found in numerous other dsRNA-binding proteins (reviewed in reference 30). Binding of dsRNA stimulates the autokinase activity of PKR, and autophosphorylation appears to lock the enzyme into an active conformation which can bind and phosphorylate eIF2 in the absence of dsRNA (16, 17, 26). Sequence analysis of phosphopeptides derived from PKR following autophosphorylation in vitro led to the identification of a cluster of autophosphorylation sites located between the dsRBMs and the kinase domain of the protein. Mutation of one site, Thr-258, reduced the efficiency of autophosphorylation and substrate phosphorylation by PKR in vitro and partially impaired kinase function in yeast and mammalian cells. Mutations at two neighboring autophosphorylation sites (Ser-242 and Thr-255) had relatively little effect on kinase function alone but exacerbated the defects associated with the Ala-258 substitution (42). Because the PKR S242A,T255A,T258A triple mutant retains significant levels of autophosphorylation and substrate phosphorylation activities, these cannot be the only autophosphorylation sites in the protein. Additional sites have been detected in the linker region between the two dsRBMs; however, it is unknown whether these sites are important for PKR function (42a).

Several protein kinases, but not all, are activated by phosphorylation of residues within the kinase domain itself, in a segment located between kinase subdomains VII and VIII called the activation loop (reviewed in references 19 and 21). As shown in Fig. 1, such activation sites are located between 5 and 10 residues N terminal to the conserved Ala-Pro-Glu (APE) motif in kinase subdomain VIII. In the crystal structures of MAP kinase Erk2 (50), cyclin-dependent kinase Cdk2 (12), and cyclic AMP-dependent protein kinase (PKA) (23), this loop faces the cleft between the N-terminal and C-terminal lobes, where the active site resides. In the case of MAP kinase and Cdk2, it has been proposed that phosphorylation of these residues alters the conformation of the activation loop to permit binding of the peptide substrate. In the case of PKA, kinetic analysis of mutant proteins has suggested that phosphorylation of the activation loop stimulates the binding of ATP and the rate of phosphoryl transfer, rather than increasing the binding of the peptide substrate (1). It has also been suggested that in all three enzymes, the phosphorylated residue in the activation loop contributes to the proper alignment of catalytic residues, particularly the conserved Asp in subdomain VI-B, or promotes the correct relative orientation of the N-terminal and C-terminal lobes of the kinase domain by interacting with basic residues in the vicinity of the catalytic center (reviewed in reference 21). In any case, phosphorylation of residues in the activation loop is a key event in stimulating substrate phosphorylation by each of these kinases. In phosphorylase kinase, it appears that the role of the phosphorylated residue is carried out by a glutamate residue in the activation loop (21).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Established phosphorylation sites in the activation loops of several protein kinases. Sequence alignments of the segments between kinase subdomains VII and VIII are shown for the protein kinases listed on the left (19, 40). Highly conserved residues common to all of the kinases are shown in bold type without underlining. Phosphorylated residues required for activation of the kinases are shown in bold type and underlined. The kinases were Erk2 (extracellular signal-regulated kinase), Cdk2 (cyclin-dependent kinase), PKA (cyclic AMP-dependent protein kinase), RSK2 (ribosomal protein S6 kinase), GSK3 (glycogen synthase kinase), dGCN2 (Drosophila GCN2 homolog), and HRI (heme-regulated inhibitor). Potential autophosphorylation sites examined in the PKR and GCN2 activation loops are indicated (positions 446, 448, and 451 and positions 882 and 887, respectively). The identification of Thr-446 in PKR and Thr-882 and Thr-887 in GCN2 as autophosphorylation sites is the subject of this report.

PKR contains two Thr residues and one Ser residue within 10 amino acids of the APE motif in subdomain VIII (SPE in PKR) and, interestingly, all of the known eIF2 kinases contain two Thr residues at exactly the same positions relative to subdomain VIII (Fig. 1). Therefore, we wished to determine whether these conserved Thr residues are additional autophosphorylation sites required for kinase catalytic function. To address this possibility, we altered each of the residues to nonphosphorylatable residues and examined the effects on kinase function in yeast and mammalian cells. The results of this analysis indicated that Thr-446 and Thr-451 in the PKR activation loop are both required for high-level kinase function in vivo and in vitro. Mass spectrometry (MS) analysis of phosphopeptides in PKR purified from yeast cells confirmed that Thr-446 is a site of autophosphorylation in vivo. Interestingly, we also found that yeast eIF2 kinase GCN2 autophosphorylates at the two conserved Thr residues in its activation loop, and both sites were found to be important for GCN2 kinase function. Thus, autophosphorylation in the activation loop is required for high-level kinase function by two different members of the eIF2 kinase family.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Plasmids and yeast strains. Construction of the PKR alleles containing the point mutations K296R (38) and T258A (42) and the triple mutation S242A,T255A,T258A (42) in the yeast expression vector pEMBLyex4 has been previously described. A PCR fusion technique (49) was used to create the following single and multiple point mutations in the PKR kinase domain. Thr-446 was mutated to Ser, Asp, or Ala by changing the Thr-446 ACA codon to TCA, GAC, or GCA, respectively. Ser-448 was mutated to Ala by changing the AGT codon to GCT. Thr-451 was changed to Ser, Asp, or Ala by changing the Thr-451 ACT codon to TCT, GAT, or GCT, respectively. Glu-490 was mutated to Gln by changing the Glu-490 GAA codon to CAA.

gcn2 ura3-52 leu2-3 leu2-112 trp1-63

ura3-52 leu2-3 leu2-112 gcn2 sui2 trp1-63

trp1-63

ura3-52 leu2-3 leu2-112 trp1-63 gcn2 gcd2::hisG

GCN2 antisera. Specific antibodies recognizing the N terminus of GCN2 were raised against a TrpE-GCN2 fusion protein bearing the GCN2 coding sequences of the EcoRI fragment of GCN2 inserted into pATH11 (ATCC 37699). A second TrpE-GCN2 fusion protein containing the C-terminal region of GCN2 was generated by subcloning the BamHI-NheI fragment of GCN2 into the pATH3 (ATCC 37697) vector, kindly provided by Matthew Marton. The fusion proteins were expressed in Escherichia coli HB101-9 and purified for use as antigens as described previously (24). Antibodies were generated by Covance Laboratories Inc.

Immunoblot analysis of protein expression in yeast cells. Transformants of the H1816 and H1817 strains containing various PKR alleles were grown in synthetic minimal dextrose (SD) medium at 30°C for ~30 h and then were shifted to inducing conditions, synthetic minimal medium containing 10% galactose and 2% raffinose (SGAL medium), for ~12 h. Whole-cell extracts were prepared by breaking the cells with glass beads in lysis buffer (100 mM Tris-HCl [pH 8.0], 20% glycerol, 1 mM -mercaptoethanol) containing complete protease inhibitor (CPI) cocktail (Boehringer Mannheim Biochemicals). For analysis of phosphatase-treated proteins, 1 µl (~400 U) of  protein phosphatase (New England BioLabs) was added to samples of whole-cell extracts (prepared as just described) containing 30 µg of total protein and 1×  protein phosphatase buffer (50 mM Tris-HCl [pH 7.8], 5 mM dithiothreitol, 2 mM MnCl₂, 100 µg of bovine serum albumin per ml); the mixtures were incubated at 30°C for 1 h. Extracts were separated by electrophoresis in sodium dodecyl sulfate (SDS)-8 to 16% polyacrylamide gradient gels and transferred to nitrocellulose. Blots were treated with blocking solution (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk. Immunodetection of PKR was done with PKR-specific monoclonal antibodies (lot 71/10; RiboGene) at a dilution of 1:1,000 in the blocking solution just described. Immune complexes were visualized with the enhanced chemiluminescence (ECL) detection system (Amersham) following the vendor's instructions.

In vitro protein kinase assays. Transformants of strain H1817 containing various plasmid-borne PKR alleles were grown for ~16 h in SD medium, diluted 1:50 in SGAL medium, and grown for another ~16 h to induce PKR expression. Whole-cell extracts were prepared, PKR was immunoprecipitated from the extracts with anti-PKR polyclonal antibodies bound to protein A-Sepharose beads (Pharmacia), and the immune complexes were assayed for kinase activity, all as previously described (38) but with the following modification. After the proteins were resolved by electrophoresis in SDS-8 to 16% polyacrylamide gradient gels, they were transferred to nitrocellulose filters, which were subjected to autoradiography with Kodak XAR film to visualize the radiolabeled proteins. Following autoradiography, the blots were treated with blocking solution and immunodetection of PKR was carried out as described above. The recombinant yeast eIF2 used in these assays was expressed in bacteria and purified as described previously (52).

In vivo labeling of PKR in yeast cells. Transformants of yeast strain H1817 expressing wild-type and mutant PKR alleles were grown for ~16 h at 30°C in synthetic low-phosphate medium containing dextrose [20 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, 15 mM (NH₄)₂SO₄, 8.5 mM NaCl, 0.1% yeast extract, 2% glucose] and then were shifted to low-phosphate medium containing 10% galactose and 2% raffinose instead of dextrose for ~12 h to induce PKR expression. About 4 units of cells at an optical density of 600 nm (OD₆₀₀) was resuspended in the same galactose-raffinose medium containing 1.5 mCi of [³²P]orthophosphate and incubated at 30°C for 4 to 6 h. Cells were broken in kinase reaction breaking buffer (KRBB; 20 mM Tris-HCL [pH 8.0], 50 mM KCl, 400 mM NaCl, 20% glycerol, 1% Triton X-100, 0.5 mM EDTA) containing phosphatase inhibitors (50 mM NaF, 20 mM -glycerol phosphate, 5 mM sodium pyrophosphate, 125 µM sodium orthovanadate) and CPI cocktail. For immunoprecipitation of PKR, 30 µl of protein A-Sepharose beads was resuspended in 200 µl of KRBB and incubated with 1 µl of PKR-specific polyclonal antiserum for 30 min at room temperature. The beads were pelleted and washed with 400 µl of KRBB. Total protein extract from 4 OD₆₀₀ units of cells in 200 µl of KRBB was added to the washed beads and incubated for 2 h at 4°C. Immune complexes were collected as described above and resuspended in Laemmli sample buffer (27). Samples were boiled for 10 min and resolved by SDS-PAGE with 10% polyacrylamide gels. Gels were dried under vacuum and subjected to autoradiography with Kodak XAR film.

Analysis of PKR expression in mammalian cells. COS1 cells were grown in 10-cm tissue culture plates to 50 to 60% confluence in Dulbecco modified Eagle medium with 10% fetal bovine serum. Plasmids bearing wild-type or mutant PKR alleles under the control of a CMV promoter (pcDNA3) were introduced into COS1 cells by the calcium phosphate precipitation procedure (39). DNA (20 µg) was dissolved in 0.25 M CaCl₂, and an equal volume of 2× HEPES-buffered saline (280 mM NaCl, 50 mM HEPES, 1.5 mM Na₂HPO4 [pH 7.1]) was added to form a precipitate. After 30 min at room temperature, the precipitate was added to the monolayer in a dropwise manner. The medium was changed at 18 h posttransfection. At 48 h posttransfection, the cells were washed twice with cold phosphate-buffered saline and lysed with 10 mM phosphate buffer (10 mM phosphate [pH 7.4], 100 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 50 µg each of aprotinin and leupeptin per ml, 2 mM phenylmethylsulfonyl fluoride, 0.1% NaN₃, 1 mM Na₃VO₄) or by a freeze-thaw-vortex procedure with 0.25M Tris-HCl (pH 8.0)-0.5% Nonidet P-40. After removal of debris, proteins were resolved in a 10% polyacrylamide-SDS gel and transferred to a 0.2 µm-pore-size nitrocellulose filter. After incubation in 5% nonfat dry milk dissolved in 20 mM Tris-HCl (pH 8.0)-150 mM NaCl-0.1% Tween 20, the blot was probed with mouse monoclonal anti-PKR antibodies (RiboGene) at a 1:300 dilution and developed with a horseradish peroxidase-conjugated anti-mouse secondary antibody by use of an ECL procedure (Amersham).

Tumorigenesis assays. Two to four nude mice (BALB/c nude; Taconic Farms, N.Y.) were injected in the left or right flank with transfected cells containing different PKR constructs at 5 × 10⁶ cells per injection per mouse (25). Tumor growth was monitored every 3 days, and tumors larger than 3 mm were regarded as positive.

Phosphoamino acid analysis of peptide substrates. In vitro kinase reaction mixtures with synthetic peptides as substrates were fractionated by electrophoresis in Tricine-SDS-10 to 20% polyacrylamide gradient gels and transferred to PVDF membranes. The membranes were exposed to film (Kodak XAR), and the labeled PKR and peptide bands were visualized by autoradiography. The strip of PVDF membrane containing the radiolabeled peptide was subjected to acid hydrolysis as described previously (22). The supernatant containing the hydrolyzed peptide was removed to a separate tube and lyophilized. Deionized water (100 µl) was added, and the sample was again lyophilized to dryness. Radiolabeled phosphoamino acids were separated by electrophoresis on thin-layer chromatography plates (EM 5716-7; Merck) at pH 3.5 (4). Phosphoserine, phosphothreonine, and phosphotyrosine standards (Sigma) were analyzed in parallel and visualized by ninhydrin staining. Radiolabeled phosphoamino acids were visualized by autoradiography.

Isolation of autophosphorylated PKR from yeast cells for phosphopeptide analysis. Plasmids pR156 and pR181 carrying six-His-tagged PKR and 6×His-PKR-K296R were introduced into yeast strain GP3299. To provide a negative control, an untagged version of wild-type PKR expressed from plasmid p1469 (38) was also transformed into yeast strain GP3299. Single colonies of each yeast strain were inoculated into 5.0 ml of SD medium and cultured overnight at 30°C. This 5.0-ml starter culture was inoculated into 100 ml of SGAL medium and grown overnight at 30°C. One liter of SGAL medium was inoculated with the 100-ml overnight SGAL culture, and the culture was grown an additional 48 h at 30°C. Cells were harvested by centrifugation at 4,500 × g for 10 min at 4°C. The pellet was resuspended in 50 ml of H₂O containing CPI cocktail and phosphatase inhibitors (50 mM NaF, 20 mM -glycerolphosphate, 125 µM Na₃VO₄). Cells were pelleted by centrifugation in a swinging-bucket rotor at 4,000 rpm for 5 min at 4°C. The pellet was resuspended in 4.0 ml of nickel column binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 0.1% Triton X-100) plus CPI cocktail and the phosphatase inhibitors just described, and the cells were broken with glass beads. An aliquot of the resulting extract containing 3.0 mg of total cell protein was incubated with 100 µl of Ni-NTA Silica resin (Qiagen) by rotation in a cold room for 1 h. The resin was pelleted and washed twice in nickel column binding buffer containing 40 mM imidazole. Proteins bound to the resin were eluted with Ni column elution buffer (20 mM sodium phosphate, 500 mM NaCl, 400 mM imidazole) containing protease and phosphatase inhibitors. Proteins eluted from the Ni-NTA Silica resin were incubated with poly(I) · poly(C)-agarose (Pharmacia) for 1 h at 4°C. The poly(I) · poly(C) agarose was pelleted and washed three times with buffer A (150 mM KCl, 20 mM HEPES, 10% glycerol, 5 mM magnesium acetate) plus CPI cocktail and phosphatase inhibitors. The pellet was resuspended in 100 µl of buffer A, and 20 µl of 6× Laemmli sample buffer was added. The samples were boiled for 5 min and separated by electrophoresis in SDS-8 to 16% polyacrylamide gradient gels. The PKR protein bands were visualized by Cu staining (Bio-Rad).

MS. In-gel digestions of the Cu-stained PKR proteins obtained from the SDS-PAGE separations just described were carried out with ~200 ng of protein and sequencing-grade modified trypsin or sequencing-grade Asp-N or Lys-C (Boehringer) at a protein/enzyme ratio of 1:1 in 50 mM NH₄HCO₃ for 2 h at 37°C, and the resulting peptides were extracted as described previously (36). The extracted peptides were redissolved in 10 µl of acetonitrile-H₂O (1:1) for MS analysis. For phosphatase treatment of the extracted peptides, 2 µl of the peptides was mixed with 2 µl (0.5 U) of calf intestinal phosphatase (CIP) (New England BioLabs) in 50 mM NH₄HCO₃ and incubated at 37°C for 30 min.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Substitutions at Thr-446 and Thr-451 impair general and GCN4-specific translational control by PKR in yeast cells. We altered the potential autophosphorylation sites Thr-446 and Thr-451 in the PKR activation loop to phosphorylatable Ser residues or nonphosphorylatable Ala or Asp residues and examined the effects on PKR function in yeast cells. cDNAs encoding the mutant proteins were expressed from a galactose-inducible GAL-CYC1 promoter in yeast strains lacking GCN2. High-level expression of PKR from this promoter on galactose medium is lethal due to extensive phosphorylation of eIF2 at Ser-51. This growth inhibition is completely eliminated by a mutation that inactivates kinase subdomain II in PKR (substitution of Lys-296 with Arg; K296R) (Fig. 2A). The degree of growth inhibition on galactose medium in strains expressing PKR from the GAL1-CYC1 promoter provides a sensitive indicator of PKR function in vivo (9, 13). Low-level expression of wild-type PKR on medium containing dextrose as a carbon source does not inhibit cell growth but is sufficient to restore the translation of GCN4 mRNA in strains lacking GCN2. gcn2 mutants are unable to grow on medium containing 3-aminotriazole (3-AT), a competitive inhibitor of histidine biosynthesis, because they cannot derepress GCN4 and its target genes in the histidine biosynthetic pathway. Consequently, the ability to complement the 3-AT sensitivity of a gcn2 mutant provides a second indicator of PKR function in yeast cells (13).

View larger version (21K): [in this window] [in a new window]   View larger version (63K): [in this window] [in a new window]   FIG. 2.   In vivo analysis of mutant PKR alleles by growth tests in yeast. (A) The schematic at the bottom represents the full-length wild-type PKR protein sequence. The boxes in the amino-terminal half represent the three regions rich in basic residues. Boxes 1 and 2 contain DRBM-1 and -2, respectively. Box 3 is a region rich in basic amino acids of unknown function. The amino acid residues spanning these regions are indicated. The black boxes in the C-terminal half (I to XI) represent conserved kinase subdomains found in the catalytic regions of all protein kinases. The locations of known (S242, T255, and T258) or suspected (T446, S448, and T451) autophosphorylation sites are indicated. Plasmids carrying the indicated PKR alleles were introduced into the gcn2 yeast strain H1816 expressing wild-type eIF2. Patches of transformants were grown to confluence in SD medium and replica plated to SD medium plus 30 mM 3-AT, SGAL medium, and SGAL medium plus 30 mM 3-AT. Plates were incubated for 3 to 4 days at 30°C, and growth was scored relative to that of the wild-type (WT) PKR transformants. Relative growth from strongest to weakest was scored as ++, +, +/, /+, and . Whole-cell lysates from the indicated strains that were either treated or not treated with  protein phosphatase ( PPase) were subjected to SDS-PAGE fractionation and immunoblot analysis as described in the legends to Fig. 3 and 4. PKR proteins whose mobilities increased when treated with phosphatase are indicated by +; proteins that showed no discernible change in mobility when treated with phosphatase are indicated by . (B) Selected transformants of H1816 bearing the indicated PKR constructs were streaked on SGAL medium and incubated for 6 days at 30°C.

Substitutions at Thr-446 and Thr-451 impair autoregulation of PKR expression in yeast cells. It has been shown that the expression of PKR is negatively autoregulated in yeast cells, such that the wild-type protein is expressed at very low levels, whereas the catalytically inactive K296R protein is expressed at about 20-fold-higher levels (13, 38). Accordingly, an independent assessment of PKR function can be achieved by comparing the in vivo steady-state levels of mutant and wild-type PKR proteins by immunoblot analysis. In addition, by measuring PKR expression in isogenic strains containing either wild-type eIF2 or eIF2 containing an S51A mutation (eIF2-S51A), we can distinguish between effects on PKR expression attributable to autoregulation (those occurring with wild-type eIF2) from differences in protein stability or immunological cross-reactivity (seen in the presence of eIF2-S51A). Figure 3 shows the effects of selected substitutions at positions 446, 451, and 258 on PKR protein levels in a strain containing wild-type eIF2, where autoregulation of PKR expression is intact. Ala substitutions at positions 446 and 451 increased PKR expression in proportion to their deleterious effects on PKR function, as judged by the growth assays described above. Thus, the inactive T451A protein was expressed at higher levels than was the T446A protein, which retained low-level activity (Fig. 3, compare lanes 5 and 8). In most experiments, the T446D and T451D proteins were expressed at slightly lower levels than were the T446A and T451A proteins, respectively (Fig. 3, lanes 12 and 13 and lanes 14 and 15) (data not shown), consistent with the idea that Asp is more functional than Ala at these two positions. The T258A mutation led to a smaller increase in PKR expression than did the T446A mutation, in agreement with the conclusion that T446 is more critical than T258 for PKR activity (Fig. 3, lanes 2 and 5). In addition, the T258A,T446A double-mutant protein was expressed at higher levels than was either the T258A or the T446A single-mutant protein, confirming that these mutations have additive effects on PKR function.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Immunoblot analysis of PKR protein levels in gcn2 yeast cells expressing wild-type eIF2. Transformants of yeast strain H1816 bearing the indicated wild-type (WT) or mutant PKR alleles were grown in SD medium at 30°C for 30 h and then shifted to SGAL medium for 12 h to induce PKR expression. Thirty micrograms of total cell protein from each strain was fractionated by SDS-PAGE with 10% polyacrylamide gels and subjected to immunoblot analysis with monoclonal antibodies against PKR (71/10) and monoclonal antibodies against PAB1 (PAB), the yeast poly(A)-binding protein. The latter were used to confirm that equal amounts of cell protein were analyzed for each strain. ECL was used to visualize immune complexes. Lanes 10 and 11, 12 and 13, and 14 and 15 contain extracts isolated from two independent transformants bearing the indicated PKR alleles.

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Immunoblot analysis of protein levels and relative mobilities of PKR proteins expressed in yeast cells containing nonphosphorylatable eIF2-S51A. (A and B) Thirty micrograms of total cell protein from yeast strain H1817 expressing wild-type (WT) PKR protein or the indicated PKR mutant proteins was subjected to SDS-PAGE with 10% (A) or 7.5% (B) polyacrylamide gels and immunoblot analysis with PKR-specific monoclonal antiserum and ECL to detect immune complexes. (C) Comparison of the relative mobilities of PKR proteins with and without phosphatase treatment. Thirty micrograms of total cell protein from transformants of the H1817 strain were treated with  protein phosphatase (+  PPase). Phosphatase-treated and untreated samples were subjected to SDS-PAGE (7.5% polyacrylamide gel) and immunoblot analysis. The broken lines in panels B and C are drawn midway through the K296R PKR bands. The displacements of the midpoints of the untreated wild-type and T451S PKR bands from these lines were 2.7 and 2.0 mm, respectively, whereas the protein bands of the phosphatase-treated samples were displaced from these lines by 1.2 and 0.5 mm, respectively.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   In vivo autophosphorylation of wild-type and mutant PKR proteins in yeast cells containing eIF2-S51A. Transformants of yeast strain H1817 (containing nonphosphorylatable eIF2-S51A) bearing the indicated wild-type (WT) or mutant PKR alleles were grown in SGAL medium to induce PKR expression and metabolically labeled with [32P]orthophosphate for 4 to 6 h (see Materials and Methods). PKR proteins were immunoprecipitated from aliquots of labeled whole-cell extracts with polyclonal antibodies against PKR, and the immune complexes were analyzed by SDS-PAGE (10% polyacrylamide) followed by autoradiography.

Substitutions at Thr-446 and Thr-451 impair PKR kinase activity in vitro. We conducted in vitro kinase assays on a subset of the mutant PKR proteins containing substitutions at position 446, 448, and 451. The proteins were immunoprecipitated from transformants of eIF2-S51A strain H1817 (in which the wild-type and mutant PKR proteins were expressed at similar levels) and incubated in the presence of [-³²P]ATP and purified eIF2. Following SDS-PAGE separation, the proteins were transferred to nitrocellulose and subjected to autoradiography to visualize the radioactivity incorporated into PKR and eIF2. The filters then were probed with PKR antibodies to measure the amounts of PKR recovered in the immune complexes. In accordance with our in vivo estimates of PKR function, the T446D, T446A, T451S, and T451D proteins showed greatly reduced autophosphorylation and eIF2 kinase activities, whereas the T451A protein was completely inactive (Fig. 6, lanes 1 to 7). Note that the Asp-451 protein had higher autokinase activity than did the Ala-451 protein and that the Asp-446 protein had higher eIF2 kinase activity than did the Ala-446 protein. In addition, Ala substitutions at the previously established phosphorylation sites Ser-242, Thr-255, and Thr-258 were less deleterious than was the Ala-446 substitution (Fig. 6, lanes 10 to 12).

View larger version (35K): [in this window] [in a new window]   FIG. 6.   In vitro kinase activities of immunopurified PKR proteins isolated from yeast strains containing eIF2-S51A. (A) Transformants of strain H1817 bearing the indicated wild-type (WT) or mutant PKR alleles were grown in SGAL medium to induce PKR expression, and the PKR proteins were immunoprecipitated from whole-cell extracts containing 150 µg of total protein with polyclonal antibodies against PKR. Immune complexes were incubated in kinase reaction buffer (KRB) (38) in the presence of [-32P]ATP and 1 µg of purified recombinant eIF2 for 15 min at 30°C. Radiolabeled samples were separated by SDS-PAGE (8 to 16% polyacrylamide gradient gel) and transferred to a nitrocellulose filter. The radiolabeled PKR and eIF2 proteins were visualized by autoradiography. It was shown previously that PKR immunopurified from yeast extracts was activated without the addition of dsRNA activators and that the addition of poly(I) · poly(C) produced no significant increase in activity for either mutant or wild-type enzymes (38); therefore, these assays were conducted without the addition of exogenous dsRNA. (B) The same nitrocellulose filter was subjected to immunoblot analysis with monoclonal antibodies against PKR as described in the legends to Fig. 3 and 4 to visualize the amounts of PKR immunoprecipitated for each sample.

Substitution of a conserved residue between kinase subdomains IX and X partially suppresses the catalytic defect imposed by the Ala-451 mutation. The crystal structure of the unphosphorylated (inactive) form of the MAP kinase ERK2 showed that the activation loop interacts with residues between kinase subdomains IX and X, located within and near the G helix. In particular, Tyr-231 of ERK2 makes two hydrogen bonds with the activation loop. Phosphorylation of Tyr-185 and Thr-183 in the ERK2 activation loop causes it to refold, resulting in a loss of contacts between the loop and neighboring segments as the kinase assumes an active conformation (7). From the yeast MAP kinase FUS3 a dominant mutation (D227N) that partially compensated for reduced phosphorylation of the activation loop of FUS3 in mutant strains containing a defective form of the MAP kinase kinase STE7 was isolated (5). Asp-227 in FUS3 is located at a position closely corresponding to that of Tyr-231 in ERK2 (10); therefore, mutation of Asp-227 to Asn in FUS3 could alter interactions between the G helix and activation loop to increase kinase activity in the hypophosphorylated form of FUS3, mimicking the effect of phosphorylation by STE7.

View larger version (39K): [in this window] [in a new window]   FIG. 7.   Substitution of Glu-490 partially suppresses the impairment of kinase activity imposed by the T451A mutation in the PKR activation loop in vivo and in vitro. (A) In vivo analysis. Plasmids carrying the indicated PKR alleles were introduced into the gcn2 yeast strain H1816 expressing wild-type eIF2. Patches of transformants were grown to confluence in SD medium and replica plated to the indicated media. Growth was scored relative to that of the strain containing wild-type (WT) PKR as described in the legend to Fig. 2. (B) In vitro analysis. Transformants of strain H1817 (expressing eIF22-S51A) bearing the indicated wild-type (WT) or mutant PKR alleles were grown in SGAL medium to induce PKR expression, and the PKR proteins were immunoprecipitated from whole-cell extracts containing 150 µg of total protein with polyclonal antibodies against PKR. In vitro kinase assays were carried out as described in Materials and Methods. Radiolabeled samples were separated by SDS-PAGE (10% polyacrylamide gel) and transferred to a nitrocellulose filter. The radiolabeled PKR proteins were visualized by autoradiography (upper panel). The same nitrocellulose filter was subjected to immunoblot analysis with monoclonal antibodies against PKR to visualize the amounts of PKR immunoprecipitated for each sample (lower panel).

Effects of Thr-446 and Thr-451 substitutions on PKR activity in mammalian cells. We wished to determine whether mutations at positions 446 and 451 would impair PKR activity in mammalian cells. It has been shown that PKR negatively autoregulates its expression in mammalian cells in a manner similar to that seen in yeast cells (2, 43). Therefore, we transfected a subset of the mutant PKR alleles into COS1 cells and measured rates of synthesis and steady-state levels of the encoded proteins as a measure of their ability to autoregulate PKR expression. The PKR cDNAs were expressed under the control of the CMV immediate-early promoter. At 48 h after transfection, the cells were metabolically labeled with [³⁵S]methionine for 1.5 h and cytoplasmic extracts were prepared and immunoprecipitated with anti-PKR antibodies (Fig. 8A). In addition, cytoplasmic extracts prepared from the transfected cells were subjected to immunoblot analysis with antibodies against PKR (Fig. 8B). The results in Fig. 8 indicate that the T446A,T451A and T446A,T451A proteins were synthesized at levels comparable to that of the K296R protein, indicating profound defects in kinase function for all three mutant proteins in COS1 cells. The T446S,T451S protein was produced at lower levels than the corresponding Ala doublemutant protein, supporting the conclusion that Ser is a functional replacement for Thr at these positions. The T258A and S242A,T255A,T258A mutant proteins were produced at levels only slightly higher than that of the wild-type protein, consistent with relatively small reductions in kinase function. These findings indicate that Thr-446 and Thr-451 are critically required for kinase function in COS1 cells and that each makes a stronger contribution to PKR activity than do Ser-242, Thr-255, and Thr-258.

View larger version (30K): [in this window] [in a new window]   FIG. 8.   Analysis of autoregulation of PKR expression in mammalian cells. The indicated PKR alleles under the control of a CMV promoter were transfected into COS1 cells (see Materials and Methods), and the cells were harvested 48 h postinfection. (A) Transfected cells were labeled with [35S]methionine, and the PKR proteins were immunoprecipitated (IP) from 500 µg of total cytoplasmic protein with anti-PKR polyclonal antibodies. Radiolabeled samples were resolved by SDS-PAGE followed by autoradiography. wt, wild type. (B) Immunoblot analysis of PKR expression levels in COS1 cells. Total cytoplasmic protein (75 µg) was subjected to SDS-PAGE followed by immunoblot analysis with monoclonal antibodies against PKR, and immune complexes were visualized by ECL.

Substitutions in PKR autophosphorylation sites lead to tumor formation in mice. As a second means of assessing the effects of mutations at positions 446 and 451 on PKR function in mammalian cells, we examined the ability of selected mutant proteins to elicit tumors in mice. Stable transfectants of NIH 3T3 cells bearing cDNAs encoding the mutant proteins were injected into nude mice, and the mice were monitored for the appearance of tumors. In accordance with previous results, the K296R and 6 alleles elicited tumor formation (Table 1). This phenomenon has been attributed to dominant interference with endogenous wild-type PKR by the catalytically inactive mutant proteins through the formation of defective heterodimers (25, 31). The T451A and S242A,T255A,T258A alleles were also effective in eliciting tumors (Table 1), indicating that their products were functionally impaired and capable of interfering with endogenous PKR.

PKR specifically phosphorylates Thr-446 and Thr-451 in synthetic peptides in vitro. To obtain biochemical evidence that Thr-446 and Thr-451 are autophosphorylation sites, we first conducted in vitro kinase assays with immunopurified PKR using synthetic peptides corresponding to residues 439 to 458 as exogenous substrates. The wild-type peptide and two mutant peptides containing single Ala substitutions at residues 446 and 451 were phosphorylated in vitro at levels higher than that seen for the Ala-446,Ala-451 double-mutant peptide (Fig. 9A). We confirmed that phosphorylation of the wild-type peptide was completely dependent on PKR by showing that no phosphorylation occurred with the K296R catalytically inactive kinase (data not shown). Analysis of the phosphoamino acids produced by PKR in the wild-type peptide revealed that the majority of phosphorylation occurred at threonine residues (Fig. 9B, left panel). Taken together, these results indicated that PKR phosphorylated both Thr-446 and Thr-451 in the synthetic peptides and did so at much higher efficiencies than it phosphorylated the two Ser residues present in the peptides at positions 448 and 456. To provide additional support for this conclusion, we analyzed the phosphoamino acids produced in a peptide containing Ser residues in place of Thr-446 and Thr-451. This mutant peptide was phosphorylated more efficiently than was the Ala-446,Ala-451 peptide (Fig. 9A), and phosphoamino acid analysis confirmed that only phosphoserine was produced in the in vitro reaction (Fig. 9B, right panel). (A larger amount of radiolabeled product was subjected to phosphoamino acid analysis for the Ser-446,Ser-451 peptide than for the Thr-446,Thr-451 peptide.) In addition, phosphoamino acid analysis of peptides containing single Ala substitutions at positions 446 and 451 showed that the bulk of the phosphorylation occurred at the sole Thr residue remaining in these peptides (data not shown). Thus, when provided with the putative activation loop peptide as an exogenous substrate, PKR showed a preference for phosphorylating threonine residues at positions 446 and 451. 

View larger version (49K): [in this window] [in a new window]   FIG. 9.   In vitro phosphorylation and phosphoamino acid analysis of synthetic peptide substrates. (A) Transformants of strain H1817 (containing nonphosphorylatable eIF2-S51A) bearing the wild-type PKR allele were grown in SGAL medium, and PKR was immunoprecipitated from whole-cell extracts as described in the legend to Fig. 6. Immune complexes were incubated in kinase reaction buffer in the presence of [-32P]ATP and 4 µg of a synthetic 20-mer peptide (lanes 2 to 6) or no added peptide (lane 1) for 15 min at 30°C. Radiolabeled samples were analyzed by Tricine-SDS-PAGE (10 to 20% polyacrylamide gradient gel). The gels were stained with Coomassie blue, dried under vacuum, and subjected to autoradiography. The wild-type peptide (whose sequence is shown on the left in panel B) was present in the reaction in lane 2. The mutant peptides analyzed in lanes 3 to 6 contained the indicated substitutions at positions 446 and 451. (B) Phosphoamino acid analysis of in vitro-labeled peptide substrates. In vitro kinase reactions were carried out as described above, and proteins were transferred to PVDF membranes and subjected to autoradiography. The strip of membrane containing the radiolabeled peptide was hydrolyzed with HCl, and soluble amino acids were resolved by thin-layer electrophoresis (see Materials and Methods), after which the phosphoamino acids were visualized by autoradiography. Unlabeled phosphoamino acid standards (phosphoserine [S], phosphothreonine [T], and phosphotyrosine [Y]) were separated along with each sample and visualized by ninhydrin staining. The positions of the standards are indicated to the right of each autoradiogram.

MS evidence for autophosphorylation of Thr-446 in PKR in vivo. To determine whether Thr-446 and Thr-451 are autophosphorylated by PKR in vivo, we used MS to evaluate the phosphorylation status of these residues in PKR expressed in yeast cells. A polyhistidine-tagged form of PKR was purified by nickel affinity and poly(I) · poly(C) affinity chromatography and SDS-PAGE (see Materials and Methods). The His-tagged PKR allele conferred on strain H1816 the same growth phenotypes in SGAL medium and SD medium containing 3-AT that we observed with wild-type PKR expressed from the same vector (data not shown), indicating that the tagged protein had wild-type eIF2 kinase activity in vivo.

Autophosphorylation sites in the activation loop of the yeast eIF2 kinase GCN2 are required for kinase function in vitro and in vivo. We noted that GCN2 contains two Thr residues at exactly the same positions relative to kinase subdomain VIII as those occupied by Thr-446 and Thr-451 in PKR (Fig. 1) and set out to determine whether these Thr residues are autophosphorylation sites required for GCN2 kinase function. Mutations made in a plasmid-borne GCN2 allele that substituted Thr-882 or Thr-887 with alanine partially or completely impaired complementation of the 3-AT-sensitive phenotype of a gcn2 strain (Fig. 10A), respectively, without reducing the steady-state level of the GCN2 protein, as measured by immunoblot analysis (Fig. 10B). Substitution of Thr-882 with Ser had no detectable effect on GCN2 function, whereas the Ser substitution at residue 887, individually or in combination with Ser-882, led to a partial reduction in complementation activity (Fig. 10A and B). Glu or Asp substitutions at both positions resulted in the same phenotypes as the corresponding Ala substitutions; however, the extent to which Asp or Glu will substitute for a phosphorylated residue in the activation loop is known to vary considerably, from no activity to partial activity for the kinases with substitutions (21). The results of immune complex assays of GCN2 autophosphorylation activity for the Ala- and Ser-substituted mutant GCN2 proteins paralleled the complementation data in showing reduced activities for the Ser-887 and Ser-882,Ser-887 mutant proteins (Fig. 10C, lanes 6 to 9) and little or no activity for proteins with single or double Ala substitutions at positions 882 and 887 (lanes 1 to 4 and 11 to 14). After normalizing the amounts of labeled GCN2 produced in these assays (Fig. 10C, top panel) for the amounts of GCN2 protein recovered in the immune complexes (bottom panel), we concluded that the Ala-882 mutant protein showed higher levels of autokinase activity than did the Ala-887 and Ala-882,Ala-887 mutant proteins (Fig. 10C, lanes 2 to 5 and 12 to 14), in agreement with the complementation data.

View larger version (26K): [in this window] [in a new window]   FIG. 10.   Analysis of substitutions at Thr-882 and Thr-887 on GCN2 function in vivo and autokinase activity in vitro. (A) In vivo analysis of GCN2 function. Patches of transformants of strain H1894 containing the indicated GCN2 alleles on low-copy-number plasmids were grown to confluence in SD medium and replica plated to SD medium or SD medium supplemented with increasing concentrations of 3-AT ranging from 7.5 to 150 mM. Growth relative to that of the wild-type (WT) control was scored as follows: ++++, +++, ++, and +, the growth of the mutant was indistinguishable from that of the wild type on 3-AT concentrations of 150, 100, 75, and 10 mM, respectively; , no growth at any of these 3-AT concentrations. The levels of in vitro kinase activities were ranked as follows: +, +/, /+, and , activities that were identical to that of wild type, somewhat reduced, or greatly reduced relative to wild type, or undetectable. (B) Immunoblot analysis of GCN2 expression. Fifteen micrograms of whole-cell extracts prepared from transformants of strain H1894 bearing the indicated GCN2 alleles on low-copy-number plasmids were separated by SDS-PAGE with 6% polyacrylamide gels and transferred to PVDF membranes. Immunoblot analysis was performed with antibodies against GCN2 and GCD6 and ECL to detect the immune complexes. (C) In vitro autokinase activities of GCN2 proteins. Transformants of strain H1894 bearing the indicated GCN2 alleles on low-copy-number (lanes 1 to 10) or high-copy-number (lanes 11 to 14) plasmids were grown in liquid SD medium to an OD600 of 1.0, and GCN2 was immunoprecipitated from whole-cell extracts containing 150 µg of total protein with polyclonal antibodies against GCN2. Immune complexes were incubated in kinase reaction buffer in the presence of [-32P]ATP for 20 min at 30°C. Radiolabeled proteins were separated by SDS-PAGE with 4 to 16% gradient gels, transferred to PVDF membranes, and visualized by autoradiography (top panels). Immunodetection of GCN2 was performed on the same membranes with anti-GCN2 polyclonal antibodies and ECL to visualize the immune complexes (bottom panels). GCN2~P, radiolabeled autophosphorylated GCN2.

View larger version (53K): [in this window] [in a new window]   FIG. 11.   Analysis of phosphoamino a