©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of the Double-stranded RNA (dsRNA) Binding Domain of the dsRNA-activated Protein Kinase, PKR (*)

(Received for publication, September 15, 1994)

Nigel A. J. McMillan Bruce W. Carpick Britton Hollis W. Mark Toone Maryam Zamanian-Daryoush Bryan R. G. Williams (§)

From the Department of Cancer Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties. Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166. We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding. Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60. Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type). We have also identified a number of other residues that influence dsRNA binding to varying degrees. Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo. However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.


INTRODUCTION

The interferon-induced kinase, PKR, (^1)is a serine/threonine kinase that has the unusual property of being dependent on double-stranded (ds) RNA for its activation(1, 2, 3) . PKR has two distinct kinase activities: 1) an autophosphorylation activity that requires the presence of dsRNA and results in the phosphorylation of several serine residues within PKR and 2) dsRNA-independent phosphorylation of substrates by phosphorylated, activated PKR(4) . The best known substrate for PKR is the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) whose phosphorylation results in the global shutdown of protein synthesis(5, 6, 7) . Other substrates have recently been described and include the NF-kappaB inhibitor, IkappaB (8) and the HIV protein Tat. (^2)

PKR becomes activated during viral infections by viral dsRNA or replicative intermediates with dsRNA-like structure, and, therefore, different viruses have developed various mechanisms to perturb PKR activity (for review, see (9) ). The role of PKR in both the antiviral and antiproliferative activities of interferon is largely modulated by the phosphorylation of eIF2alpha(10, 11, 12, 13) . The expression of PKR in Saccharomyces cerevisiae results in a growth-suppressive phenotype that can be reversed by the co-expression of the N-terminal half of the protein(13) . The loss of PKR activity through the overexpression of a catalytically inactive form of PKR (K296R) has been shown to result in the malignant transformation of NIH3T3 cells and formation of tumors in nude mice. However, the exact mechanism by which PKR mediates this tumor-suppressor function was not established(11, 12) .

Previous work by several groups has shown that the dsRNA binding site of PKR is present as two nearly identical domains comprising amino acids 55-75 (domain I) and 145-166 (domain II)(13, 14, 15, 16, 17, 18) . The PKR genes from human, mouse, and rat all share a high degree of homology within these domains and have the same overall domain structure. Furthermore, several other dsRNA-binding proteins from a wide variety of organisms also share this domain(19) . These include the human TAR-binding protein, Escherichia coli RNase III, Drosophila staufen protein, vaccinia virus E3L protein, Xenopus RNA binding protein A, and the Schizosaccharomyces pombe pac1 protein. These dsRNA binding domains contain many basic amino acids and have a high probability of forming alpha helices.

Domain I in human PKR has been found to be essential for the binding of dsRNA while domain II was not absolutely required(14) . More refined approaches have clarified the dsRNA binding motifs further. Feng et al.(15) utilized block deletion analysis to show that the deletion of amino acids 39-50 or 58-69 resulted in an almost complete loss of dsRNA binding ability (3% of wild type). Green and Mathews (14) used linker scanning mutagenesis, in which blocks of 3 amino acids were mutated to Gly-Ala-Leu, to perform a detailed study of the PKR N-terminal region and found that the mutants that had the most deleterious effects to binding were clustered around domain I. McCormack et al.(20) found that the mutation of lysine 64 to glutamic acid totally ablated dsRNA binding.

While these studies provided a general outline of the dsRNA binding domain, they did not identify the individual residues that may be important for binding dsRNA. In order to more fully understand the structure-function relationship in the binding of PKR to dsRNA, we investigated the contribution of individual amino acids to dsRNA binding. Utilizing both alanine-scanning and random mutagenesis, we found 2 critical residues that were absolutely required for dsRNA binding (glycine 57 and lysine 60). Mutations of 2 additional residues (lysine 64 and leucine 75) resulted in less than 10% binding compared to wild type. We also identified a number of other residues that affected dsRNA binding to various degrees. In vitro and in vivo functional assays of the various mutants using bacterial, yeast, and mammalian cell expression systems confirmed the importance of these residues for PKR function.


MATERIALS AND METHODS

Mutagenesis of PKR

Site-directed mutagenesis was performed using the method of Deng and Nickoloff(21) . Briefly, 1 selection and 11 mutant oligonucleotides (21-24-mers) were synthesized. The selection oligonucleotide (5`-GATTATCCCTCGGAATTAC-3`) mutates the unique NcoI site 289 base pairs downstream from the PKR ATG and changes methionine 98 to leucine. Each mutant oligonucleotide along with the selection oligonucleotide (50 ng each) was added to 200 ng of PKR cDNA (cloned into pBluescript KS-) and annealed by brief boiling followed by cooling on ice. Following annealing, the DNA strands were elongated and ligated using T4 DNA polymerase and T4 DNA ligase before being transformed into the E. coli strain mutS (thi, supE, Delta(lac-proAB), [mutS::Tn10][F`proAB, lac^q ZDeltaM15]), which is deficient for mismatch repair. Transformants were pooled and plasmid DNA was digested with NcoI before being transformed into E. coli strain DH5alpha. Plasmids were sequenced to confirm mutagenesis.

For random mutagenesis, a modification of the method of Busby et al.(22) was used. Briefly, a 1 µg/ml solution of PKR cDNA encoding the first 183 amino acids of PKR cloned into the yeast expression vector pEMBLyex4 was treated with 1 M hydroxylamine hydrochloride, 50 mM sodium pyrophosphate, and 2 mM EDTA for 4 h at 75 °C. Hydroxylamine reacts with dsDNA to create N-4-hydroxycytosine which, upon plasmid replication, results in cytosine-to-thymine and guanine-to-adenine substitutions. This protocol yielded mutations at a frequency of 3% as measured by uracil auxotrophy. The mutagenized DNA was transformed directly into the yeast strain H86 as described previously(13) , and loss of dsRNA binding was measured as a result of the inability to inhibit PKR activity.

In Vitro Transcription and Translation

Linearized plasmid DNA (5 µg) was transcribed in vitro with phage T7 RNA polymerase (Life Technologies, Inc.) according to the manufacturer's instructions (Promega), and the complementary RNA yield was measured at A. For in vitro translation, 1 µg of RNA was mixed with 35 µl of nuclease-treated rabbit reticulocyte lysate (Promega), 1 µl of RNasin ribonuclease inhibitor (at 40 units/µl), 1 µl of 1 mM amino acids mixture (minus methionine), 4 µl of [S]methionine at 10 µCi (370 kBq)/ml, and H(2)O to 50 µl. The reaction was performed at 30 °C for 60 min.

Purification of Bacterially Expressed PKR

For wild type PKR, the cDNA was amplified by polymerase chain reaction and inserted into the NdeI/BamHI site of the expression vector pET15b (Novagen Inc). For mutant PKRs, the HindIII/PstI fragment was subcloned into the BamHI site of the vector pET15b and transformed into the E. coli strain BL21(DE3). Transformed bacteria (500 ml) were grown to A = 0.6 before gene expression was induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside to 2 mM, and incubation continued for 3 h. Bacteria were pelleted by centrifugation at 3000 times g and resuspended in 8 ml of binding buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 5 mM imidazole). Cells were lysed by sonication (5 times 60 s at 20,000 cycles), the lysate was centrifuged at 14,000 times g, and the supernatant was filtered through a 0.45-µm filter before being loaded upon a nickel-agarose column. The histidine-tagged PKR protein was eluted according to the manufacturer's instructions and concentrated using a Centricon®-3 ultrafiltration unit (Amicon Inc.) prior to gel filtration chromatography on a Superose-12 FPLC column (Pharmacia Biotech Inc.). The PKR fractions were pooled, diluted in buffer DBGA (10 mM Tris-HCl, pH 7.6, 50 mM KCl, 2 mM magnesium acetate, 20% glycerol, and 7 mM 2-mercaptoethanol), concentrated by ultrafiltration as above, and stored at -80 °C.

Activation Assays

PKR protein (400 ng) was brought up to a volume of 30 µl in buffer DBGA before the addition of 20 µl of buffer DBGB (DBGA plus 2.5 mM MnCl(2)) and [-P]ATP (50 Ci/mmol) to 2 mM for a final volume of 60 µl. The reactions were incubated at 30 °C for 15 min before the addition of SDS loading buffer and then boiled for 3 min prior to loading on 10% SDS-PAGE gels and autoradiography.

Poly(I):Poly(C) Binding Assay

In vitro translated products were tested for poly(I):poly(C) binding using poly(I):poly(C)-agarose (Pharmacia). In vitro translated protein (5 µl) was added to 50 µl of poly(I):poly(C)-agarose beads, which had previously been washed three times in binding buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM magnesium acetate, 1 mM dithiothreitol, 0.5% Nonidet P-40, and 10% glycerol). The beads and protein were incubated at 30 °C for 60 min with intermittent mixing before being washed three times in 1 ml of binding buffer and resuspended in 50 µl of SDS-PAGE buffer. Bound proteins were analyzed by SDS-PAGE and quantitated by densitometery of autoradiographs. The percentage of bound protein was calculated as a percentage of the total amount of protein added in the assay (5 µl).

Expression of PKR Mutants in Yeast

Mutant PKR cDNAs were cloned into the yeast expression vector pEMBLyex4 and expressed in S. cerevisiae as described previously(13) . The expression plasmids were transformed into the haploid yeast strain W303a and grown at 30 °C on agar plates containing synthetic medium lacking uracil and with 2% glucose as the sole carbon source. Transformants were then grown to stationary phase in liquid synthetic media containing 0.1% glucose and lacking uracil. Following washing, the cells were resuspended to A = 0.1 in synthetic media containing 2% galactose and lacking uracil, and incubation continued at 30 °C. At various time intervals, cell growth was monitored by measuring absorbance at 600 nm and by direct cell counting.

Immunopurification of PKR from Yeast

Yeast cells (20 ml) expressing mutant PKR proteins were harvested 24 h after induction of PKR with galactose by centrifugation at 1000 times g. Protein was extracted using the glass bead technique as described(23) .

Expression in Mammalian Cells

The reporter plasmid used was pBI which contained the NF-kappaB enhancer site from the human tumor necrosis factor alpha promotor (positions 635 to 621 as a single copy in the forward orientation) (24) upstream of the minimal tk promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene. Mutant PKR cDNAs were cloned into the vector pcDNA1neo (Invitrogen) and expressed constitutively under the cytomegalovirus promoter. The vector RSV-betagal was used to standardize transfection efficiency. The plasmids were transiently transfected into RAW 264.7 (mouse macrophage) cells, previously starved for 4 h with 0.1% fetal bovine serum, with the DEAE-dextran/chloroquine method as described previously (8) using 20 µg of both pBI and pcDNA1neo-PKR plasmid and 5 µg of RSV-betagal plasmid per 5 times 10^6 cells. Following 24 h of incubation, cells were washed twice with phosphate-buffered saline and the medium replaced with or without poly(I):poly(C) (100 µg/ml). The cells were incubated for an additional 8 h before being harvested in 1 ml of TEN (40 mM Tris, pH 8, 1 mM EDTA, and 150 mM NaCl) and centrifuged in a Microfuge at 14,000 times g for 10 s and 4 °C. The cell pellets were resuspended in 100 µl of 0.25 M Tris, pH 8, and sonicated at 20,000 cycles for 10 s. Lysates were centrifuged for 10 min at 4 °C, and the supernatants were used for both CAT (nonchromatographic) and beta-galactosidase assays as described previously(25) .


RESULTS

Alanine Scanning Mutagenesis of PKR

We and others have previously identified two conserved dsRNA binding motifs located within the N-terminal half of PKR(13, 14, 15, 16, 18, 19, 20) . The most conserved regions of these domains are found in two 20-21 amino acid stretches, comprising residues 55-75 and 145-165 of human PKR. Alignment of the human, mouse, and rat PKR protein sequences identified a core motif within the dsRNA binding domain (Fig. 1A). Among these three proteins, 17 out of 20 amino acids are conserved while 15 out of 20 are identical. Using this information we designed 11 alanine-scanning mutants (Table 1) and tested each for the ability to bind dsRNA. Alanine was chosen as a substitute because it eliminates the amino acid side chain beyond the beta-carbon without altering the main chain conformation (as a glycine or proline substitution would). Furthermore, alanine would not impose large steric or electrostatic effects and has an intermediate hydrophobicity.


Figure 1: Alignment of various dsRNA binding domains. A, human(34) , mouse(15) , and rat (GenBank L29281) PKR protein sequences were aligned, and the conserved residues are shaded. The consensus domain is indicated. B, the domains of other dsRNA binding proteins were aligned, and the conserved residues are shaded. The sequences are the vaccinia virus E3L protein(35) , the E. coli RNase III(36) , the Drosophila staufen protein(37) , the human TAR-binding protein(38) , and the Xenopus RNA-binding protein A(19) . The consensus domain is indicated.





Mutant proteins were synthesized using in vitro transcription/translation reactions (using [S]methionine) and then tested for dsRNA binding activity using poly(I):poly(C) agarose in the presence of high salt (300 mM NaCl) as described previously(16) . The results were analyzed by taking input protein as 100% and comparing the amount bound as a percentage of input. Interestingly, the mutant G57A migrated with an electrophoretic mobility 5-6 kDa less than that of wild type and all other mutant proteins (Fig. 2A, lane G57A). As with all mutants, sequencing was performed to confirm the correct mutation, and, in the case of G57A, three independent experiments yielded the same result. Green and Mathews (14) have previously noted a similar phenomenon with a mutant that changed residues 58-60 from Arg-Ser-Lys to Gly-Ala-Lys.


Figure 2: Poly(I):poly(C) binding assays of PKR mutants. A, in vitro translated PKR mutants. Five µl of in vitro transcribed/translated and S-labeled PKR proteins were analyzed by SDS-PAGE and autoradiography as indicated. B, poly(I):poly(C) agarose binding assays of PKR mutants from A. The mutant kinase proteins from A (5 µl) were added to poly(I):poly(C) agarose beads and incubated at 30 °C for 30 min before extensive washing with binding buffer. Bound protein was analyzed by SDS-PAGE. Quantitation is presented in Table 1.



The results of the binding assay showed a marked variation in the dsRNA binding abilities of the mutants ( Fig. 2and Table 1). Two mutants were totally deficient in binding (G57A and K60A) while two others (K64A and L75A) had binding efficiencies below 10% of wild type. Four other mutants had intermediate binding activities (S59A, K61A, E62A, and K69A) ranging from 28-68%, while three mutations had no effect on binding as compared to wild type (G55A, V72A, and E73A). The importance to dsRNA binding of the basic lysine residues at positions 60 and 64, but not 61 or 69, indicates that not all lysine residues contribute equally to binding.

Random Mutagenesis of PKR

The region of homology among the various dsRNA-binding proteins extends beyond domains I and II (19) although the homologies within these regions are low. Patel et al.(26) have shown that certain amino acids outside the core domains are important to dsRNA binding, most likely by contributing to protein stability rather than directly to binding. To investigate the possibility that these other conserved residues play a role in dsRNA binding, we randomly mutagenized the entire dsRNA binding region of human PKR. DNA encoding the first 183 amino acids of PKR was cloned into the yeast expression vector pEMBLyex4 and treated with N-4-hydroxycytosine to induce random mutations.

Previous work from our laboratory had shown that the expression of PKR in an inducible yeast cell system resulted in a growth suppressor phenotype which could be reversed by co-expressing the N-terminal half of PKR(13) . The mechanism by which this dominant-negative phenotype occurs is thought to involve the sequestering of dsRNA, thus depriving wild type PKR of an activating species. Hence, by co-expressing mutant N-terminal PKR with the wild type protein, we could assay for plasmids that no longer suppressed PKR activity. Using this approach, we analyzed 900 colonies, of which 64 gave a growth phenotype, a mutation rate of 7.1%. Sequencing of these 64 mutants revealed 7 which contained detectable amino acid mutations (Table 2).



Two of the 7 random mutations, A63V and L75F, fell within domain I. Both alanine 63 and leucine 75 are highly conserved among dsRNA binding proteins (Fig. 1) and therefore would be expected to be critical to binding. The latter result confirms data from the alanine scanning mutations (Fig. 2) which indicated that leucine 75 is a critical amino acid for dsRNA binding. The other random mutations fall outside the highly conserved core domain, indicating that these regions also contribute to the ability to bind dsRNA. Three of the seven mutants were found to contain the same mutation (R40S). Arginine 40 is conserved among PKR genes but not among the other dsRNA binding proteins, although there appears to be a requirement for a basic amino acid at position 39(19) . The two other random mutant sites (V24G and S83C) are not conserved in the dsRNA binding proteins and perhaps involve changes in secondary structure.

Functional Assays of Mutants

In order to test whether the loss of the ability to bind to dsRNA correlated with the loss of activation, three of the most severely affected mutants (G57A, K60A, and K64A) along with wild type PKR were subcloned into the bacterial expression vector pET15b and expressed as polyhistidine-tagged fusion proteins. The various PKR proteins were purified from crude bacterial lysates by nickel affinity chromatography followed by gel filtration FPLC using a Superose-12 column and tested in activation assays (Fig. 3). Coomassie staining of the purified proteins confirmed that equal amounts of PKR were present in each sample (Fig. 3A). Wild type PKR was active while the mutant PKRs were all profoundly deficient in their ability to become activated (Fig. 3B). These results indicate that the ability to bind dsRNA is directly related to the ability to become activated. It should be noted that no dsRNA was added into this activation assay as it was found to increase activity only marginally. (^3)The fact that wild type PKR is activated in the absence of dsRNA is a phenomenon that has previously been observed with GST-PKR fusion proteins expressed in bacteria and may be due to PKR activation by bacterial dsRNA or incorrect folding of the fusion protein(8) . The GST-PKR response to dsRNA was able to be restored by denaturation/renaturation indicating that changes in secondary structure may accompany activation. (^4)


Figure 3: Activation assays of bacterially expressed PKR mutants. Bacterially expressed histidine-tagged wild type and mutant (G57A, K60A, and K64A) PKR proteins were purified from bacteria by nickel agarose and gel filtration chromatography before being assayed for activity. A, Coomassie stain. PKR from activation assays were stained to ensure that equal amounts of PKR were present. B, activation assays. PKR proteins were activated in the presence of [-P]ATP for 15 min at 30 °C before analysis by SDS-PAGE and autoradiography.



Expression of Mutants in Yeast Cells

The expression of PKR in a yeast system has been used previously to investigate the functional activity of PKR(13, 27, 28) . PKR expression results in its activation and the subsequent phosphorylation of the yeast eIF2alpha (which shares a high degree of homology with human eIF2alpha)(13, 28) . The species responsible for activating PKR in yeast is unknown, although co-expression of the PKR dsRNA binding domain restores normal growth. Furthermore, Feng et al.(15) have previously shown that a PKR mutant containing an 11 amino acid deletion in domain I (Delta58-69), which could not bind dsRNA, was unable to suppress growth in yeast cells. We therefore decided to utilize this system to assay the function of the various alanine-substituted PKR mutants in order to correlate dsRNA binding ability with PKR activity. The mutants were subcloned into the vector pEMBLyex4 and transformed into the yeast strain W303a. PKR protein expression was controlled via the GAL10-CYC1 hybrid promotor, whereby growth on galactose as the sole carbon source activated expression. Following induction, cell growth was monitored by measuring absorbance at 600 nm as well as by direct cell counting. The results from six of the mutants, representing the entire spectrum of dsRNA ability, are shown (Fig. 4A).


Figure 4: Expression of PKR mutants in yeast cells. A, growth curves of yeast cells expressing PKR mutants. Selected mutant PKR cDNAs were subcloned into the galactose-inducible expression vector pEMBLyex4, and the plasmids were transformed into the yeast strain W303a. Expression was induced by growth on galactose and growth was monitored by measuring A and direct cell counting. The PKR mutants were chosen so as to represent the entire spectrum of dsRNA binding activity. B, Western blot of PKR expressed in yeast cells. The yeast cells expressing mutants that grew in the presence of PKR (G57A, K60A, and K64A) as well as a control strain (E62A) were lysed and PKR protein immunopurified using monoclonal antibodies to PKR. Following blotting, PKR protein was detected using a PKR polyclonal antibody.



The results indicate that the ability to bind dsRNA correlates with the ability to inhibit cell growth. Mutants with as little as 28% of wild type binding activity (K61A) were able to totally repress yeast cell growth. Yeast strains containing the mutants previously shown not to bind dsRNA (G57A, K60A, and K64A) did not have any major growth-suppressive phenotypes, although K64A, which had 5.3% dsRNA binding activity, grew at a reduced rate compared to K60A. All other mutants had growth-suppressed phenotypes indicating that dsRNA binding activity as low as 28% (K61A) allows enough active PKR to give a complete suppression of growth. Western blot analysis was performed to ensure that the growth suppression was not the result of differing levels of PKR (Fig. 4B). The results indicate that those strains that were not growth-suppressed (G57A, K60A, and K64A) had PKR levels similar to a strain that was growth-suppressed (E62A). Therefore, PKR mutants retaining low dsRNA binding activity were nonetheless potent inhibitors of cell growth.

Expression of Mutants in Mammalian Cells

The loss of PKR function in NIH3T3 cells through overexpression of the catalytically inactive PKR mutants (including K296R) has been shown to result in the formation of tumors in nude mice, leading to the suggestion that PKR is a tumor suppressor gene(11, 12) . The manner in which K296R has a dominant-negative effect on wild type PKR is unknown. Recent work from our laboratory has shown that PKR is involved in the dsRNA-mediated regulation of the transcription factor NF-kappaB(8, 29) . PKR was shown to phosphorylate the NF-kappaB inhibitor, IkappaB, resulting in the release of NF-kappaB. An in vivo system was developed in which the treatment of mouse macrophage cells with dsRNA resulted in the activation of endogenous PKR which in turn activated NF-kappaB and resulted in the up-regulation of a choramphenicol acetyltransferase (CAT) reporter gene. This system allows for a direct assay of PKR activity in cells. Wild type PKR activity was ablated when K296R was expressed in these cells, as is the case for yeast.

As stated earlier, the mechanism by which the K296R mutant acts in a dominant-negative manner over wild type PKR is unknown. We considered three possibilities: (i) K296R is able to bind to and sequester an activating species, possibly dsRNA; (ii) K296R is able to heterodimerize with wild type PKR and thus inhibit its activation; or (iii) K296R binds to kinase substrates, protecting them from phosphorylation. The mutants generated in this study allow us to investigate the first possibility. Consequently, we decided to utilize the in vivo system to test the possibility that K296R regulates PKR by binding dsRNA.

The mutants K60A, K64A, and K296R were cloned into the vector pcDNA1neo downstream of the cytomegalovirus promotor. Plasmids containing the mutant kinase cDNAs were transiently transfected into murine macrophage cells (RAW 264.7) along with the reporter plasmid, pBI, which contains the CAT gene downstream of a minimal thymidine kinase promotor and an NF-kappaB enhancer site derived from the human tumor necrosis factor alpha promotor. After 24 h of transfection, cells were treated plus or minus dsRNA (100 µg/ml) for 8 h, and the level of CAT activity was measured (Table 3). dsRNA treatment of cells transfected with pBI and pcDNAIneo increased CAT activity 6-fold compared to cells not treated with dsRNA. Co-transfection with the K296R mutant reduced the CAT induction to a 1.9-fold increase, as previously observed(8) , while the mutants K60A and K64A gave intermediate inductions (3.7- and 4.2-fold, respectively). Since K60A and K64A, which do not bind dsRNA, were found to have an effect on PKR activity (Fig. 3B), we conclude that dsRNA binding is most likely not the mechanism by which mutant forms of the kinase are trans-dominant over wild type.




DISCUSSION

The results presented in this study extend and refine previous investigations of the dsRNA binding domain of PKR. First, we have defined 2 residues which are absolutely required for dsRNA binding (Gly-57 and Lys-60) as well as 2 other residues (Lys-64 and Leu-75) whose mutation to alanine is highly detrimental to binding. Second, domain I is more important to dsRNA binding than domain II as all our mutations were in domain I. Third, the ability to bind dsRNA correlates directly with PKR function in vitro and in vivo. Finally, dsRNA binding is not the mechanism by which mutant PKR is trans-dominant over wild type PKR.

The purpose of the alanine-scanning mutagenesis was to replace amino acids without altering the backbone structure of the polypeptide chain. In such a way, the contribution of individual amino acid side chains could be analyzed. Our results indicate that the lysine residues at positions 60, 61, 64, and 69 are important for binding dsRNA, particularly lysine 60, which is absolutely required for binding activity. These basic residues fall within a region of PKR which has a high probability of existing in an alpha helical conformation(30) . Helical wheel projections of this region indicate that the side chains of lysines 60, 61, 64, and 69 would all be oriented on one side of a putative helix. Other groups have postulated that the presence of multiple positive charges on one side of a helix would assist in the interaction of this motif with dsRNA(14, 18) , and our data support this hypothesis. The importance of the positively charged lysine residues within this domain may be reflected by the fact that PKR possesses a relatively nonspecific affinity for dsRNA and other polyanions.

Our results also indicate the importance of stable secondary structure within the dsRNA binding domain of PKR. For example, the mutant G57A had an electrophoretic mobility of 5-6 kDa less than all other mutants. Computer-derived secondary structure predictions indicate a high probability of alpha helix formation within the sequence beginning with amino acid 58. Substitution of glycine 57 (a strong helix-breaking residue) to alanine (a strong helix-former) might function to stabilize or extend the secondary structure in this region, resulting in the altered electrophoretic mobility. The mutant G55A had normal mobility, further indicating a change involving the alpha helix to be likely. Green and Mathews (14) found that mutating amino acids 58-60 (Arg-Ser-Lys to Gly-Ala-Lys) also had an altered mobility of M(r) 4000.

Another mutant which shows a greatly reduced dsRNA binding capacity is L75A. The leucine at this position is highly conserved among dsRNA binding proteins, and, indeed, those proteins which do lack leucine at this site have in its place the bulky hydrophobic residues valine, phenylalanine, or methionine (Fig. 1). There are 4 alanine residues that are highly conserved in the dsRNA binding region (Fig. 1) which we did not mutate yet may still be important in dsRNA binding. The random mutagenesis approach clearly indicates that Ala-63 is important. Mutation of alanine 174 to valine in the vaccinia dsRNA binding protein E3L (equivalent to Ala-67 in PKR) abrogated binding indicating that this is also an important residue(31) .

The work presented here also confirms the importance of domain I for dsRNA binding over domain II. All our mutations were in domain I and resulted in dramatic changes in binding ability even though domain II was intact. Others have shown that mutations in residues in domain II have a less deleterious effect than mutation to similar residues in domain I(14) .

The region of homology between PKR and the family of dsRNA binding proteins extends beyond the highly conserved binding domains to encompass a less homologous region. St. Johnston et al.(19) observed this region to extend from amino acids 11-77 and 101-167. The random mutation data presented here indicate that at least 3 amino acids outside domain I, but within these regions of lesser homology, also contribute to dsRNA binding, possibly by stabilizing the secondary structure. Patel et al.(26) also found that nonconserved mutations at amino acids 18 and 19 were deleterious to binding.

While many studies have investigated in vitro dsRNA binding activity, little has been done to examine the effects of these mutations in vivo. Expression levels of the catalytically inactive mutant K296R have been reported to be 30-50-fold higher than wild type PKR at the translational level(27, 32) . The dsRNA binding mutant, K64E, has also been reported to be expressed at higher levels than wild type PKR, and this was thought to be due to the disruption of PKR function. The results presented here using the yeast expression system indicate that the ability of PKR to bind dsRNA correlates strongly with its ability to function in growth suppression. Mutants G57A, K60A, and K64A, all severely deficient in dsRNA binding, were also unable to inhibit the growth of yeast (Fig. 4). The activating species in yeast has never been identified although yeast extracts have been shown to activate PKR from mammalian cell extracts(13) . The data presented here indicate that dsRNA, or single-stranded RNA possessing dsRNA secondary structure (perhaps in a fashion similar to HIV-1 TAR mRNA(33) ), is most likely the activating species rather than a polyanionic species that could act as nonspecific activator.

Using the mutants generated in this study, we have investigated the mechanism by which mutant PKR is trans-dominant over wild type PKR. The loss of PKR activity through the overexpression of the catalytically inactive PKR mutant, K296R, has been shown to result in the malignant transformation of NIH3T3 cells and formation of tumors in nude mice(11, 12) . While the antiproliferative and antiviral actions of PKR have been shown to be mediated through eIF2alpha phosphorylation, the mechanism of its tumor-suppressing activity remains unknown. K296R-expressing cells are still able to phosphorylate eIF2alpha after interferon treatment and encephalomyocarditis virus infection (10) indicating that the antiviral response is still intact in these cells.

We considered three possible mechanisms: (i) K296R was able to bind to and sequester an activating species (possibly dsRNA); (ii) K296R was able to heterodimerize with wild type PKR and inhibit its activation; and (iii) K296R bound to other kinase substrates thus protecting them from phosphorylation. Our results indicate that mutants K60A and K64A had a moderate effect on PKR function, and, therefore, we conclude that the sequestering of dsRNA is not the mechanism of trans-dominance, leaving the other two possibilities. The means by which PKR becomes active is still not clear, and evidence for either inter- and intramolecular phosphorylation is not conclusive. Furthermore, the substrate by which PKR mediates tumor suppression is also unknown so no conclusions as to either possibility can be drawn at the present time. We are currently testing these two mutants for their ability to form tumors in nude mice. Finally, the mutants generated in this study will be useful in investigating the mechanism of PKR activation enabling us to investigate the mechanism of tumor suppression mediated by PKR.


FOOTNOTES

*
This work was supported in part by NIAID Grant R01 A1 34039-02 from the National Institutes of Health and a grant from the Human Frontiers Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Cancer Biology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9652; Fax: 216-445-6269.

(^1)
The abbreviations used are: PKR, protein kinase, RNA-activated; CAT, chloramphenicol acetyltransferase; ds, double-stranded; eIF, eukaryotic initiation factor; poly(I):poly(C), polyinosonic acid:polycytidylic acid; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

(^2)
N. A. J. McMillan, D. P. Siderovski, J. Galabru, W. M. Toone, T. W. Mak, A. G. Hovanessian, and B. R. G. Williams, submitted for publication.

(^3)
N. A. J. McMillan, B. W. Carpick, B. Hollis, W. M. Toone, M. Zamanian-Daryoush, and B. R. G. Williams, unpublished observation.

(^4)
A. Kumar and B. R. G. Williams, unpublished observation.


ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. Glen Barber and Michael Katze for PKR polyclonal antibody. We also would like to thank Dr. Jahar Haque and Aseem Kumar for helpful discussions and Drs. Robert Silverman and Ara Hovanessian for critical reading of this manuscript.


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