(Received for publication, September 15, 1994)
From the
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.
The interferon-induced kinase, PKR, ()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
subunit of eukaryotic initiation factor
2 (eIF2
) whose phosphorylation results in the global shutdown of
protein synthesis(5, 6, 7) . Other substrates
have recently been described and include the NF-
B inhibitor,
I
B (8) and the HIV protein Tat. (
)
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
eIF2(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 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.
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.
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.
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.
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.
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.
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-B enhancer site
derived from the human tumor necrosis factor
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.
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 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 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
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
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
eIF2 phosphorylation, the mechanism of its tumor-suppressing
activity remains unknown. K296R-expressing cells are still able to
phosphorylate eIF2
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.