From the Department of Biochemistry and Molecular
Biology and § Graduate School of Biomedical Sciences,
New Jersey Medical School, University of Medicine and Dentistry of New
Jersey, Newark, New Jersey 07103
Received for publication, August 11, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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The interferon-inducible double-stranded RNA
(dsRNA)-activated protein kinase PKR is regulated by dsRNAs that
interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus.
The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and
cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and
the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has
RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs
have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1).
Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and
strongly inhibits the dimerization of the intact PKR N terminus (p20)
containing both dsRBMs. dsRBM1, like p20, activates reporter gene
expression in transfection assays, and it plays a determinative role in
localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2
has weak or no activity in dsRNA binding, stimulation of gene
expression, and PKR localization, but it strongly enhances these
functions of dsRBM1 when contained in p20. However, dsRBM2 does not
enhance the annealing activity of dsRBM1. This study shows that the
dsRBMs of PKR possess distinct properties and that some, but not all,
of the functions of the enzyme depend on cooperation between the two motifs.
PKR1 is a
serine/threonine kinase found in a latent state in most cells. It plays
an important part in cellular anti-viral defense as well as apoptosis,
signal transduction, cell growth, and differentiation (1, 2). Recently
it was also found to be involved in RNA splicing of the tumor necrosis
factor- Two double-stranded RNA-binding motifs, dsRBMs 1 and 2, occupy the
regulatory N-terminal portion of PKR. The dsRBM is a ~70-amino acid
motif that has been found in various proteins in almost all organisms
from Escherichia coli to humans (16). Sequence analysis of
completed genomes revealed 3 dsRBMs in 2 proteins of the yeast Saccharomyces cerevisiae, 24 dsRBMs in 9 proteins of the
nematode Caenorhabditis elegans, and 54 dsRBMs in 14 proteins of Drosophila melanogaster (17). The
three-dimensional structure of the dsRBM has been solved by nuclear
magnetic resonance and crystallographic techniques (18-20). The x-ray
crystal structure of the second dsRBM of XlrbpA in complex with dsRNA
revealed three regions of dsRBM forming direct contacts with dsRNA.
Sequence alignments of dsRBMs display the existence of variations in
these regions, suggestive of variations in dsRNA binding. Several
biochemical studies have indicated that dsRBMs can exhibit wide
differences in dsRNA binding (21, 22). In particular, the deletion or
mutation of dsRBM1 and dsRBM2 of PKR exerts different effects on its
dsRNA binding ability and kinase activity (23-26), even though the
three-dimensional folding of these two motifs is similar (19).
The dsRBMs play a key role in PKR function and regulation. Although
their exact role in PKR activation is still not clear, it appears that
dsRNA binding leads to autophosphorylation of PKR facilitated by
protein homodimerization (27, 28). It has been suggested that latent
PKR exists in an inactive dimer conformation and changes to an active
dimer form upon binding to activators, possibly by releasing the kinase
domain from inhibitory interactions with the protein's N terminus (2,
29). An intrinsic dimerization activity has been mapped within this
region (30-32). The dsRBMs have also been implicated in further
aspects of PKR function. For example, mutations in the kinase domain of
PKR usually show dominant negative effects, including the ability to
enhance the expression of reporter genes (23, 28, 33). The mechanism for the dominant negative effect is not fully understood, but it is
thought that the mutants either dimerize with wild-type PKR, thereby
blocking its function, or bind to and sequester cellular PKR activators
such as dsRNA (23). Moreover, PKR localizes in the cytoplasm and
nucleolus of the cell (34-36) and associates with ribosomes (37-39).
The ribosome association is tightly related to the dsRNA-binding
activity of PKR, but the region of PKR that is responsible for its
subcellular localization is not clear. Finally, a dsRNA-annealing
activity has been reported for the dsRBMs of Xlrbpa (40). Whether this
feature is shared by the dsRBMs of PKR is unknown.
To understand the functional differences between the two dsRBMs of PKR,
and their involvement in the biochemical activities and cytological
properties of the full-length protein, we have studied protein-RNA
interactions, RNA annealing, protein dimerization, reporter gene
activation, and protein localization. Our results suggest that, with
the exception of oligomerization, the differences between dsRBM1 and
dsRBM2 reflect their different dsRNA binding affinities. Nevertheless,
the two dsRBMs cooperate to achieve optimal dsRNA binding and to
accomplish both reporter gene activation and correct protein localization.
Plasmids--
Plasmids SRG5
p10A-EGFP, containing amino acids 1-95 of PKR, was constructed using
the primer set PKR-N and p10A-C. Similarly, p10B-EGFP (amino acids
88-186) was constructed using p10B-N and p20-C; p20-EGFP (amino acids
1-186) using PKR-N and p20-C, PKR-EGFP and K296R-EGFP (full-length,
amino acids 1-551) using PKR-N and PKR-C, and Protein Purification--
Transformed E. coli were
grown overnight in 3-ml cultures and transferred to 500 ml of LB medium
with kanamycin. Cultures were grown at 37 °C until the absorbance
A600 reached 0.5 then 1 mM
isopropyl-1-thio- RNA Synthesis and Purification--
RNAs for dsRNA production
and annealing assays were obtained by cutting plasmid pBSKII
(Strategene) with PvuII and transcribing with T3 and T7 RNA
polymerases. The T3 and T7 transcripts are 245 and 335 nucleotides
long, respectively, and complementary over 132 nucleotides. dsRNAs were
generated and purified as described previously (4).
Gel Mobility Shift Assay--
The gel mobility shift assay was
described previously (24). Briefly, purified dsRNAs and proteins were
mixed on ice in gel mobility shift buffer and run on 5% tris-glycine
polyacrylamide gel.
Poly(rI·rC) Pull-down Assay--
The poly(rI·rC) pull-down
assay was described previously (43). Briefly, poly(rI·rC)-Sepharose
was incubated with the proteins and washed with different
concentrations of salt. Proteins were eluted by mixing with gel loading
buffer, briefly boiled and loaded onto SDS-polyacrylamide gels.
Filter Binding Assay--
Poly(rI·rC) at 200 µg/ml was
5'-end-labeled using [ Annealing Assay--
For each reaction, a 15-µl solution
containing proteins or KCl, labeled T3 and T7 RNA substrates, and 1×
annealing buffer (200 mM KCl, 1 mM magnesium
acetate, 1 mM DTT, 100 µg/ml bovine serum albumin, 10 mM HEPES, pH 7.6) was incubated at 30 °C for 1 h.
Digestion mix (35 µl) containing 25 unit T1 RNase (Roche Molecular
Biochemicals) and 10 µl 5× T1 digestion buffer (5 mM EDTA, 750 mM NaCl, 50 mM Tris-HCl, pH 8.0) was
then added. After incubation at 37 °C for 1 h, the reaction was
stopped by adding 150 µl of precipitation mix (80 µg/ml yeast total
RNA and 266 µM NaCl) and 200 µl of
phenol:chloroform:isoamyl alcohol (1:1:1, v/v). The RNAs were
precipitated with ethanol and resolved in an 8 M urea/10%
TBE polyacrylamide gel.
Protein Cross-linking--
Proteins and poly(rI·rC) in
phosphate-buffered saline (PBS) were incubated on ice for 20 min and
then mixed with cross-linking buffer (2 mM dimethyl
suberimidate (Pierce), 100 mM NaCl, 10 mM HEPES, pH 8.0). After incubation at room temperature for
90 min, the reaction was stopped by adding 1 M glycine and
2× gel loading buffer. Samples were run in 12.5% SDS-polyacrylamide
gels, transferred to a polyvinylidene difluoride membrane (Millipore),
blotted with anti-His-tag antibody (CLONTECH), and
visualized with ECL (PerkinElmer Life Sciences).
GST Pull-down Assay--
GST fusion PKR was expressed in
E. coli and purified using glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Dialyzed purified GST-PKR was immobilized on the beads, His-tagged p20
and different concentrations of poly(rI·rC) were added, and the
slurry was incubated on ice for 30 min. The beads were then washed five
times with PBS containing 1 mM DTT and briefly boiled in
2× gel loading buffer. Samples were run in 12.5% SDS-polyacrylamide
gels, followed by Western blotting using anti-His antibody.
Cell Transfection and Luciferase Reporter Assay--
Human 293 cells grown in 12-well plates were transfected with plasmids
using the calcium phosphate method as described previously (41). Cells
were harvested 40 h after transfection by washing once with PBS
and incubating with 150 µl of lysis buffer (0.2% Triton X-100, 100 mM potassium phosphate buffer, pH 7.8) at 4 °C for 30 min. Cell lysates were centrifuged briefly to remove cell debris.
Supernatant (20 µl) was used for luciferase assay (luciferase assay
kit, Promega) and monitored using a Lumicount luminometer (Packard).
Subcellular Localization--
HeLa cells were grown on
coverslips (No. 2, 18-mm circle, VWR) until 70% confluence and then
transfected using the calcium phosphate method as described above.
Cells were grown for 36 h after transfection, washed once with
PBS, and fixed with freshly made 4% paraformaldehyde/PBS. Cells were
visualized under a fluorescence microscope (Olympus) using a
100×-magnification objective.
dsRNA Binding--
Earlier mutational and deletion analysis
indicated that the two dsRBMs of PKR differ in their dsRNA binding
affinity and their importance for PKR function (23, 25, 26, 44). To
examine their properties and behavior at the biochemical level, we
cloned dsRBM1 and dsRBM2 of PKR into vectors that allowed expression in
E. coli either as unmodified or His-tagged proteins (Fig.
1). In accordance with their approximate
molecular weights and following the previous convention (4, 24), the
recombinant proteins p10A (previously called p10), p10B, and p20 carry,
respectively, the first dsRBM, second dsRBM, and the intact N terminus
of PKR containing both dsRBMs.
We used three methods to compare the binding affinities of the dsRBMs
for dsRNA, namely a poly(rI·rC)-Sepharose pull-down assay, a
nitrocellulose filter-binding assay, and a gel mobility shift assay.
First, the proteins were incubated with poly(rI·rC)-Sepharose in low
salt concentration (25 mM KCl). Aliquots of the beads were washed with buffer containing increasing concentrations of KCl, and the
protein remaining associated with dsRNA was assayed by gel
electrophoresis and staining. Fig.
2A shows that the three proteins bound to the resin and were eluted at varying salt
concentrations. p20 resisted elution to 0.75-1 M KCl,
whereas p10A was eluted at ~0.4 M KCl and p10B at ~0.2
M. These data confirm that the binding of p20 to
poly(rI·rC) is stronger than that of p10A (24) and show that p10B
also binds but more weakly. For a more quantitative comparison of the
relative affinities, we conducted filter-binding assays with
end-labeled poly(rI·rC). As estimated from the data of Fig.
2B, the Kd for p20 binding to
poly(rI·rC) is about 5 × 10
In gel mobility shift assays with purified dsRNA of defined size, p20
gives rise to a series of bands corresponding to the sequential binding
of p20 molecules until the RNA is fully saturated. This occurs at ~11
base pairs/p20 molecule (4, 24). Using dsRNA of 132 base pairs in the
presence of competitor tRNA, low concentrations of p20 gave rise to
several distinct bands (Fig. 3A, lane 5). At
higher concentrations, p10A caused a mobility shift without giving rise
to a very discrete banding pattern (lanes 9-12), whereas
p10B did not elicit a mobility shift even at the highest concentrations
tested (lanes 6-8). These assays indicate that p10A binds
to dsRNA with a higher affinity than p10B and that there is
cooperativity between the two dsRBMs in binding dsRNA. Mixing
experiments (lanes 2-4) show that this cooperativity is
achieved only when the two dsRBMs are tandemly located on the same
protein molecule.
The failure to detect an interaction between p10B and dsRNA by the
mobility shift technique, in contrast to the matrix-binding techniques
used with poly(rI·rC) in Fig. 2, prompted us to carry out assays in
the absence of tRNA competitor. Under such conditions, p10B gave rise
to a mobility shift at high concentrations (Fig. 3B,
lanes 5-9), reaffirming the conclusion that dsRBM2
interacts with dsRNA with lower affinity than dsRBM1 and implying that
the dsRBM2 interaction is not entirely specific for dsRNA.
dsRNA Annealing Activity--
Hitti et al. (40)
demonstrated that the dsRBMs of Xlrbpa have RNA annealing activity.
However, this activity did not correlate with the affinity of the
Xlrbpa dsRBMs for dsRNA. Using the protocol diagrammed in Fig.
4A, we assayed the ability of
the dsRBMs of PKR to catalyze the annealing of two partially
complementary single-stranded RNAs. As expected, a high concentration
of KCl caused annealing (Fig. 4B, lane 7) but
bovine serum albumin was ineffective (lanes 2-4). Annealing
activity was displayed by p10A (lanes 14-16) and p20
(lanes 8-10) but not by p10B (lanes 11-13).
Thus, the annealing activity of the PKR dsRBMs correlates with their
dsRNA binding affinity, suggesting that dsRNA binding is important for
the annealing function, but cooperativity between the two dsRBMs is not
evident in this assay.
Protein Dimerization--
The activation of PKR requires that it
undergo dimerization and autophosphorylation (27, 28). Dimerization has
been observed both in vivo and in vitro and is
attributed to sequences within (30, 46) as well as outside (31) of the
dsRBMs. In the case of dimerization via sequences in the dsRBM region,
it has been questioned whether the dimerization is an intrinsic
activity of the protein or is mediated by dsRNA (46). To address this
issue, we used DMS, a chemical cross-linking reagent reactive with
primary amine groups (30). Cross-linking was carried out with p20,
p10A, and p10B in the presence or absence of poly(rI·rC). Fig.
5A (top panel)
shows that p20 dimerizes in the absence of poly(rI·rC), confirming
the intrinsic dimerization activity of the PKR N terminus (30). p20
dimerization was inhibited by high concentrations of poly(rI·rC),
suggesting a competition between dsRNA binding and protein
dimerization. It could be that residues involved in dsRNA binding also
participate in protein·protein interactions, or perhaps dsRNA binding
causes a conformational change in the protein that disfavors
dimerization. Both p10A and p10B were also cross-linked by DMS (Fig.
5A, middle and bottom panels) but with somewhat different characteristics from p20 and each other. Like p20,
p10B predominantly gave rise to dimers, whereas p10A multimerized. The
dimerization of p10B was not inhibited by poly(rI·rC), however, and
the multimerization of p10A was only slightly reduced at high concentrations of poly(rI·rC). These observations can be interpreted in terms of the distribution of dimerization and dsRNA binding sites
(see "Discussion").
These results were obtained with proteins purified under denaturing
conditions (followed by a renaturation step) to exclude possible
bacterial dsRNA contaminants from the protein preparations. In a
reconstruction experiment, the purification protocol reduced dsRNA
binding to undetectable levels (data not shown). To verify our
conclusions, we studied the cross-linking of mutant forms of p20 that
are defective in dsRNA binding (44). All four mutants were cross-linked
by DMS to similar extents in the absence of poly(rI·rC), but the
ability of a high concentration of poly(rI·rC) to inhibit
cross-linking was determined by the mutants' ability to bind dsRNA
(Fig. 5B). The two mutants in which dsRNA binding is only
mildly impaired (LS14 and LS17: dsRNA binding ~20 and 45% of
wild-type, respectively) behaved like wild-type p20: Their cross-linking was sharply decreased by poly(rI·rC). In contrast, protein·protein cross-linking was unaffected by poly(rI·rC) for the
two mutants in which dsRNA binding is essentially ablated (LS16 and
LS19:
The ability of p20 to dimerize in the absence of dsRNA appears to
contradict our previous observation (46). Instead of monitoring the
existence of preformed oligomers as above, we therefore conducted pull-down assays to monitor the formation of protein·protein
interactions. Full-length PKR tagged with GST was immobilized on
glutathione-Sepharose beads, which were incubated with p20 in the
presence of various concentrations of poly(rI·rC). GST-PKR interacted
weakly with p20 in the absence of poly(rI·rC). In the presence of
increasing concentrations of poly(rI·rC), p20 binding increased to a
maximum then declined. High concentrations of poly(rI·rC) effectively eliminated the binding of p20 to PKR (Fig. 5C). Consistent
with our earlier findings (46), these results indicate that the
formation of p20·PKR heterodimers is largely dependent on the
presence of dsRNA. Evidently, under these conditions, the pre-existing
p20 dimers efficiently dissociate and heterodimerize with GST-PKR only
in the presence of dsRNA.
Activation of Reporter Gene Expression--
Several inactive
mutants of PKR function as dominant negative inhibitors and enhance the
expression of reporter genes in trans. However, the
mechanism of this effect is uncertain. We therefore examined the
ability of p10A, p10B, and p20 to stimulate gene expression.
EGFP-tagged dsRBM constructs were transfected into human 293 cells
together with a firefly luciferase reporter gene. At the highest
concentrations of EGFP-dsRBM fusion constructs tested, reporter gene
expression was enhanced about 7-fold by p20 and 4-fold by p10A, but no
effect was observed with p10B or with EGFP alone (Fig.
6). Similar results were obtained with
the DHFR reporter gene by Wu and Kaufman (33), who also concluded that
dsRNA binding rather than dimerization correlates with PKR activation.
The enhancements seen with p20 and p10A were
dose-dependent, and the maximal effect attained by p20-EGFP
was similar to that obtained with the dominant negative mutant of PKR,
K296R (data not shown). Thus, the results obtained in these reporter
gene expression assays correlate with the dsRNA binding activity of the
dsRBMs but not with their dimerization ability.
Subcellular Localization--
By immunofluorescence and electron
microscopy, PKR is detected in the cytoplasm and nucleoli as well as
diffusely throughout the nucleoplasm (34, 35, 47). To define the
contribution of the dsRBMs to the intracellular distribution of PKR, we
expressed EGFP-tagged constructs in human HeLa cells (Fig.
7). As expected, PKR-EGFP elicited clear
signals in both the cytoplasm and nucleoli. The inactive kinase mutant
K296R-EGFP exhibited the same distribution as wild-type PKR, indicating
that the kinase activity of PKR is not necessary for its proper
localization. EGFP alone gave a diffuse signal throughout the cell,
especially in the nucleus. This pattern of EGFP distribution was
unaffected by the attachment of dsRBM2 (p10B-EGFP), the kinase domain
( It has been reported that members of the dsRBM motif family differ
in their dsRNA binding affinity (16, 22, 48). Here we have examined the
two dsRBMs of PKR to explore differences between dsRBMs with respect to
several additional functions and to assess whether these functions are
related to dsRNA binding. The additional functions include RNA
annealing, protein dimerization, stimulation of gene expression, and
intracellular localization. We confirmed that dsRBM1 has a greater
affinity for dsRNA than dsRBM2 and found that it also has a much
greater ability to anneal single-stranded RNAs, to enhance gene
expression in transfection assays, and to participate in PKR
localization. As with dsRNA binding, dsRBM2 facilitated the activity of
dsRBM1 in all these assays except for RNA annealing. Cross-linking
assays showed that dsRBM1 is able to oligomerize, whereas dsRBM2
dimerizes under the same conditions.
The x-ray crystal structure of dsRBM2 of Xlrbpa, a dsRBM-containing
protein found in Xenopus laevis, in complex with dsRNA showed that three regions within the dsRBM are involved in dsRNA binding (20). NMR studies of PKR indicated that dsRBM1 and -2 have
nearly the same three-dimensional protein folding (19). The differences
in dsRNA binding between the dsRBMs of PKR are therefore attributable
to differences between the side chains of the two dsRBMs. Inspection of
the alignment of the two PKR dsRBMs with the consensus sequence (Fig.
1) suggests that regions 1 and 3 are similar between dsRBM1 and -2, whereas region 2 of dsRBM2 differs from that in dsRBM1 and the
consensus sequence. The histidine and arginine residues in region 2 participate in protein·RNA interactions (20), implying that the lack
of homology in this region may contribute to the weaker activity of
dsRBM2 in dsRNA binding. dsRBMs have been divided into two groups based on their homology to the consensus sequence (21, 22). Group A dsRBMs,
including dsRBM1 of PKR, are homologous to the consensus throughout the
sequence, whereas group B dsRBMs like PKR dsRBM2 display conservation
only in the C-terminal area where region 3 is located. Our observation
that p10B exhibits weak dsRNA binding affinity correlates with its
relatively low level of homology in region 2.
In various assays for dsRNA binding, we observed that dsRNA has a
higher affinity for p20 than for p10A or p10B. This implies that dsRBM2
is able to strengthen the dsRBM1:RNA interaction considerably due to
the cooperativity between the two dsRBMs as suggested previously (44).
Cooperation requires that the dsRBMs are covalently joined (Fig.
3A), possibly indicating that the presence of two dsRBMs on
the same molecule reduces ligand off-rate. The fact that eukaryotic dsRBM-containing proteins frequently contain more than one dsRBM suggests that cooperative binding might be a commonly used device to
augment the protein's affinity for dsRNA. Another possibility is that
multiple dsRBMs confer sequence specificity upon the interactions with
RNAs, particularly in the case of proteins that contain more than three
dsRBMs. Conceivably PKR has evolved to have two dsRBMs to allow it to
bind dsRNA with high affinity yet low specificity. A notable feature of
our filter binding assays is the observation that at the saturation
point more end-labeled poly(rI·rC) was bound to p20 than to p10A and
p10B (Fig. 2). This may indicate that p20 can bind certain structured
RNAs, e.g. tailed duplexes, that exist in poly(rI·rC) and
are not bound efficiently by p10A or p10B.
It was reported previously that the dsRBMs of Xlrbpa can facilitate
dsRNA annealing and that this annealing activity is independent of
dsRNA binding, because some dsRBMs that failed to bind dsRNA still
catalyzed annealing (40). In PKR, annealing activity seems to
correspond more closely to dsRNA binding activity, although cooperativity between the two dsRBMs is not beneficial toward the
reaction (Fig. 4). This would be expected if the protein functions by
facilitating the matching up of two complementary strands. The
correlation of RNA binding with annealing is supported by the
observation that the binding of one dsRBM-containing molecule induces a
more uniformly double-stranded conformation in an imperfect duplex,
thereby facilitating the binding of further protein molecules (49). The
annealing activity of the dsRBM suggests that PKR and other
dsRBM-containing proteins may play a role as chaperones, facilitating
the folding of cellular RNAs (40). This property of PKR may sensitize
the cellular response to viral infection by annealing two complementary
viral RNAs, which can then activate the PKR-mediated anti-viral defense mechanism.
The ability of PKR to dimerize has been demonstrated by several assays,
including yeast two-hybrid, gel filtration, chemical cross-linking,
far-Western, and various pull-down protocols (30, 46, 50). At least two
regions of the molecule have been implicated in dimerization (30, 31).
Dimerization involving the N-terminal region is complicated by the
possibility that the interactions may be bridged by dsRNA as well as
due to direct protein·protein interactions. In our experiments, we
excluded dsRNA contamination by purifying the proteins under stringent
denaturing conditions followed by a renaturing step: Such preparations
still formed dimers (p20 and p10B) and higher multimers (p10A).
Furthermore, p20 dimerization was unaffected by mutations that severely
impair dsRNA binding (Fig. 5B), indicating that the
N-terminal dsRBMs have an intrinsic ability to interact. Interestingly,
the dimerization of p20 was inhibited by poly(rI·rC) at high
concentration. Two possible explanations for this observation can be
considered. One is that amino acids involved in dsRNA binding are also
engaged in dimerization, resulting in competition between dsRNA binding and dimerization for the same interaction site. This view is supported by our finding that competition correlates with dsRNA binding affinity.
When dsRNA binding is strong, as in the case of p20, dimerization is
efficiently competed, whereas when the dsRNA binding is weaker (p10A)
or very weak (p10B, LS16, LS19) there is less or no detectable
competition (Fig. 5, A and B). An alternative interpretation is that the binding of dsRNA changes the structure of
the protein, thereby disfavoring protein-mediated dimerization. Support
for this view can be drawn from mutational data such as that in Fig.
5B, indicating that the dsRNA-binding activity is separable
from protein dimerization (28, 33, 51). Furthermore, residues
responsible for dimerization and dsRNA binding lie on distinct faces of
the C-terminal Mutations in the kinase domain of PKR enhance the expression of
reporter genes, presumably by down-regulating the activity of cellular
wild-type PKR. Two mechanisms have been advanced to explain this
dominant negative effect: It could result from the interaction of the
mutant kinase with wild-type kinase resulting in the formation of
inactive homodimers or the sequestration of PKR activators such as
dsRNA in the cell (23, 53). Our results show that the N-terminal region
of PKR (p20) and even the first dsRBM alone (p10A), are able to
stimulate reporter gene expression, whereas dsRBM2 (p10B) lacks this
activity (Fig. 6). The magnitude of the effect correlates with the
ability of the dsRBMs to bind to dsRNA but not with their ability to
dimerize, supporting the view that dsRNA sequestration underlies the
dominant negative effect.
PKR localizes in the cytoplasm, strongly in the nucleolus, and
diffusely throughout the nucleoplasm (34, 35, 47). Its cellular
localization does not change after interferon induction or adenovirus
infection (34). By immunoelectron microscopy, PKR is found mainly in
the dense fibrillar component of the nucleolus, which is believed to be
the site of nascent rRNA synthesis and processing (54), suggesting that
PKR might be involved in ribosome biosynthesis (34). Our study
indicates that the dsRBMs are required for PKR's localization and that
this activity correlates with dsRNA binding (Fig. 7). However, the
exact mechanism for this localization is still not clear. Because
ribosomes are assembled and processed in the nucleolus and PKR
associates with ribosomes (35, 37, 55), it is tempting to speculate
that ribosome binding is responsible for PKR's subcellular
localization. In extracts of yeast expressing human PKR, the kinase was
found to be associated with the 40 S ribosomal subunit (37), although, in mammalian extracts, PKR was shown to interact with the L18 protein
of the 60 S ribosome subunit and to compete for dsRNA binding (38).
This discrepancy notwithstanding, in both cases the dsRBMs seemed to be
important for ribosome binding. Because PKR's localization correlates
with its dsRNA-binding activity, its ribosome association is likely to
be due to the binding of PKR to structured RNA such as ribosomal RNA
(rRNA). This view is supported by the fact that the NMR structure of
dsRBM has many features in common with the N-terminal domain of the
prokaryotic ribosomal S5 protein (18). Furthermore, several other
dsRBM-containing proteins also localize in the nucleolus (56,
57).3
A related question raised by this study is how PKR is transported into
the nucleus. Although the localization of proteins within the nucleus
may be largely driven by diffusion and retention, the process of moving
proteins into the nucleus is believed to be carried out by an elaborate
transport system (58). For the relatively small protein molecules in
this study, such as EGFP itself and its fusions with p10A, p10B and
p20, movement into the nucleus may be through diffusion, whereas for
larger proteins such as the fusions of EGFP with full-length PKR, In conclusion, the RNA-annealing, reporter gene activation, and
subcellular localization properties of PKR are related to the
dsRNA-binding activity of its dsRBMs, which is predominantly dependent
on dsRBM1, whereas oligomerization appears to be at least partially
independent of dsRNA binding. Of the RNA-related properties, only
annealing is not discernibly influenced by cooperation between the two
dsRBMs. Based on this study and previous findings, such as the
observation that cytosolic PKR is predominantly in a dimeric form,
whereas ribosome-associated PKR is monomeric (50, 59) and that PKR in
the nucleus is predominantly basic (i.e. unphosphorylated)
(35), we propose the model shown in Fig.
8 for the activation and localization of
PKR. In the latent state, PKR exists in the cell in two forms: an
inactive dimer in the cytoplasm, and an inactive monomer associated
with ribosomes. It is actively transported to the nucleus by unknown
mechanisms and moves to the nucleolus by binding to structured RNA,
probably rRNA. When dsRNA is present, as during viral infection, PKR in the cytoplasm switches from an inactive, protein-mediated homodimer form to an active, dsRNA-mediated dimer. It is unclear whether ribosome-associated PKR can be directly activated by dsRNA. The binding
of short structured RNAs, such as adenovirus VA RNAI, leads
to the formation of inactive PKR monomers in the cytoplasm. We also
speculate that protein activators such as PKR-activating protein and
nuclear factor 90, which, respectively, contain three and two
dsRBMs, activate PKR by a mechanism involving dsRBM interactions, which
allow PKR to switch from an inactive dimer to an active dimer.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gene (3). The role of PKR in protein synthesis is well
studied. It inhibits translation initiation by phosphorylating the
subunit of initiation factor eIF2. Phosphorylated eIF2
binds with
high affinity to a second initiation factor, eIF2B, that is usually present in limiting amounts. Sequestration of eIF2B prevents it from
promoting GTP/GDP exchange on the eIF2 complex, a critical step in
forming the ternary complex eIF2·GTP·Met-tRNAi
that is essential for initiation. Kinase activity of PKR is activated by autophosphorylation upon binding to its activators, which include most notably, perfect duplex dsRNAs (4) as well as other highly structured RNAs (5-8), polyanionic molecules such as heparin or
polyglutamine, and protein regulators such as PKR-activating protein (9) and nuclear factor
90.2 Inhibitors of PKR are
even more numerous and diverse than activators; they include viral and
cellular RNAs and proteins, such as adenovirus virus-associated
RNAI, Epstein-Barr virus EBER RNA, human Alu RNA, human p58
protein, TAR RNA-binding protein, and vaccinia virus E3L and K3L
proteins (2, 10-15).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
LBN and SRG5
Lp10 (24) were used
to construct 6xHis-tagged p10A, p10B, and p20. SRG5
LBN was cut with
NdeI and BamHI to give a fragment encoding p20,
or with NcoI and BamHI for p10B. SRG5
Lp10 was
cut with NdeI and BamHI to give a fragment encoding p10A. Using complementary restriction sites, the fragments were ligated into pET28b (Novagen) yielding His-p10A and His-p20, and
into pET30LIC for His-p10B. For EGFP constructs, the EGFP expression
vector pcEGFP (BE) was first constructed by inserting the EGFP coding
fragment from pBIEGFP (CLONTECH) into pcDNA3.1 (Invitrogen) using BamHI and EcoRI. pcEGFP was
cut with NheI and BamHI and ligated with
PKR fragments generated by PCR using the following primers:
PKR-N, 5'-TAGCGCTAGCATGGCTGGTGATCTTTCAG-3'; PKR-C,
5'-AGCTGGATCCACATGTGTGTCGTTCATTTT-3'; p10B-N,
5'-TAGCGCTAGCATGACAACAACGAATTCTTC-3'; p10A-C,
5'-CAGCTGGATCCTTCTGAAGAATTCGTTG-3'; and p20-C,
5'-CAGCTGGATCCACACGTAGTAGCAAAAGA-3'.
1-EGFP (amino acid
88-551) using p10B-N and PKR-C. The primer set PKR-N and PKR-C was
also used for constructing
2-EGFP, and
N-EGFP. PCR was carried
out with pfu DNA polymerase (Stratagene) using the following templates:
human wild-type pcDNA3-PKR (41) for p10A-EGFP, p10B-EGFP, p20-EGFP,
1-EGFP, and PKR-EGFP; pcDNA3-K296R mutant for K296R-EGFP;
pSRG
2 (42) for
2-EGFP; sub1:1 (44) for
2'-EGFP; or
PKR-
14-257 (37) for
N-EGFP. For point mutants in p20,
templates were from Green et al. (44). PCR products were
purified using a PCR purification kit (Qiagen), cut with NheI and BamHI, and ligated into the pcEGFP
vector that was cut with the same enzymes. For His-EGFP-tagged
proteins, fragments cut from various EGFP constructs were inserted into
pET28b using NheI and NotI. For luciferase
reporter assays, the luciferase plasmid pBILuc(+) was constructed by
ligating the luciferase fragment excised from pSPLUC(+) (Promega) by
NheI and XbaI into pcDNA3.1(+) cut with
NheI.
-D-galactopyranoside (final
concentration) was added to induce protein expression at 30 °C for
2.5 h. Cells were collected by centrifugation and resuspended in
buffer A with protease inhibitors (5 mM imidazole, 0.5 M NaCl, 0.1% Nonidet P-40, and 20 mM HEPES, pH
7.5). Cells were briefly sonicated and centrifuged at 10,000 × g for 30 min. The supernatant was used for protein
purification. For His-tagged protein purification, the POROS-MC (PE
Applied Biosystems) column matrix was used on a BioCAD Sprint perfusion
chromatography system (PE Applied Biosystems). The column was charged
with 50 column volumes of 0.1 M NiCl2, washed,
and loaded with bacterial extract. After extensive washing with
buffer A, His-tagged proteins were eluted by buffer B (500 mM imidazole, 0.5 M NaCl, and 20 mM
HEPES, pH 7.5). Proteins were dialyzed overnight against dialysis
buffer (20% glycerol, 150 mM NaCl, 1 mM DTT,
0.5 mM EDTA, and 20 mM HEPES, pH 7.5) and
stored at
80 °C until use. For protein purification under
denaturing conditions, cell extracts were suspended in buffer DA (6 M guanidine-HCl, 5 mM imidazole, 0.5 M NaCl, and 20 mM HEPES, pH 7.6), loaded onto the column, washed with buffer DA then buffer RA (6 M urea,
0.5 M NaCl, 20% glycerol, 20 mM HEPES, pH
7.5), and renatured on the column at room temperature for 90 min by
gradually changing the proportions of buffer RA (from 100% to 0%) and
buffer RB (150 mM NaCl, 20% glycerol, and 20 mM HEPES, pH 7.5; from 0% to 100%). Proteins were eluted
with buffer DB (0.5 M imidazole, 150 mM NaCl, 20% glycerol, and 20 mM HEPES, pH 7.5) and dialyzed as
above. The denaturation step reduced the binding of
32P-labeled poly(rI·rC) with His-tagged p20-EGFP
to background levels.
-32P]ATP (ICN) and T4
polynucleotide kinase (New England BioLabs) at 37 °C for 45 min,
heated at 68 °C for 5 min, cooled down slowly to room temperature,
and passed through a Bio-Gel p-30 polyacrylamide gel column (Bio-Rad).
Labeled poly(rI·rC), about 50,000 cpm, was incubated with proteins on
ice for 20 min in binding buffer (5 mM MgCl2, 1 mM DTT, 0.1 mg/ml E. coli tRNA, 0.1 mg/ml bovine
serum albumin, and 20 mM HEPES, pH 7.5). The filter binding
assay was carried out as described previously (4) using washing buffer (50 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 20 mM HEPES, pH 7.5) and a 0.45-µm
nitrocellulose membrane (Schleicher & Schuell). Retained poly(rI·rC)
was quantified using an InstantImager (Packard).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structures of PKR, p10A, p10B, and p20.
A, map of PKR, p10A, p10B, and p20 used in this study,
showing amino acid residue numbers. B, purified 6xHis-tagged
proteins were separated in 12.5% SDS gels and stained with Coomassie
Brilliant Blue. Positions of marker proteins (sizes in kilodaltons
(KD)) are indicated. C, sequence alignment of
dsRBM1 and dsRBM2 of PKR using BestFit (GCG Package) (vertical
lines, identical residues; colons, homologous residues;
periods, weakly homologous residues) together with a dsRBM
consensus sequence (20). Secondary structures are marked (h,
-helix; b,
-sheet) based on the published NMR
structure (19). Three regions of dsRBM shown to interact with dsRNA are
denoted 1, 2, and 3.
9 M, in
agreement with previous measurements by gel mobility shift analysis
(24, 45). The affinities of p10A and p10B were lower, with
Kd values about 5 × 10
8 and
2 × 10
7 M, respectively. The affinity
of p10A for perfectly duplexed RNA measured by gel mobility shift
analysis, 3.8 × 10
7 M (24), was
somewhat lower than the estimate obtained here, possibly implying that
single-stranded tails present in poly(rI·rC) contribute to the
binding.
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Fig. 2.
Protein-poly(rI·rC) interactions.
A, poly(rI·rC)-Sepharose pull-down assay. Proteins were
bound to poly(rI·rC)-Sepharose beads, which were then washed with the
concentrations of KCl indicated (from 25 to 1000 mM).
Retained proteins were dissociated in gel sample buffer, resolved in
SDS gels, and stained with Coomassie Brilliant Blue. The amount of
protein used for this assay is indicated in the lane marked
Load. B, filter binding assay with end-labeled
poly(rI·rC). Complexes formed with His-tagged proteins were filtered
onto nitrocellulose membrane using a slot-blot apparatus, and the
membrane was subjected to autoradiography (bottom panel) and
quantitation using an InstantImager. The graph depicts binding to p20,
; p10A,
; and p10B,
. Protein concentrations were as follows:
1, 1.2 ng/ml; 2, 2.4 ng/ml; 3, 4.9 ng/ml; 4, 9.8 ng/ml; 5, 19.5 ng/ml; 6,
39.1 ng/ml; 7, 78.1 ng/ml; 8, 156 ng/ml;
9, 313 ng/ml; 10, 625 ng/ml; 11, 1.25 µg/ml; 12, 2.5 µg/ml; and 13, 5 µg/ml.
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Fig. 3.
Gel mobility shift assays. A,
gel shift assay with labeled dsRNA (132 bp) in the presence of
competitor tRNA. Protein concentrations were as follows: p20, 80 ng/ml
(lanes 3-5); p10A, 6.25 µg/ml
(lanes 2, 4, and 12), 3.13 µg/ml (lane 11), 1.6 µg/ml (lane
10), and 0.8 µg/ml (lane 9); p10B,
12 µg/ml (lanes 2, 3, and
8), 6 µg/ml (lane 7), and 3 µg/ml
(lane 6). Lane 1 contained dsRNA only.
B, gel shift assay in the absence of competitor RNA.
Concentrations of His-tagged proteins were 24 ng/ml (lanes
1, 6, and 11), 195 ng/ml
(lanes 2, 7, and 12), 1.56 µg/ml (lanes 3, 8, and
13), 12.5 µg/ml (lanes 4,
9, and 14), and 0.1 mg/ml (lanes
5, 10, and 15). Lane 16 contained dsRNA only.
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Fig. 4.
RNA annealing. A, schematic
outline of the assay. B, products of the annealing assay
were resolved in an 8 M urea/5% TBE polyacrylamide gel,
and subject to autoradiography. Protein concentrations for the assay
were 0.666 µg/ml (lanes 2, 8,
11, and 14), 6.66 µg/ml (lanes
3, 9, 12, and 15), and 66.6 µg/ml (lanes 4, 10, 13,
and 16). The KCl concentrations in lanes
5-7 were 13.3 mM, 133 mM, and 1.33 M. No protein or KCl was added to the reaction in
lane 1.
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Fig. 5.
Protein·protein interactions.
A, chemical cross-linking assays. Each assay contained 8 µg/ml His-tagged protein and 2 mM DMS, except
lane 1 where no DMS was added, together with
poly(rI·rC) at the following concentrations: none (lanes
1 and 2), 20 ng/ml (lanes 3), 0.2 µg/ml (lanes 4), 2 µg/ml (lanes
5), 20 µg/ml (lanes 6), and 200 µg/ml (lanes 7). B, cross-linking of His-tagged
p20-EGFP and its mutants by DMS in the presence (solid bars)
or absence (open bars) of poly(rI·rC) at 200 µg/ml.
Results are expressed as percentages of the input proteins that
cross-linked to form dimers. C, GST-PKR pull-down assay with
His-tagged p20 (1.0 µg of p20/assay). Reactions in lanes
4-8 contained poly(rI·rC) at 0.05, 0.5, 50, and 500 µg/ml, respectively. The lane marked 1/2 Load contains
half of the amount of protein used in this assay. Proteins were
resolved in a 12.5% SDS gels and detected using anti-6xHIS
antibody.
5% of wild-type dsRNA binding), confirming that the intrinsic
capacity of p20 to dimerize is reduced by interaction with high
concentrations of dsRNA.
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Fig. 6.
Trans-activation assay. Human 293 cells
growing in 12-well plates were transfected with a plasmid expressing
the firefly luciferase reporter gene together with constructs
expressing EGFP, p10A-EGFP, p10B-EGFP, or p20-EGFP at the following
concentrations: 80 ng (bars 1, 4, 7,
and 10), 200 ng (bars 2, 5,
8, and 11), and 500 ng (bars 3,
6, 9, and 12). pUC119 was used to
bring the total amount of the transfected plasmid to 500 ng. The
luciferase activities given by EGFP constructs at each concentration
were arbitrarily set at 100%. Error bars represent standard
deviations. Transfections were done in duplicate and the experiment was
repeated twice.
N-EGFP), or both (
1-EGFP). However, the intact N terminus of PKR
(p20-EGFP) conferred a localization indistinguishable from that of the
wild-type PKR construct, indicating that this region is sufficient for
the proper localization of PKR. Similar distributions were seen with
p10A-EGFP and
2'-EGFP (deletion of amino acids 104-185), which
contains dsRBM1 plus the kinase domain. These constructs were
concentrated in the nucleolus, although they gave an increased
nucleoplasmic signal relative to PKR-EGFP, p20-EGFP, and K296R-EGFP.
Thus, dsRBM1 plays an important role in PKR distribution, and it is
nearly sufficient alone for the proper localization of PKR. Presumably,
the failure of these fusion proteins to clear fully from the
nucleoplasm is due to their decreased affinity for RNA relative to PKR
forms containing both dsRBMs. One construct,
2-EGFP (deletion of
amino acids 104-158), gave an anomalous pattern. This fusion protein,
which lacks most of dsRBM2 but retains its C-terminal third, was
restricted to the cytoplasm possibly because it is misfolded or binds
preferentially to cytoplasmic components such as ribosomes (38).
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Fig. 7.
Subcellular localization of PKR-EGFP fusion
proteins. A, schematic of constructs used in this
study. B, HeLa cells growing on coverslips were transfected
with 200 ng of plasmids expressing the indicated EGFP fusion proteins.
Protein localization was visualized by fluorescence microscopy using a
lens with × 100 magnification.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix of the dsRBM (52). It is possible that dsRNA
can switch PKR from an inactive dimer mediated by protein·protein
interactions to an active dimer mediated by dsRNA. Comparison of p20
homodimers (whose existence is independent of dsRNA binding) with the
formation of GST-PKR·p20 heterodimers (which is
dsRNA-dependent: Fig. 5C) suggests that the
efficiency or rate of protein·protein dimerization (p20·PKR) is
considerably less than that of dsRNA-mediated dimerization. Consistent
with this notion, no heterodimers were detected in pairwise
cross-linking reactions between p10A, p10B, and p20 (data not shown).
Apparently, pre-existing dsRBM oligomers do not readily dissociate and
heterodimerize in the absence of dsRNA. This observation may help
explain conflicting reports regarding the RNA dependence of PKR
dimerization (30, 46).
1,
2', and
N, nuclear penetration might be facilitated by an active
transport process. No nuclear localization signal has been found in
PKR, however. Remarkably,
2-EGFP failed to localize in the nucleus. One possible explanation is that PKR lacking dsRBM2 is misfolded in the
cell and cannot pass through the nuclear pores. Consistent with this
explanation, PKR
2 is highly deficient in dsRNA binding (42).
Alternatively, it is possible that the presence of the C terminus of
dsRBM2 together with an intact dsRBM1 might cause the protein to bind
to some component in the cytoplasm with very high affinity, so that it
is effectively sequestered in the cytoplasm. In this case, however, the
RNA binding properties of the
2 mutant make it unlikely that the
ligand is RNA.
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Fig. 8.
Model for PKR activation and
localization. See "Disussion" for details.
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ACKNOWLEDGEMENTS |
---|
We thank L. Manche for help in the early
stages of this work, B. R. Williams for the GST-PKR plasmid, and
P. R. Romano for the PKRN mutant.
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FOOTNOTES |
---|
* This study was supported in part by Grant AI34552 from the National Institutes of Health.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.
¶ Supported by predoctoral fellowship 981005T from the American Heart Association. Present address: R. W. Johnson Pharmaceutical Research Inst., 3210 Merryfield Row, San Diego, CA 92121.
To whom correspondence should be addressed. Tel.:
973-972-4411; Fax: 973-972-5594; E-mail: mathews@umdnj.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M007328200
2 L. Parker, I. Fierro-Monti, and M. B. Mathews, manuscript in preparation.
3 B. Tian and M. B. Mathews, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PKR, interferon-induced dsRNA-activated protein kinase; dsRNA, double-strand RNA; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DTT, dithiothreitol; GST, glutathione S-transferase; dsRMB, dsRNA-binding motif; eIF2, eukaryotic initiation factor 2.
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