From the Laboratory of Gene Regulation and
Development and the ¶ Laboratory of Molecular Growth Regulation,
NICHHD, National Institutes of Health, Bethesda, Maryland 20892, the
§ Jefferson Center for Biomedical Research, Thomas Jefferson
University, Doylestown, Pennsylvania 18901, and the ** Department of
Biochemistry and Molecular Biology, University of Medicine and
Dentistry of New Jersey, New Jersey Medical School,
Newark, New Jersey 07103
Received for publication, March 8, 2001, and in revised form, April 26, 2001
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ABSTRACT |
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Protein kinase PKR is activated by
double-stranded RNA (dsRNA) and phosphorylates translation initiation
factor 2 The human double-stranded RNA
(dsRNA)1-dependent
protein kinase PKR is transcriptionally induced by interferon and
activated in virus-infected cells by dsRNAs produced during the virus
life cycle. PKR interferes with virus replication by phosphorylating the PKR kinase activity is stimulated in vitro by dsRNA, and the
N-terminal 168 amino acids of the protein contain two copies of a
dsRNA-binding motif (DRBMs I and II; Fig.
1A) that is also present in other dsRNA-binding proteins
(5). Point mutations in the DRBMs which impair dsRNA binding by PKR
in vitro reduce the ability of PKR to phosphorylate eIF2 to inhibit protein synthesis in virus-infected mammalian
cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required
for activation by dsRNA. There is strong evidence that PKR activation
requires dimerization, but the role of dsRNA in dimer formation is
controversial. By making alanine substitutions predicted to remove
increasing numbers of side chain contacts between the DRBMs and dsRNA,
we found that dimerization of full-length PKR in yeast was impaired by
the minimal combinations of mutations required to impair dsRNA binding
in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to
abolish dimerization without affecting dsRNA binding, destroyed both
activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on
PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the
activation loop were found here to be critical for kinase activity in
yeast. Using an antibody specific for phosphorylated Thr-451, we showed
that Thr-451 phosphorylation is stimulated by dsRNA binding. Our
results provide strong evidence that dsRNA binding is required for
dimerization of full-length PKR molecules in vivo, leading
to autophosphorylation in the activation loop and stimulation of the
eIF2
kinase function of PKR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of translation initiation factor 2 (eIF2
),
converting eIF2 from a substrate to an inhibitor of its guanine
nucleotide exchange factor, eIF2B. This reduction in recycling of eIF2
by eIF2B leads to a general inhibition of translation that limits viral
protein synthesis (1). The yeast Saccharomyces cerevisiae harbors an eIF2
kinase known as GCN2 which is activated by uncharged tRNA when cells are starved for amino acids. Limited phosphorylation of
eIF2
by GCN2 under starvation conditions leads to increased translation of GCN4 mRNA, encoding a transcriptional
activator of amino acid biosynthetic genes (for review, see Ref. 2). Low level expression of PKR in yeast produces a moderate level of
eIF2
phosphorylation that is sufficient to induce GCN4
expression without inhibiting general protein synthesis (3). When PKR is expressed at higher levels, eIF2
phosphorylation increases to the
point where general translation and yeast cell growth are strongly
inhibited (3, 4).
in yeast cells, supporting the idea that the DRBMs mediate the
stimulatory effect of dsRNA on PKR kinase activity (4, 6). dsRNA
stimulates the autokinase activity of PKR (7-9), and multiple
autophosphorylation sites have been identified in the dsRNA binding and
kinase domains of PKR (10-13). Alanine substitution of the
autophosphorylation site at Thr-258 (T258A), located between
the DRBMs and kinase domain, produced a modest reduction in PKR
function in yeast and mammalian cells (10). In contrast, substitution
of the autophosphorylation site at Thr-446 (T446A) in the
activation loop of the kinase domain substantially reduced kinase
activity. The T451A mutation in the activation loop
completely destroyed kinase function, but the evidence that this site
is autophosphorylated was inconclusive (11).
View larger version (35K):
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Fig. 1.
Locations of mutations made in PKR.
A, schematic representation of the wild-type PKR
protein showing the locations of DRBMs I and II between amino acid
residues 10-78 and 101-168, respectively, and the third basic domain
between residues 232 and 261. The kinase domain spans residues 267-551
and contains the conserved subdomains labeled I-XI. A
deletion mutant ( 244-296) lacking a predicted
dimerization interface (25) is indicated below the schematic.
B, amino acid alignment of Xlrbpa-2 with PKR DRBMs I and II.
Three dsRNA contact regions in Xlrbpa-2 which interact independently
with dsRNA (35) are indicated, with residues making side chain or
backbone interactions with the RNA shown in boldface.
Residues in PKR homologous to those in Xlrbpa-2 making side chain
interactions with dsRNA were replaced by Ala in this study and are
indicated by (a dot) as is the replacement of Ala-67 by Glu
(A67E mutation). The locations of
-helices and
-sheets
in the structure of the PKR DRBMs are indicated below the sequence (17,
34). C, FLAG-tagged PKR alleles
(FL-PKR) analyzed in this study. The alleles with mutations
in the DRBMs are designated by the number of Ala substitutions in DRBM
I or II, and the specific substitutions are indicated in
parentheses. WT, wild-type.
It has been proposed that dsRNA binding overcomes a negative effect of the DRBMs on PKR catalytic function, based on the finding that extensive deletions in the N-terminal region of the protein that removed both DRBMs did not abolish (14), and even enhanced (15) kinase activity. The N-terminal and C-terminal halves of PKR physically interacted in the two-hybrid assay (16), and NMR data indicate that DRBM-II, but not DRBM-I, binds to the kinase domain (17). Additionally, dsRNA binding induces a conformational change in PKR (18, 19). These results have led to models in which the DRBMs (or just DRBM-II) bind to the kinase domain and interfere with its enzymatic function, and this inhibitory interaction is eliminated by a conformational change in PKR elicited by dsRNA binding to the DRBMs (17, 20).
There is also evidence that dsRNA activates PKR by promoting dimerization of the enzyme and intermolecular autophosphorylation. The fact that PKR activation exhibits second-order kinetics with respect to protein concentration suggests that the active form of PKR is a dimer (9), and the enzyme was purified as a phosphorylated dimer (21). The observation that two inactive PKR alleles containing deletions of DRBM-I or DRBM-II functionally complemented was most readily explained by formation of active heterodimers (6). It was also shown that an N-terminal segment of PKR containing the DRBMs could be coimmunoprecipitated with full-length PKR from transfected COS cells (14), and N-terminal segments were found to interact with themselves and with full-length PKR in various interaction assays (22-25).
PKR activation is inhibited by high concentrations of dsRNA, leading to a bell-shaped dsRNA activation curve (26). This behavior has been explained by proposing that dimerization requires binding of two PKR molecules to the same dsRNA, as high dsRNA concentrations would favor dissociation of dimers into inactive monomers bound to different dsRNA molecules (9). Consistently, high level activation of PKR requires a minimum length of dsRNA (~40 base pairs) which is considerably larger than the binding site for two DRBMs in a single PKR molecule (11-16 base pairs) (18, 27, 28). These data, combined with the observation that PKR can autophosphorylate in trans (29, 30), have led to the idea that binding of two PKR molecules to the same dsRNA promotes dimerization and intermolecular autophosphorylation events required for substrate phosphorylation.
Although there is some evidence that PKR dimerization is strongly dependent on dsRNA binding (19, 22), many studies suggest that dimerization is relatively unaffected by point mutations in the DRBMs that impair dsRNA binding in vitro (14, 22-24, 30). The results of the latter studies suggest that protein-protein interactions can mediate PKR dimerization in the absence of dsRNA binding. Wu and Kaufman (20) found that dimerization by isolated DRBMs could occur independently of dsRNA binding but that dimerization of full-length PKR with the isolated DRBM was dsRNA-dependent. To account for these findings, they proposed that dsRNA binding to full-length PKR was required primarily to dissociate the DRBMs from the kinase domain and unmask a dimerization surface in the DRBMs. However, recent evidence that only DRBM-II interacts with the kinase domain implies that DRBM-I is exposed and available for dimerization constitutively (17). A second dimerization domain in PKR was localized to the 244-296 interval, overlapping the N-terminal portion of the kinase domain (25). Nanduri et al. (17) proposed that this region becomes available for dimerization after dissociation of DRBM-II from the kinase domain upon dsRNA binding and provides the critical dimerization surface in the enzyme. However, the importance of the 244-296 interval for dimerization by full-length PKR has not been addressed experimentally.
An alternative model for PKR activation was proposed by Patel and Sen (31) in which dimerization occurs independently of dsRNA binding, and dsRNA (or alternative stimulatory ligands) activate a preformed PKR dimer by producing a conformational change in the protein. It was found that mutating residue Ala-67 to Glu in DRBM-I abolished dimerization of the N-terminal domain of PKR without affecting dsRNA binding. This suggested that Ala-67 is required for a critical protein-protein contact between the dimerized DRBMs. The fact that A67E had a much greater effect on kinase function than did a mutation (K60A) that abolished dsRNA binding without impairing dimerization led these workers to propose that dimerization precedes dsRNA binding and that PKR dimers can be activated in yeast independently of dsRNA.
The solution structure of the N-terminal domain of PKR showed that DRBMs I and II have topologies highly similar to that of other DRBMs (32, 33), separated by a flexible linker of about 20 residues (34). The crystal structure of a complex between dsRNA and a single DRBM from the Xenopus laevis RNA-binding protein A (Xlrbpa-2) revealed three different contact regions in the DRBM which interact with two successive minor grooves and the intervening major groove on one face of the dsRNA molecule (35). The same three RNA contact regions were identified in the NMR structure of the DRBM-3 of Drosophila Staufen bound to an RNA stem-loop (36). In view of these findings, we considered the possibility that certain PKR mutations studied previously which altered only one of three RNA contact regions in DRBM-I (e.g. K60A) might not completely impair dsRNA binding. In this event, conclusions about the dependence of dimerization and kinase activation on dsRNA binding based on such mutations would be questionable. In addition, the conclusion that dimerization by full-length PKR is dsRNA-dependent derived from analysis of a single mutation (K64E) in DRBM-I (20). With the finding that only DRBM-II interacts with the kinase domain (17), we wished to determine whether dsRNA binding by DRBM-II is required for dimerization.
Accordingly, we constructed point mutations designed to destroy side
chain interactions in one, two, or all three predicted dsRNA contact
regions in DRBMs I and II and examined their effects on dimerization by
full-length PKR molecules expressed in yeast cells. Our results provide
strong support for the idea that dimerization by full-length PKR is
dependent on dsRNA binding, but they show that it occurs independently
of the dimerization domain between residues 244 and 296. We also
employed the DRBM point mutants to investigate whether dsRNA binding
and dimerization stimulate autophosphorylation in the PKR activation
loop. Using an antibody specific for phosphothreonine 451, we provide
evidence that phosphorylation of this residue is stimulated by dsRNA
binding. Finally, we show that Ser-33 and multiple autophosphorylation
sites in the flexible linker between the DRBMs, and within kinase
subdomain V, are largely dispensable for PKR function in yeast. Our
data provide strong support for a model in which dsRNA binding enables
dimer formation, enhancing autophosphorylation of Thr-451 in the
activation loop with attendant activation of the eIF2 kinase
function of PKR.
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MATERIALS AND METHODS |
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Plasmids and Yeast Strains-- Descriptions of the plasmids employed are given in Table I, and details of their construction will be provided upon request.
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Immunoblot Analysis of PKR Expression and eIF2 Phosphorylation
in Yeast Cells--
Transformants of strains H1894 (a ura3-52
leu2-3, -112, gcn2
trp1-
63) (37), J82
(a ura3-52 leu2-3, -112 gcn2
trp1-
63 sui2
p1098 [SUI2-S51A
LEU2] (37), and GP3299 (a ura3-52 leu2-3, -112 gcn2
trp1-
63
gcd2
::hisG pAV1033 [GCD2-K627T
TRP1] (38) containing different PKR alleles were grown
in SC medium overnight, diluted 1:50 in SC medium (39) and grown to
A600 of 0.6-1.0, and then shifted to
synthetic medium containing 10% galactose and 2% raffinose (SGAL) for
~12 h. Whole cell extracts (WCEs) were prepared by
breaking cells with glass beads in lysis buffer (20 mM
Tris, pH 8.0, 50 mM KCl, 400 mM NaCl, 20%
glycerol, 0.5 mM EDTA, 0.1% Triton X-100) supplemented
with 1 mM phenylmethylsulfonyl fluoride, 10 mM
2-aminopurine, 10 mM NaF, 50 mM
-glycerolphosphate, 125 µM sodium orthovanadate, and
complete protease inhibitor (CPI) mixture (Roche). For analysis of
phosphatase-treated proteins, samples of WCE containing 25 µg of
protein, prepared as above except that 10 mM NaF, 50 mM
-glycerolphosphate, and 125 µM sodium orthovanadate were omitted from the lysis buffer, were treated with 2 units of calf intestinal alkaline phosphatase (CIP) (New England
BioLabs) for 30 min at 37 °C. A phosphatase inhibitor mixture
containing 10 mM sodium pyrophosphate, 5 mM
EDTA, 5 mM EGTA, and 125 µM sodium
orthovanadate was added to selected samples prior to CIP treatment.
Proteins were separated by SDS-PAGE and transferred to nitrocellulose
membranes. Membranes probed with pT451 antibodies were blocked in TBS-T
(20 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween
20) containing 3% bovine serum albumin, whereas all other membranes
where blocked in a solution of TBS-T containing 5% non-fat dry milk.
Immunodetection of FLAG-tagged PKR (FL-PKR), triple HA-tagged PKR
(HA3-PKR), and untagged PKR was conducted as indicated in
the figure legends using a monoclonal antibody against the FLAG epitope
(Sigma), polyclonal antibodies against the HA-epitope (BabCo), and
polyclonal antibodies specific for the N terminus (N-18) or C terminus
(K-17) of PKR (Santa Cruz Biotechnology). Immunodetection of PKR
phosphorylated at Thr-451 was conducted using phosphospecific
polyclonal antibodies (BIOSOURCE International) in
blocking solution containing 3% bovine serum albumin. Immunoblot
analysis of eIF2
phosphorylation was conducted using polyclonal
antibodies specific for pS51 (Research Genetics) and polyclonal
antibodies against total eIF2
(CM-217) (40). Immune complexes were
visualized with the enhanced chemiluminescence (ECL) detection system
(Amersham Pharmacia Biotech) according to the vendor's instructions
and quantified by video image densitometry of the resulting
autoradiograms using NIH Image 1.61 software.
Poly(I-C) Binding Assays-- Yeast WCEs were prepared by breaking cells with glass beads in the yeast lysis buffer described above supplemented with CPI and phosphatase inhibitor mixtures. Samples of WCEs containing 200 µg of protein were incubated with poly(I-C)-agarose beads (Amersham Pharmacia Biotech) in 200 µl of binding buffer (150 mM KCl, 20 mM Hepes, 10% glycerol, 5 mM magnesium acetate) plus CPI mixture and phosphatase inhibitors for 1 h at 4 °C. The beads were collected by centrifugation, washed three times with lysis buffer, resuspended in 30 µl of 2 × Laemmli sample buffer (41), and boiled for 5 min. Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis as described above.
Coimmunoprecipitation of Mutant and Wild-type Epitope-tagged PKR Proteins from Yeast Extracts-- Yeast WCEs were prepared by breaking yeast cells with glass beads in immunoprecipitation lysis buffer (IP buffer) (20 mM sodium phosphate, pH 7.0, 500 mM NaCl, 0.1% Triton X-100) supplemented with CPI and phosphatase inhibitor mixtures. Aliquots containing 500 µg of protein were diluted to 100 µl in IP buffer and incubated with anti-FLAG M2-agarose (Sigma) at 4 °C overnight with rocking. The beads were collected by centrifugation, washed three times with IP buffer, resuspended in 30 µl of 2 × Laemmli sample buffer, and boiled for 5 min. The proteins were resolved by SDS-PAGE and subjected to immunoblot analysis as described above.
Analysis of PKR Expression and Activity in Mammalian
Cells--
HeLa cells grown in Dulbecco's modified Eagle's medium
with 10% fetal bovine serum were treated with interferons (IFN)
/
and poly(I-C) as described in Fig. 6A. Cells
were washed twice with cold phosphate-buffered saline containing CPI
and phosphatase inhibitor mixtures described above and lysed in
extraction buffer (20 mM Tris, pH 7.5, 150 mM
NaCl, 1% Triton X-100) containing CPI mixture and phosphatase
inhibitors. For immunopurification of PKR, an aliquot of WCE containing
500 µg of protein was incubated with PKR monoclonal antibody 71/10
(Ribogene/Questcor) for 2 h at 4 °C with rocking. Immune
complexes were collected with protein G-Sepharose beads (Amersham
Pharmacia Biotech) and washed three times with extraction buffer plus
CPI mixture and phosphatase inhibitors. Proteins were separated by
SDS-PAGE, and immunodetection was conducted as described above.
In vitro kinase assays were carried out as described
previously (6) except for details stipulated in the legends to Fig. 6,
B-D. PKR was purified as described previously (9) except
that SP-Sepharose was used in place of S-Sepharose, and a heparin
column replaced the hydroxylapatite and Mono S columns.
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RESULTS |
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Mutations Destroying Predicted Amino Acid Side Chain-RNA
Interactions in PKR DRBMs I and II Produce Additive Reductions in
Kinase Activity in Yeast--
To investigate whether dimerization of
PKR is dependent on dsRNA binding by one or both DRBMs, we made Ala
substitutions predicted to destroy side chain interactions in all three
regions that contact dsRNA in one or both DRBMs (Fig. 1,
A-C). These mutations were introduced into a FLAG
epitope-tagged allele of PKR (FL-PKR) and expressed from a galactose-inducible promoter in a gcn2
yeast strain (H1894). We also made the A67E substitution in
DRBM-I reported to abolish dimerization (31), and a deletion of
residues 244-296 containing the second dimerization domain in PKR
(Fig. 1A) (25), to address the importance of these residues
for dimerization by full-length PKR. The effects of the mutations on
PKR function in vivo were determined by analyzing the growth
rates of the resulting yeast transformants on different media (Table
II, fourth through seventh columns).
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When induced at high levels on galactose medium, wild-type PKR
inhibits cell growth via a general inhibition of translation resulting
from high level eIF2 phosphorylation. Thus, the strain bearing the
FL-PKR allele, encoding FLAG-tagged wild-type PKR, failed to
grow on galactose medium, whereas that expressing catalytically inactive FL-PKR-K296R grew well on this medium (Table II, fifth column). A low level of PKR expression under noninducing conditions (glucose medium) complements the failure of gcn2
cells to
grow on medium containing 3-aminotriazole (3-AT), an inhibitor of
histidine biosynthesis. (Induction of GCN4 mRNA
translation by eIF2
phosphorylation derepresses histidine
biosynthetic enzymes and permits cell growth on 3-AT medium (37).)
Thus, the strain bearing FL-PKR grew on glucose medium
containing 3-AT, whereas the FL-PKR-K296R transformant did
not (Table II, sixth column). As expected, the transformants bearing
FL-PKR-K296R or vector alone grew indistinguishably on both
media (Table II).
By comparing the growth of the various DRBM point mutants on these two media, and also on galactose medium containing 3-AT, we could infer their relative kinase activities in vivo (6) (Table II, fourth column; for details, see legend). The double Ala substitution of Ser-59 and Lys-60 in region 3 of DRBM-I (the FL-PKR-2AI allele) led to moderate reductions in kinase activity (Table II), whereas Ala substitutions in the corresponding residues of DRBM-II (Thr-149 and Lys-150) in the FL-PKR-2AII allele had no discernible effect on kinase function. (The 2AI and 2AII allele names designate two Ala substitutions in DRBM- I or II, respectively.) A more substantial defect was observed when all four of these mutations were combined in PKR-2AI+2AII (Table II), consistent with the previous conclusion that DRBMs I and II cooperate in binding dsRNA (42, 43). Combining the Ala substitution of His-37 in region 2 of DRBM-I with the two mutations just described in region 3 of DRBM-I (producing FL-PKR-3AI) led to a greater reduction in kinase activity than was seen for the FL-PKR-2AI allele containing only region 3 mutations. This finding supports the idea that regions 2 and 3 make additive contributions to dsRNA binding by DRBM-I (35). By contrast, the corresponding triple mutation in DRBM-II present in FL-PKR-3AII had no effect on kinase function, consistent with the previous conclusion that DRBM-I is more critically required than DRBM-II for dsRNA binding and kinase function (6, 42, 44). The allele that combined all six of these mutations in both DRBMs (FL-PKR-3AI+3AII) was more defective than FL-PKR-3AI and had very low (but detectable) activity in these assays (Table II). Thus, DRBM-II becomes essential for PKR activity only when DRBM-I is severely impaired.
Combining two mutations in region 1 (N15A and T16A) and a third
mutation in region 3 (K61A) with the aforementioned mutations in
regions 2 and 3 of DRBM-I (yielding FL-PKR-6AI) reduced PKR function further than did the latter 3 mutations alone. Similarly, the
FL-PKR-6AII allele containing the corresponding 6 substitutions in DRBM-II showed impaired kinase function, whereas the
parental allele FL-PKR-3AII with only 3 mutations in DRBM-II
had wild-type function. As expected, the FL-PKR-6AI+6AII
allele containing all 12 mutations in both DRBMs I and II had very low
activity. It was surprising, however, that it showed slightly greater
activity than did FL-PKR-3AI+3AII bearing fewer mutations.
This last observation could be explained by proposing that the
6AI+6AII mutations reduce the negative effect of the DRBMs
on kinase activity (15, 20), reducing the requirement for dsRNA binding
and yielding higher kinase activity than that of FL-PKR-3AI+3AII. In
agreement with previous results (31), the A67E mutation led
to a substantial reduction in kinase activity, comparable to the
effects of multiple substitutions in the contact points for dsRNA in
DRBM-I (Table II). The 244-296 mutation
abolished kinase activity, as expected from the fact that it removes
conserved residues in the kinase domain of PKR.
To eliminate the possibility that the reductions in kinase activity
conferred by these mutations resulted from reductions in PKR
expression, we conducted Western analysis on WCEs from the yeast
strains described above following growth on galactose medium. In
agreement with previous results, the mutant enzymes with reduced kinase
activities were generally more abundant than those with higher
activities (Fig. 2A). This
phenomenon has been attributed to negative autoregulation of PKR
expression which depends on its eIF2 kinase activity (6). Thus, the
2AI product was expressed at higher levels than the
2AII product, the 3AI product was more abundant
than the 3AII product, and the 6AII product was
more highly expressed than the 3AII product. Furthermore, the 2AI+2AII product was expressed at higher levels than
either the 2AI or 2AII product. These
measurements of protein expression for the different mutant alleles
shown in Fig. 2 are consistent with our assignments of their in
vivo kinase activities based on cell growth assays (Table II,
fourth column). However, the inverse relationship between kinase
activity and protein expression did not strictly apply to the most
heavily mutated FL-PKR alleles containing 6 or 12 substitutions in the DRBMs. Thus, although the 6AI,
6AI+6AII, and 3AI+3AII products were expressed at
much higher levels than wild-type FL-PKR, they accumulated to lower levels than the relatively more active 3AI product (Fig.
2A). In the case of the 6AI+6AII product, this
discrepancy may arise from the intrinsic instability of the protein
resulting from 12 Ala substitutions (see below).
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We also measured the levels of wild-type and mutant FL-PKR proteins by
Western analysis in a different yeast strain (J82) containing an Ala
substitution for Ser-51 in eIF2, the site of phosphorylation by PKR
and GCN2. In agreement with previous results (6), FL-PKR-K296 and
FL-PKR were expressed at similar levels during growth on galactose
medium because of loss of autoregulation of PKR expression in this
strain. All of the other mutant FLAG-tagged PKR proteins were expressed
at comparable levels, except for the 6AI+6AII product that
accumulated to only ~30% of the level of wild-type FL-PKR (data
summarized in Table II, last column).
Finally, we analyzed the level of eIF2 phosphorylation on Ser-51 for
selected FL-PKR mutants in another strain that is also defective for
PKR autoregulation. This strain (GP3299) contains wild-type eIF2
but
harbors a mutation in the GCD2-encoded subunit of eIF2B
which eliminates the inhibitory effect of eIF2
phosphorylation on
translation. Because the levels of wild-type and mutant PKR proteins
are similar in this strain, the amounts of eIF2
phosphorylation observed in these transformants should be roughly proportional to the
specific activities of the PKR proteins being expressed. To measure the
relative levels of eIF2
phosphorylated on Ser-51, we carried out
Western analysis of whole cell extracts using antibodies specific for
phosphorylated Ser-51 (pS51) and also with polyclonal eIF2
antibodies. The ratios of Western signals from these two blots were
calculated and normalized for the amount of PKR in the extract. As
expected, the wild-type FL-PKR transformant had readily
detectable amounts of pS51, whereas none was observed in the strain
containing catalytically inactive FL-PKR-K296R (Fig. 2B, lanes 1-4). The strains containing the
3AI+3AII and 6AI+6AII mutant alleles had
normalized ratios of pS51 to total eIF2
that were ~60% of that
observed for wild-type FL-PKR, indicating a defect in
eIF2
kinase activity in these mutants. The
FL-PKR-2AI+2AII transformant showed a smaller defect,
consistent with the results of growth tests shown in Table II. These
reductions in pS51 levels may seem modest in relation to the
substantial increases in cell growth and PKR expression associated with
these DRBM mutations; however, small increases in the proportion of
total eIF2
that is phosphorylated on Ser-51 can have a large impact
on the rate of translation in vivo (37). It also should be
noted that the 3AI+3AII and 6AI+6AII alleles
induced GCN4 expression on galactose medium (Table II, Gal
3-AT), consistent with the residual eIF2
phosphorylation present in
the transformants (Fig. 2B).
PKR Dimerization in Vivo Is Strongly Correlated with dsRNA Binding
Activity--
We verified that the mutations made in the DRBMs reduce
dsRNA binding by comparing mutant and wild-type FL-PKR proteins in cell
extracts for binding to poly(I-C)-agarose beads. To ensure that similar
amounts of mutant and wild-type proteins were being compared, we used
extracts from the J82 transformants described above in which PKR
autoregulation was abolished. The bound proteins were eluted from the
poly(I-C)-agarose beads and resolved by SDS-PAGE along with aliquots of
the input extracts (Fig. 3). The results of replicate binding assays were quantified and are summarized in Table
II (second column).
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As expected, the wild-type FL-PKR product bound to
poly(I-C)-agarose, whereas the PKR-14-257
product, lacking DRBMs I and II, showed no binding activity in this
assay (Fig. 3, lanes 2 and 3). Surprisingly, the
catalytically inactive protein FL-PKR-K296R showed higher levels of
dsRNA binding than did wild-type FL-PKR. Somewhat elevated levels of
dsRNA binding also were observed for the catalytically inactive
244-296 product and for the 2AII
and 3AII products. At present, we do not understand the
basis for the elevated levels of dsRNA binding observed for these
mutants. Most of the remaining mutants showed reductions in poly(I-C)
binding which correlated well with the extent of kinase impairment.
Thus, the 2AI and 2AII products showed high level
dsRNA binding, whereas the combination of these mutations in the
2AI+2AII construct produced low levels of dsRNA binding
(Fig. 3, lanes 4-6; Table II). The 3AI product
showed less dsRNA binding than did the 2AI product, and the
3AI+3AII protein had lower binding activity than did the 3AI product. (The amount of 3AI product that
bound to poly(I-C) in the particular assay shown in Fig. 3 was
considerably lower than that observed in several replicate assays not
shown (for details, see Table II).) As expected, the 6AII
product had reduced dsRNA binding activity compared with wild-type but
showed less binding activity than did the 6AI product (Table
II and Fig. 3). It is noteworthy that the A67E mutation in
DRBM-I completely abolished poly(I-C) binding by FL-PKR, at odds with a
previous report indicating that this mutation had little impact on
dsRNA binding in vitro (31).
We next examined the ability of the mutant FL-PKR proteins to form
heterodimers with a wild-type PKR protein tagged with three copies of
the HA epitope (HA3-PKR) that was coexpressed from the galactose-inducible promoter in the same yeast transformants. The
FL-PKR proteins were purified from cell extracts using immobilized FLAG
antibodies and probed with HA antibodies for the presence of
copurifying HA3-PKR. As shown in Fig.
4, a large proportion of the
HA3-PKR was coimmunoprecipitated with wild-type FL-PKR, whereas none was recovered from an extract containing untagged PKR
(compare lanes 3 and 6). Thus, the presence of
HA3-PKR in the immunopurified fraction was dependent on the
presence of FL-PKR. The amount of HA3-PKR that
coimmunoprecipitated with each FL-PKR mutant was measured and corrected
for the immunoprecipitation efficiency of the FLAG-tagged mutant
protein itself (Fig. 4). These normalized amounts of
coimmunoprecipitating HA3-PKR were taken as a measure of
the relative dimerization activities of the mutant proteins. The
results obtained from the assays shown in Fig. 4 (and replicate
experiments not shown) are summarized qualitatively in Table II (third
column; for details, see legend).
|
All of the FL-PKR mutants with >50% of wild-type dsRNA binding
activity were indistinguishable from wild-type FL-PKR in the ability to
form heterodimers with HA3-PKR in vivo. This
category included the 244-296,
2AI, 2AII, 3AII, and 6AII
products (Table II, second and third columns). The remaining mutants
with more serious defects in dsRNA binding displayed reduced ability to heterodimerize with HA3-PKR. Thus, the 3AI and
2AI+2AII products had moderate and low level dimerization
activities, respectively, whereas the 3AI+3AII, 6AI, and
6AI+6AII products formed only trace amounts of heterodimers
with HA3-PKR. Considering the good correlation we observed
between dsRNA binding and heterodimer formation, we conclude that
formation of stable PKR dimers in yeast depends strongly on dsRNA
binding. In keeping with its failure to bind dsRNA (Fig. 3), the
A67E product was nearly incapable of dimerizing with
HA3-PKR (Fig. 4).
dsRNA Binding by PKR Is Required for High Level Autophosphorylation
of Thr-451 in the Kinase Activation Loop--
Previously, we presented
strong genetic evidence that autophosphorylation of PKR on Thr-451 is
required for kinase activity in vitro and in yeast cells;
however, we could not detect phosphorylated Thr-451 (pT451) by mass
spectrometry sequencing of phosphopeptides from PKR purified from yeast
(11). Here we obtained physical evidence for Thr-451 phosphorylation by
Western analysis of PKR expressed in yeast using antibodies specific
for pT451. As shown in Fig. 5A
(upper panel), the pT451 antibody reacted with wild-type PKR
and with PKR-S242A, T255A, T258A containing Ala substitutions in three
other autophosphorylation sites (10). However, it did not react with
catalytically inactive PKR-K296R, nor with PKR-T451A and PKR-T446A. All
five proteins were readily detected on the same blot using polyclonal
antibodies against PKR (Fig. 5A, lower panel). We
verified that detection of wild-type PKR by Western analysis with the
pT451 antibodies was abolished by the addition of a phosphorylated
peptide containing pT451, but not by the unphosphorylated version of
the same peptide (data not shown). The fact that T446A eliminated the reactivity of PKR with pT451 antibodies suggests that
autophosphorylation of Thr-446 is a prerequisite for phosphorylation of
Thr-451. It could be argued that T446A does not prevent
phosphorylation of Thr-451 but instead abolishes recognition of pT451
by the phosphospecific antibody. This seems unlikely because the pT451
antibody was raised against a synthetic phosphopeptide that does not
include Thr-446.
|
To corroborate these findings, we sought to demonstrate that reactivity of wild-type PKR with pT451 antibodies was eliminated by treatment of the extract with a protein phosphatase. As shown in Fig. 5B, wild-type PKR in untreated extracts had a lower electrophoretic mobility than did PKR-K296R (PKR panel, lanes 1 and 4), which is attributable to extensive autophosphorylation of the wild-type enzyme (10). Treatment of extracts with CIP increased the mobility of wild-type PKR to nearly that of PKR-K296R, whereas inclusion of phosphatase inhibitors reversed the effect of CIP on PKR mobility. As expected, treatment with CIP in the presence or absence of inhibitors had no effect on the mobility of the defective kinase (Fig. 5B, PKR panel). Thus, it appears that CIP treatment removed the majority of phosphorylated residues from wild-type PKR. Importantly, CIP treatment also greatly decreased the reactivity of wild-type PKR to pT451 antibodies, in a manner reversed by phosphatase inhibitors (Fig. 5B, pT451 panel, lanes 1-3). We conclude that the pT451 antibodies specifically recognize PKR autophosphorylated on Thr-451.
We next investigated whether autophosphorylation of Thr-451 is stimulated by dsRNA binding. In the first approach, the pT451 antibodies were employed in Western analysis of yeast extracts containing FL-PKR proteins bearing the multiple mutations in both DRBMs which eliminated dsRNA binding and dimerization. The products of the 2AI+2AII, 3AI+3AII, and 6AI+6AII mutant alleles contained pT451 at levels only ~25% of that observed for wild-type FL-PKR (Fig. 5C, lanes 1-10). The 6AI allele also showed a clear reduction in the amount of pT451 compared with that present in wild-type (Fig. 5C, lanes 11-13); however, we could not detect a significant reduction in pT451 content for any other mutant proteins bearing fewer substitutions in the DRBMs (data not shown). These findings are consistent with the idea that autophosphorylation of Thr-451 is stimulated by dsRNA binding and dimerization of PKR. Below, we discuss the possible origin of the residual pT451 observed for the heavily mutated proteins that cannot bind dsRNA.
To obtain independent evidence that Thr-451 autophosphorylation is
stimulated by dsRNA binding, we sought to demonstrate that phosphorylation of Thr-451 by wild-type PKR could be stimulated by
dsRNA. PKR isolated from yeast cells is highly active and cannot be
stimulated further by treatment with dsRNA (6). Hence, we immunopurified PKR from cultured human (HeLa) cells that were treated
with IFN- and
(IFN
/
) to induce PKR and also with poly(I-C)
to activate the enzyme prior to purification. Western blot analysis of
the resulting extracts confirmed that PKR expression was induced by
IFN
/
and that phosphorylation of eIF2
on Ser-51 was stimulated
by poly(I-C) treatment of the cells (Fig.
6A). PKR was immunopurified,
and the immune complexes were incubated with [32P]ATP and
analyzed by SDS-PAGE and autoradiography. As expected, PKR from the
cells treated with both IFN
/
and poly(I-C) had higher autokinase
activity than did the enzyme immunopurified from cells treated with
IFN
/
alone (Fig. 6B). In a separate experiment, the
immune complexes were incubated with nonradioactive ATP and subjected
to Western blot analysis with antibodies against PKR pT451. The results
showed that only the activated form of PKR isolated from
poly(I-C)-treated cells reacted strongly with the pT451 antibodies, and
this occurred only when PKR was incubated in the presence of ATP (Fig.
6C, lane 6). Thus, reactivity with pT451
antibodies was correlated with high level autokinase activity of the
immunopurified enzyme. To explain the fact that pT451 was detected in
this last experiment only after incubation of the in vivo
activated enzyme with ATP, we suggest that pT451 was dephosphorylated either prior to or during isolation of the enzyme from the
poly(I-C)-treated cells and was regenerated in vitro on
addition of ATP.
|
Finally, we wished to demonstrate that phosphorylation of Thr-451 could be stimulated by dsRNA treatment of purified PKR. Toward this end, PKR isolated from 293 cells was immunoprecipitated, incubated with ATP in the presence or absence of poly(I-C), and probed by Western analysis using pT451 antibodies. As shown in Fig. 6D, we detected pT451 only when the PKR was incubated with poly(I-C), supporting the idea that Thr-451 autophosphorylation is stimulated by dsRNA. Unexpectedly, we found that immunoprecipitation of the PKR was a prerequisite for poly(I-C)-dependent Thr-451 phosphorylation in these last experiments (data not shown). Several ways to account for the stimulatory effect of immunoprecipitation on Thr-451 phosphorylation are considered below.
Mutations in Other Autophosphorylation Sites Located between the DRBMs or within the Kinase Domain Insert Have Little Impact on PKR Function in Yeast-- Previously, mass spectrometric analysis of PKR expressed in yeast identified phosphorylation sites at Ser-33 in DRBM-I, a cluster of six sites in the linker region between the DRBMs (residues 81-107) and a cluster of six sites in the 315-356 interval located in a large insert between kinase subdomains IV and V (12). There are 8 Ser and Thr residues in the 315-356 interval, and only six autophosphorylation sites were detected by mass spectrometry; hence 2 of the Ser or Thr residues in this interval may not be autophosphorylated. Similarly, there are 7 Ser and Thr residues in the 81-107 interval, and only six autophosphorylation sites were detected by mass spectrometry. Direct chemical sequencing of phosphopeptides derived from PKR purified from mammalian cells and autophosphorylated in vitro confirmed the presence of multiple autophosphorylation sites between DRBMs I and II, identifying Ser-83, Thr-88, Thr-89, and Thr-90 as specific phosphorylated residues in this interval (13).
To address the importance of these additional phosphorylation sites, we
made a single Ala substitution at Ser-33 (S33A) and 7 simultaneous Ala substitutions at all of the potential sites located
between DRBMs I and II (7Ala). Additionally, 10 simultaneous Ala substitutions were made to remove all eight potential
phosphorylation sites in the kinase domain insert plus residues Ser-357
and Ser-359 located immediately upstream (10Ala). The
17Ala allele combines all of the mutations present in the
10Ala and 7Ala alleles. The mutant proteins were
expressed in the gcn2 yeast strain H1894 and compared
with wild-type PKR for their effects on cell growth and resistance to
3-AT, as described above. Surprisingly, the S33A,
7Ala, 10Ala, and 17Ala mutations had
no detectable effect on PKR function (Fig.
7A). Consistently, combining
the 17Ala substitutions with the S242A,
T255A, T258A mutations (3Ala)
(producing the 20Ala allele), did not reduce kinase activity
any further than the 3Ala substitutions alone (Fig.
7A).
|
In accordance with our results on the 7Ala allele, Taylor et al. (13) reported recently that Ala substitutions in residues Ser-83, Ser-88, Ser-89, and Thr-90 had no effect on PKR function in yeast. The results we obtained for the 10Ala mutation were unexpected, however, in light of a previous report that the single S355A substitution destroyed PKR function in yeast using the same growth assays employed here (45). We constructed the PKR-S355A allele and found that it too was indistinguishable from wild-type PKR in our growth assays (data not shown). We have no explanation for the discrepancy between our results and those published previously.
We analyzed the mutations in multiple phosphorylation sites using a more stringent test for PKR function by combining them with the T446A substitution in the activation loop of the kinase domain. The latter mutation substantially impairs, but does not abolish, PKR activity in the growth assays. As shown previously, the 3Ala substitution exacerbated the phenotype of T446A (11) (Fig. 7A), as did the 10Ala and 17Ala substitutions, leading to enhanced growth on galactose medium compared with that seen for the PKR-T446A single mutant (Fig. 7, A and B). Thus, autophosphorylation of one or more sites located between kinase subdomains IV and V may be important for kinase activity when autophosphorylation in the activation loop is eliminated. In contrast, the S33A and 7Ala substitutions had little or no effect on cell growth when combined with T446A (Fig. 7, A and B). Moreover, the 10Ala mutation was indistinguishable from the 17Ala mutation in exacerbating the phenotype of T446A, consistent with the idea that autophosphorylation of sites between DRBMs I and II (inactivated by 7Ala) makes little or no contribution to PKR activity in yeast.
To provide biochemical evidence that these additional phosphorylation
sites are utilized in yeast, we examined the electrophoretic mobilities
of the mutant proteins by Western analysis. In the first experiment
shown in Fig. 8A, the extracts
were treated with CIP in the presence and absence of phosphatase
inhibitors prior to SDS-PAGE. In the presence of CIP inhibitors, the
mobilities of the PKR proteins containing 7Ala,
10Ala, 17Ala, or 20Ala substitutions were greater than that of wild-type PKR and more similar to that of
catalytically inactive PKR-K296R, as would be expected if these mutations remove autophosphorylation sites (Fig. 8A,
+ lanes). The 7Ala mutations led to a
greater increase in mobility than did the 10Ala
substitutions (lanes 2, 4, and 6),
suggesting that sites located between the DRBMs are phosphorylated more
extensively than those between kinase subdomains IV and V (13).
Consistently, treatment with CIP in the absence of inhibitors led to
smaller increases in mobility for the 7Ala,
17Ala, and 20Ala mutants than for the
10Ala and wild-type enzymes (Fig. 8A, compare
+ and lanes). However, even PKR-20Ala
migrated more slowly than did PKR-K296R in the presence of CIP
inhibitors (lanes 19 and 21), and the mobility of
PKR-20Ala, but not that of PKR-K296R, was increased by CIP treatment in
the absence of inhibitors (lanes 19-22). These last
findings imply the presence of additional phosphorylated residues in
the 20Ala protein (at a minimum, pS33, pT446, and pT451), in keeping
with its relatively high kinase activity in vivo (Fig.
7A).
|
Combining the T446A substitution with the 20Ala mutations increased the mobility of the mutant PKR protein to the point where it was indistinguishable from catalytically inactive PKR-K296R (Fig. 8B), suggesting the absence of nearly all phosphorylation sites in PKR-T446A,20Ala. Given that T446A abolishes phosphorylation of Thr-451 (Fig. 5A), Ser-33 is the only known phosphorylation site remaining in this mutant protein. As shown in Fig. 7A, PKR-T446A,20Ala has readily detectable kinase activity in vivo, but Ser-33 makes no detectable contribution to kinase function. Thus, either autophosphorylation is not essential for PKR activity, or additional autophosphorylation sites remain to be identified which are required for the residual kinase function of PKR-T446A,20Ala.
We showed previously that Ala replacement of Thr-451 in the activation
loop abolishes the autokinase and eIF2 kinase activities of PKR
in vitro and in yeast cells (11). If autophosphorylation of
Thr-451 is stimulated by dsRNA binding and dimerization, as concluded
above, then mutations in the DRBMs which diminish Thr-451 phosphorylation should reduce bulk autophosphorylation by PKR. As shown
in Fig. 8C, the 2AI+2AII, 3AI+3AII, and 6AI+6AII mutants all
migrated more rapidly than did wild-type PKR, and more similarly to
PKR-K296R, as expected if the DRBM mutations reduce autophosphorylation of many sites in PKR. Consistently, treatment with CIP in the absence
of inhibitors led to smaller increases in mobility for the three DRBM
mutants (particularly 6AI+6AII) than it did for the wild-type enzyme
(Fig. 8D, compare + and
lanes
for each protein). These findings confirm that dsRNA binding and
dimerization are required for autophosphorylation by PKR on many sites
in addition to Thr-451.
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DISCUSSION |
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---|
The dsRNA Binding Activity of PKR Is a Prerequisite for Stable Dimerization by Full-length PKR Molecules in Vivo-- An important goal of this study was to examine thoroughly the relationship between dsRNA binding and dimerization by full-length PKR molecules under physiological conditions. To this end, we introduced Ala substitutions at all three predicted regions of contact with dsRNA in DRBMs I and II and analyzed their effects on dsRNA binding and dimerization by PKR proteins expressed in yeast. A nearly complete loss of dsRNA binding in our in vitro assays required alanine substitutions made simultaneously in contact region 3 of both DRBMs (FL-PKR-2AI+2AII) or simultaneous alterations in all three contact regions of DRBM-I (FL-PKR-6AI). Importantly, we observed a dramatic defect in dimerization only for these and more extensively mutated constructs (FL-PKR-3AI+3AII and FL-PKR-6AI+6AII) which completely lacked dsRNA binding activity in vitro. Additionally, the construct with alterations in contact regions 2 and 3 of DRBM-I (FL-PKR-3AI) showed partial reductions in both dsRNA binding and dimerization. Thus, we observed a strong correlation between the ability of PKR molecules to bind dsRNA in vitro and to dimerize in vivo.
Our mutations were designed to disrupt side chain interactions between
the DRBM and dsRNA without affecting the overall topology of the DRBM.
Indeed, contact regions 2 and 3 of Xlrbpa-2 reside in loops located
between strands 1 and 2, or
3 and
-helix 2, respectively
(35). Hence, the 3AI and 3AII mutations should impair dsRNA binding without disrupting the structures of the DRBMs.
Accordingly, the fact that PKR-3AI+3AII cannot dimerize is a strong
indication that formation of stable PKR dimers is critically dependent
on binding to dsRNA. This conclusion is at odds with several previous
reports indicating that dimerization can occur independently of dsRNA
binding (14, 22-24, 30). At least in some of these cases, single
substitutions made in DRBM-I probably did not completely eliminate
dsRNA binding. To avoid this complication, we progressively removed
predicted dsRNA contacts in both DRBMs I and II. In accordance with our
results, Wu and Kaufman (20) showed that the K64E mutation
in DRBM-I impaired the ability of full-length PKR-K296P to dimerize
with an N-terminal fragment containing wild-type DRBMs (the BD
fragment). Our study extends their findings in providing evidence that
stable dimer formation by two full-length functional PKR molecules is
dependent on dsRNA binding by both subunits of the dimer.
Because introducing the K64E mutation into the BD fragment did not impair its ability to dimerize with the wild-type BD segment, Wu and Kaufman proposed that the isolated DRBMs can dimerize independently of dsRNA through protein-protein contacts (14). To account for their subsequent finding that dsRNA binding was required for dimerization of full-length PKR with the BD segment, they suggested that dsRNA binding was required primarily to dissociate the DRBMs from the kinase domain and unmask a dimerization surface in the DRBMs (20). Having found that only DRBM-II binds tightly to the kinase domain, Nanduri et al. (17) proposed that dsRNA binding to DRBM-I is required for dimerization because it stimulates cooperative dsRNA binding to DRBM-II, dissociating the latter from the kinase domain and unmasking the 244-296 dimerization surface in the kinase domain. Our finding that residues 244-296 are dispensable for dimerization refutes the idea that this segment is the essential dimerization surface in PKR. If dsRNA stimulates dimerization by eliminating interaction of DRBM-II with the kinase domain, then destroying dsRNA binding by DRBM-II might be expected to impair dimerization. Inconsistent with this prediction, the six Ala substitutions in DRBM-II in the 6AII allele produced no reduction in PKR dimerization (Table II and Fig. 4). To maintain this aspect of the model proposed by Nanduri et al., it seems necessary to propose that binding of dsRNA to DRBM-I can indirectly disrupt interaction of DRBM-II with the kinase domain in the absence of dsRNA contacts in DRBM-II.
In contrast to the models of Wu and Kaufman (20) and Nanduri
et al. (17), it was proposed by Patel and Sen (31) that PKR
can dimerize in the absence of dsRNA and that preformed PKR dimers can
be activated either by dsRNA or other ligands. This idea was based on
their finding that the A67E mutation in DRBM-I destroyed
dimerization with little effect on dsRNA binding and that
A67E impaired kinase activity much more than did the
K60A mutation, which impaired dsRNA binding but not
dimerization. These findings implied that protein-protein contact
between DRBMs dependent on Ala-67 was more important than dsRNA binding
for PKR dimerization. In our assays, however, A67E abolished
dsRNA binding. Hence, the failure of FL-PKR-A67E to dimerize may result
from impaired dsRNA binding rather than loss of a critical
protein-protein contact. Given its location in -helix 2 of DRBM-I,
and the strong conservation of this residue (34), the A67E
mutation probably disrupts the secondary or tertiary structure of
DRBM-I. The fact that dimerization and kinase function were not
strongly impaired by the K60A mutation (31) can be explained
by proposing that this single substitution in DRBM-I is not severe
enough to destroy dsRNA binding by full-length PKR in
vivo.
Although our results provide strong support for the idea that dsRNA
binding is required to form stable PKR dimers in vivo, the
number and relative importance of the molecular interactions that
stabilize PKR dimers remain unclear. We favor the idea that binding of
two PKR molecules to the same molecule of dsRNA contributes substantially to the stability of a PKR dimer. Protein-protein interactions are also likely to be important, including the proven association involving residues 244-296 (25). Because this last interaction is dispensable for dimerization by full-length PKR, interactions between the DRBMs, or additional interactions between the
two kinase domains in the PKR dimer, also are predicted. Indeed, we
recently observed dimerization by isolated PKR DRBMs purified under
conditions that exclude dsRNA
contamination.2 Although none
of these interactions alone may be sufficient to support stable dimer
formation under physiological conditions, in aggregate they could
produce a stable ternary complex consisting of two PKR molecules bound
to the same molecule of dsRNA (Fig. 9).
We reached the same conclusion previously for the composition of a
stable heterodimer between PKR and the DRBM-containing protein E3,
wherein both proteins are bound to the same molecule of dsRNA (46).
|
It was surprising that the 2AI mutation in dsRNA contact region 3 of DRBM-I (S59A,K60A) did not impair dsRNA binding in our assays. This finding is at odds with previous results indicating that PKR-K60A completely lacked dsRNA binding activity (31). Likewise, we reported previously that a cluster of mutations in residues 58-60 reduced dsRNA binding activity to <5% of wild-type, although it impaired PKR activity in yeast by the same moderate amount observed here for the 2AI mutation (6, 42) (Table II). The fact that the 2AI mutation significantly reduced PKR function in yeast (Table II) makes us suspect that it produces a small reduction in dsRNA binding in vivo which our poly(I-C) binding assays were not sensitive enough to detect. One difference between our binding assays and those described previously is that we employed full-length PKR proteins made in yeast rather than in rabbit reticulocyte lysates. Perhaps PKR is associated with different proteins or contains different post-translational modifications which affect dsRNA binding when synthesized in these two systems. Because our assays for dsRNA binding, dimerization, and kinase activity all used the same source of PKR proteins expressed in yeast, they should allow a direct comparison of the effects of DRBM mutations on these different activities of PKR.
It was also unexpected that the A67E mutant had greater kinase activity than did the 3A+3AII mutant, even though both displayed the same severe defects in dsRNA binding and dimerization. Perhaps A67E perturbs the structure of DRBM-I in a way that indirectly impairs the postulated inhibitory interaction of DRBM-II with the kinase domain (17, 20), permitting substantial kinase activity in the absence of dsRNA binding or dimerization.
Autophosphorylation of Thr-451 Is Dependent on Thr-446 and Stimulated by dsRNA Binding-- Autophosphorylation in the activation loop of PKR is critical for PKR function (11), and a second goal of this study was to provide evidence that dsRNA binding and dimerization stimulate autophosphorylation of the activation loop. Using an antibody specific for pT451, we obtained the first physical evidence for autophosphorylation of this important residue. Unexpectedly, pT451 was eliminated from PKR expressed in yeast by the T446A mutation, suggesting that autophosphorylation of Thr-446 is a prerequisite for pT451. This last conclusion seems at odds with the fact that PKR-T446A has detectable kinase activity whereas PKR-T451A is inactive (11). Presumably, part of the defect associated with T451A derives from structural perturbation of the activation loop, unrelated to the function of Thr-451 as a phosphorylation site. In this view, T446A prevents autophosphorylation at both 446 and 451, and the T446A phenotype reveals the true impairment of PKR function associated with a failure to autophosphorylate the activation loop.
The phosphorylation of Thr-197 in the activation loop of protein kinase A is extremely stable and considered to be a step in protein maturation rather than a regulatory event (47, 48). In contrast, phosphorylation of Thr-183 and Thr-185 in the activation loop of mitogen-activated protein kinase ERK2 by MEK2 (49) is highly regulated and represents a key event in the activation of ERK2 by mitogenic agents (50). Given the critical importance of Thr-451 for PKR activity, if autophosphorylation of this residue is dependent on dsRNA binding, it would represent an important molecular marker of PKR activation. We found that the amount of pT451 in PKR expressed in yeast was reduced to about 25% of the wild-type level by the multiple mutations in both DRBMs which abolished dsRNA binding and dimerization (Fig. 5). These data strongly suggest that phosphorylation of Thr-451 is stimulated by dsRNA binding. Additional evidence for this conclusion came from our finding that Thr-451 could be phosphorylated in vitro by PKR immunopurified from HeLa cells, provided that the cells were pretreated with dsRNA. Presumably dsRNA treatment induced autophosphorylation of Thr-451 in vivo, but the pT451 was eliminated by a protein phosphatase before or during immunopurification of the enzyme. We also observed autophosphorylation of Thr-451 by highly purified PKR, dependent on dsRNA treatment and immunoprecipitation of the enzyme. The immunoprecipitation of PKR may have removed a copurifying phosphatase; alternatively, binding of PKR to antibodies may promote irreversible dimerization that maintains a high level of Thr-451 phosphorylation in the presence of phosphatase activity. The relative ease of detecting pT451 in yeast cells (without immunoprecipitation of PKR) may reflect the absence of a pT451 phosphatase activity or a higher level of dimerization produced by naturally occurring dsRNAs in this organism, compared with treatment of HeLa cells or purified enzyme with poly(I-C).
Taken together, our findings support the idea that binding of PKR to dsRNA increases autophosphorylation in the activation loop of the kinase domain (Fig. 9). Because dsRNA binding promotes dimerization, this would facilitate trans-autophosphorylation of Thr-446 and Thr-451 by the two kinase moieties in a PKR dimer (29, 30). In addition, dimerization of the kinase domains seems to be required for efficient substrate phosphorylation by PKR (6, 23). As described above, dsRNA binding may also produce a conformational change that releases DRBM-II from inhibitory interactions with the kinase domain (Fig. 9). This rearrangement could overcome a negative effect of DRBM-II on autophosphorylation of Thr-446 and Thr-451 in the activation loop. Based on studies of other kinases, autophosphorylation in the activation loop would promote proper alignment of key catalytic residues, or the correct orientation of the two lobes of the PKR kinase domain, required for substrate binding or phosphoryl transfer (51-54).
With this model, how can we explain the fact that Thr-451 was still
autophosphorylated, albeit at reduced levels, by mutant proteins like
FL-PKR-3AI+3AII which cannot bind dsRNA or form stable dimers? One
possibility is that unstable PKR dimers can form transiently in the
absence of dsRNA binding and persist long enough for intermolecular
autophosphorylation to occur at a reduced rate. Alternatively, low
level intramolecular autophosphorylation of Thr-451 by PKR monomers may
occur. This second mechanism could account for the relatively high
level of pT451 found in the FL-PKR-A67E product (data not
shown), which is severely impaired for dimerization. Thus, by reducing
the negative effect of the DRBMs on kinase function, A67E
may allow Thr-451 autophosphorylation to occur in the absence of
dimerization. Even though pT451 can be generated by the A67E mutant, the autophosphorylated monomers still exhibit reduced eIF2
kinase activity (Table II), presumably because dimerization of the
kinase domains is also required for efficient eIF2
phosphorylation (6).
Autophosphorylation Sites in the Kinase Domain Insert Are Required for High Level PKR Function Only When Autophosphorylation in the Activation Loop Is Impaired-- We and others (13) have shown that Ala substitutions of the phosphorylation sites located between the DRBMs had little or no effect on kinase activity when introduced into wild-type PKR or PKR-T446A. In contrast, we showed here that mutations replacing all of the phosphorylation sites in the kinase domain insert (10Ala) did significantly reduce PKR activity when combined with the T446A mutation. The additive effects of the 10Ala and T446A mutations suggest that Thr-446 and one or more autophosphorylation sites in the kinase domain insert make independent contributions to an activated conformation of the enzyme. No additional reduction in PKR function occurred when the 10Ala and 3Ala substitutions were combined with T446A in the PKR-20Ala,T446A allele, implying that the sites affected by 10Ala and 3Ala mutations promote PKR activity by the same mechanism.
The S83A, S88A, S89A, and T90A substitutions between the two DRBMs
exacerbated the deleterious effect of the 3Ala substitutions on PKR function when the mutant proteins were expressed transiently in
COS-1 cells, but not in yeast (13). Presumably, the phosphorylation sites between the DRBMs promote PKR activation independently of Ser-242, Ser-255, and Thr-258 in a manner that depends on mammalian factors that are absent in yeast cells. Similarly, the
autophosphorylation sites in the kinase domain insert may play a more
important regulatory role in mammalian cells than in yeast, for example
functioning as recognition sites for a protein phosphatase or kinase
that modulates PKR activity independently of dsRNA.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Laboratory of Gene Regulation and Development for many useful suggestions, and Bobbie Felix for help in preparing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01-AI34552 (to M. B. M.) and American Heart Association Predoctoral Fellowship 9810005T (to B. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Cell Processing, Institute of
Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
To whom correspondence should be addressed. Tel.: 301-496-4480;
Fax: 301-496-6828; E-mail: ahinnebusch@nih.gov.
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M102108200
2 B. Tian and M. B. Mathews, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: dsRNA, double-stranded RNA; DRBM, dsRNA-binding motif; Xlrbpa-2, X. laevis RNA-binding protein A second dsRNA binding domain; WCE, whole cell extract; CPI, complete protease inhibitor; CIP, calf intestinal alkaline phosphatase; PAGE, polyacrylamide gel electrophoresis; FL-PKR, FLAG-tagged PKR; HA3-PKR, triple hemagglutinin-tagged PKR; IFN, interferon; 3-AT, 3-aminotriazole.
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REFERENCES |
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