(Received for publication, May 12, 1995; and in revised form, October 12, 1995)
From the
The 58-kDa protein, referred to as P58, is a cellular inhibitor
of the interferon-induced, double-stranded RNA-activated protein
kinase, PKR. The P58 protein inhibits both the autophosphorylation of
PKR and the phosphorylation of the PKR natural substrate, the
subunit of eukaryotic initiation factor eIF-2. Sequence analysis
revealed that P58 is a member of the tetratricopeptide family of
proteins. Utilizing experimental approaches, which included
coprecipitation or coselection of native and recombinant wild-type and
mutant proteins, we found that P58 can efficiently complex with the PKR
protein kinase. Attempts to map the P58 interactive sites revealed a
correlation between the ability of P58 to inhibit PKR in vitro and bind to PKR. The DnaJ sequences, present at the carboxyl
terminus of P58, were dispensable for binding in vitro, while
sequences containing the eIF-2
similarity region were essential
for efficient complex formation. Furthermore, not all tetratricopeptide
motifs were necessary for PKR-P58 interactions. Initial experiments to
map the binding domains present in PKR showed that P58 complexed with
PKR molecules that lacked the first RNA binding domain but did not bind
to a PKR mutant containing only the amino terminus. These data, taken
together, demonstrate that P58 inhibits PKR through a direct
interaction, which is likely independent of the binding of
double-stranded RNA to the protein kinase.
PKR ()(for protein kinase RNA-dependent) is a 68-kDa
serine-threonine kinase that is induced in cells upon interferon
treatment(1, 2, 3, 4) . In the
presence of double-stranded RNA (or other polyanions), PKR becomes
autophosphorylated and phosphorylates the
subunit of eukaryotic
translation initiation factor 2 (eIF-2)(5, 6) .
Phosphoryation of eIF-2 by PKR prevents recycling of eIF-2
GDP to
eIF-2
GTP, and as a result, protein synthesis initiation is
globally blocked within the cell(7, 8) . PKR
activation and phosphorylation of eIF-2 therefore can represent a major
effector of the interferon antiviral response. Interestingly, PKR, due
to its translationally repressive functions(9, 10) ,
also may be involved in the control of cell growth. In this regard,
introduction of catalytically inactive and select regulatory domain
mutants of PKR into NIH 3T3 cells results in their malignant
transformation(11, 12, 13) . In certain cases
this may be due to dominant negative inhibition of the endogenous
murine PKR by the mutants, leading to deregulated protein
synthesis(11, 13) .
Both RNA and DNA viruses
produce RNA intermediates that can activate PKR. As a result, a number
of these viruses encode or activate proteins or RNAs that specifically
inhibit the action of
PKR(14, 15, 16, 17) . If this
down-regulation of PKR failed to occur, viral protein synthesis would
become severely compromised. We have shown previously that PKR
activation is inhibited in influenza virus infected cells by a cellular
protein(18, 19, 20, 21, 22) .
The PKR inhibitor, termed P58 based on its molecular weight of 58 kDa,
was purified to homogeneity from Madin-Darby bovine kidney cells, and
subsequently the gene was molecularly cloned using reverse
genetics(19, 20) . Recombinant P58 is capable of
inhibiting PKR activation and eIF-2 phosphorylation(20) .
Sequence analysis indicated that P58 is a member of the
tetratricopeptide (TPR) family of proteins, which is characterized by
internal 34 amino acid repeats. Moreover it was found that P58
possessed sequence similarity to the DnaJ heat shock family of proteins
and more limited similarity to the eIF-2 subunit.
We found
that overexpression of P58 in NIH 3T3 cells caused their malignant
transformation, demonstrating the oncogenic potential of the PKR
inhibitor(23) . The molecular mechanisms underlying P58-induced
down-regulation of PKR and transformation of murine cells remain
unknown. Earlier work from our laboratory demonstrated that P58 did not
retain ATPase, phosphatase, or protease
activity(20, 21) . Furthermore, P58 did not sequester
dsRNA activator nor did the inhibitor degrade dsRNAs. ()The
current study was initiated to determine whether, like the adenovirus
VAI RNA inhibitor (24) and the vaccinia virus K3L PKR
inhibitor(25) , P58 functioned through a direct interaction
with PKR. Utilizing recombinant wild-type and mutant proteins, we now
show that P58 and PKR form a complex in vitro. Moreover, P58
variants, which fail to inhibit PKR in vitro, do not interact
with PKR. Finally we demonstrate that P58 can interact with a PKR
variant lacking RNA binding domain 1 but not to a mutant containing
both dsRNA binding domains.
Figure 1:
P58 and PKR recombinant proteins. Panel A, at the top of the panel is depicted the P58
gene with the nine TPR motifs. The regions of eIF-2 and DnaJ
similarities are highlighted. The histidine-tagged wild-type
and mutant are depicted below and are described under ``Material
and Methods.'' Panel B, the GST-P58 fusion protein (and
GST tag alone as control) expressed from vector pGEX2T-P58 are shown. Panel C, the wild-type PKR is depicted, which contains the two
dsRNA binding domains (RBD-1 and -2) in the regulatory amino terminus
and the 11 catalytic domains found at the carboxyl terminus. Also
depicted in panel C is a mutant PKR, which lacks the first
amino-terminal 97 amino acids and RNA binding domain 1. As described in
the text, this mutant was cloned and purified as a histidine fusion
protein.
To determine whether the cellular P58 PKR inhibitor
functioned by directly complexing with the PKR protein kinase, the
following series of experiments was performed. Initially, purified, in vitro activated, P-labeled native PKR was
mixed with increasing amounts of crude lysates prepared from E.
coli overexpressing either the recombinant fusion protein,
GST-P58, or, as a control, GST alone. After incubation at 30 °C in
the presence of PBS, 1% Triton X-100, GST-P58 and GST control proteins
in the lysates were selected on glutathione-agarose beads as described
under ``Materials and Methods,'' and subjected to gel
electrophoresis. Coselected proteins were then visualized either by
autoradiography or by Coomassie Brilliant Blue staining of the
nitrocellulose filter obtained after blotting the SDS-polyacrylamide
gel. Staining of the filter demonstrated that the predominant bound
proteins, as expected, were either GST-P58 or GST (Fig. 2A). Most significantly, autoradiography of the
identical nitrocellulose filter revealed that radiolabeled PKR was
coselected on glutathione-agarose beads containing GST-P58 but not GST,
despite the large molar excess of the latter. The binding of PKR to
GST-P58 increased with increasing concentrations of lysates (Fig. 2B, lanes 2-4). PhosphorImager
quantitation revealed that up to 9-15-fold more PKR was selected
on beads containing GST-P58 compared with beads containing GST alone.
Furthermore, no PKR binding to the glutathione-agarose beads alone was
observed (Fig. 2B, lane 1). Approximately 13% of
the input radiolabeled PKR was estimated to bind to GST-P58 at the
highest levels of extracts tested.
Figure 2:
GST-P58 complexes with P-labeled PKR. GST-P58 and GST expression was induced in E. coli, and cell lysates were prepared as described under
``Materials and Methods.'' Increasing amounts of GST-P58 and
GST containing crude lysates were mixed with in vitro activated,
P-labeled PKR (2 pmol) for 15 min at 30
°C. The samples were diluted in PBS, 1.0% Triton X-100 and
subjected to selection on gluathione-agarose beads. Samples were
washed, resuspended in SDS disruption buffer, and electrophoresed by
SDS-polyacrylamide gel electrophoresis. Gels were then blotted onto
nitrocellulose filters. Panel A, Coomassie Brilliant Blue
staining of nitrocellulose filter after blotting of polyacrylamide gels
containing glutathione-agarose selected proteins. Amounts of crude
bacterial lysates subjected to selection are shown at the top of the panel. Migration of GST-P58 and GST proteins are indicated
by arrows on the right and molecular weight markers
are shown on the left. Panel B, nitrocellulose filter
described in panel A was subjected to autoradiography to
detect binding of radiolabeled PKR to either GST-P58, or GST. Lanes
1-8 are therefore identical to those in panel A. Arrow shows position of PKR. An aliquot of the
P-labeled PKR added to the binding reaction is shown in lane 9. Phosphorimager quantitation revealed that at the
highest level of extracts added approximately 13% of input radiolabeled
PKR bound to GST-P58. Quantitation also revealed that PKR bound to
GST-P58 9-15-fold more efficiently than PKR bound to GST alone
over the range of concentrations tested.
To provide additional evidence
for this PKR-P58 association, a reciprocal type experiment was
performed with [S]methionine-labeled GST-P58 and
unlabeled heparin-activated native PKR. Following the addition of PKR
to a preparation of purified radiolabeled GST-P58 (or radiolabeled GST
alone as control) and incubation in the presence of PBS, 1% Triton
X-100, the mixture was immunoprecipitated using a polyclonal antiserum
specific for PKR. There was a molar excess of both GST-P58 and GST
added to the binding reaction: at the highest concentrations (lanes
3 and 6) 12 pmol of GST-P58 and 40 pmol of GST were mixed
with 2 pmol of PKR as described in detail in the figure legend.
Increasing amounts of [
S]methionine-labeled
GST-P58 (approximately 10% of input) were coprecipitated with PKR,
whereas no detectable radiolabeled GST was coprecipitated with PKR (Fig. 3, compare lanes 1-3 with lanes
4-6). We also determined that the radiolabeled GST-P58
itself was not recognized by the PKR polyclonal antibody (data not
shown). Taken together with the previous experiments, these results
show that PKR interacts with P58 and that this interaction is specific
and not dependent on the GST tag.
Figure 3:
[S]Methionine-labeled
GST-P58 complexes with PKR. Increasing amounts of either
[
S]methionine-labeled purified GST-P58 (lanes 1-3, representing 1.0, 2.0, and 12.0 pmol,
respectively) or GST (lanes 4-6, representing 4.0, 8.0,
and 40.0 pmol, respectively) were mixed with unlabeled,
heparin-activated PKR (2 pmol), and immunoprecipitated with polyclonal
PKR antiserum derived from baculovirus expressed PKR. An aliquot of
radiolabeled purified GST-P58 or GST is shown in lanes 7 and 8, respectively. The migration of GST-P58 and GST are shown by arrows on the right. Phosphorimager quantitation
revealed that approximately 10% of input radiolabeled GST-P58 bound to
PKR.
The next group of experiments
attempted to map the sites on the P58 protein, which were required for
PKR binding. We analyzed wild-type and variant histidine fusion P58
recombinant proteins that had been previously analyzed for their
ability to inhibit PKR autophosphorylation activity in
vitro(20) . Purified P58 histidine fusion proteins were
incubated with P-labeled native PKR at approximately a
15-20-fold molar excess followed by immunoprecipitation with
either P58 specific monoclonal antibody or an irrelevant monoclonal
antibody prepared against IgG1. Coprecipitation of radiolabeled PKR
with the P58-specific antibody would indicate that a specific
interaction between PKR and P58 occurred. Four mutant P58 recombinant
proteins were tested in these studies (Fig. 1). (i) SER241
contains a serine to alanine mutation at amino acid 241 within an ELS
tripeptide in the P58 protein. A similar ELS tripeptide, containing
serine 51, the residue phosphorylated by PKR, is found within the eIF-2
subunit. Although this sequence similarity suggested an important
role of this serine for P58 function, the variant nonetheless retained
full PKR inhibitory activity(20) . (ii) Mutant 8-1 lacked amino
acids 167-504 and was nonfunctional in our in vitro PKR
autophosphorylation assay(20) . (iii) Mutant 8-2 lacked amino
acids 278-504 but contained the region of similarity with the
eIF-2
subunit and retained kinase inhibitory
function(20) . (iv) Finally, mutant 9-1 lacked the
amino-terminal 167 amino acids and also retained PKR inhibitory
function(20) . We first verified that the specific P58
monoclonal antibody immunoprecipitated each of these recombinant
proteins. [
S]Methionine-labeled extracts were
prepared from E. coli overexpressing each of these four
mutants (Fig. 4A). The P58 specific monoclonal antibody,
2F8, efficiently immunoprecipitated SER241, 8-1, and 8-2 (Fig. 4A) but could not immunoprecipitate 9-1, which lacked
the amino terminus (data not shown). Mutant 9-1 was efficiently
immunoprecipitated, however, with monoclonal antibody 3D7, which we
previously determined recognized the histidine-thrombin tag present at
the very amino terminus of the fusion protein (Fig. 4A). Previously the functional activity of these
variants were assayed by the inhibition of PKR
autophosphorylation(20) . Before performing the binding
experiments, we tested the ability of the P58 variants to inhibit PKR
activity as measured by the phosphorylation of exogenously added
histones (Fig. 4B). Both P58, 8-1, and SER241 inhibited
PKR function and reduced histone phosphorylation (40-50%), while
8-1 failed to significantly inhibit PKR activity (<10%) compared
with the control. Variant 9-1 also inhibited PKR-mediated histone
phosphorylation (data not shown).
Figure 4:
Binding
of wild-type and mutant histidine fusion P58 proteins to P-radiolabeled PKR and analysis of P58 function. Panel
A, recombinant proteins were labeled in the presence of
[
S]methionine as described under
``Materials and Methods.'' Mutants SER241, 8-1, and 8-2 were
immunoprecipitated by 2F8 mAb, while 9-1 was immunoprecipitated by 3D7 (lanes A). As control, all extracts were immunoprecipitated
with an irrelevant IgG1 mAb (lanes B). A profile of total
cellular proteins derived from extracts containing SER241, 8-1, 8-2,
and 9-1 proteins is shown on the right of panel A. Panel B, approximately equimolar amounts of the P58 wild-type
and variants were tested for their ability to inhibit PKR-mediated
histone phosphorylation as described under ``Materials and
Methods.'' As a negative control, we tested the PKR inhibitory
activity of material which bound to and eluted from a nickel column
exposed to extracts from E. coli, which expressed the
histidine fusion vector alone (CON). The histone bands were
subjected to phosphorimager analysis for quantitation. Relative to the
controls, P58 wild-type inhibited 51%, SER241 inhibited 40%, 8-2
inhibited 41%, and 8-1 inhibited approximately 8%. Panel C,
radiolabeled PKR (2 pmol) was reacted with purified histidine fusion
proteins and coprecipitated with 2F8 mAb for SER241 (20 pmol added),
8-1 (55 pmol), 8-2 (33 pmol), and 3D7 mAb for mutant 9-1 (30 pmol) (lanes A), or as a control each was coprecipitated with the
IgG1 mAb (lanes B). As an additional control (CON)
coprecipitations with 2F8 (A) or IgG1 (B) mAbs were
performed on a mixture containing radiolabeled PKR and eluate from a
nickel column to which was added extracts from E. coli expressing the vector alone. PhosphorImager quantitation revealed
that approximately 7% of input radiolabeled PKR bound to SER241 and 8-2
and approximately 4% to 9-1. Quantitation showed that compared with the
control, 8-fold more PKR bound to SER241, 5-fold more PKR bound to 8-2,
4-fold more PKR bound to 9-1, and 0.25-fold more PKR bound to the
negative 8-1 mutant.
The binding or coprecipitation experiments were then executed using 3D7 mAb for 9-1, and 2F8 mAb for the other three constructs (and the irrelevant IgG1 antiserum as a negative control). As an additional negative control, we tested the binding of PKR to material, which bound to and eluted from a nickel column exposed to extracts from E. coli, which expressed the histidine fusion vector alone (Fig. 4C, CON). For binding experiments using the histidine fusion proteins, we determined that, although binding did occur in the presence of PBS/Triton, the specificity of binding was greatly enhanced utilizing a binding buffer containing 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 0.5% SDS. The binding of mutants 8-1, 8-2, and 9-1 was compared with SER241, which we earlier demonstrated bound to PKR with equal efficiency to the wild-type PKR (data not shown). Radiolabeled PKR bound to both 8-2 and 9-1 but failed to specifically interact with mutant 8-1 (Fig. 4C). We estimate that approximately 7% of input PKR bound to SER241 and 8-2, whereas 4% bound to mutant 9-1. PKR failed to react with control extracts eluted from the nickel column and, furthermore, was not coprecipitated with the irrelevant monoclonal antibody. In addition, we determined that radiolabeled PKR alone failed to react with the P58-specific mAb (data not shown). It is unclear whether the slightly reduced binding to 9-1 relative to the other mutants is due to the difference in monoclonal antibody used or a bona fide difference in the affinity between 9-1 and PKR. It is clear, however, that the binding of the P58 variants correlated well with their ability to inhibit PKR in vitro since SER241, 8-1, and 9-1 are functional while 8-1 is unable to inhibit PKR in vitro.
The final series of experiments were intended to gain
additional knowledge of the molecular mechanisms by which P58 inhibits
PKR. We were particularly interested in determining whether P58 bound
to the dsRNA binding sites on PKR. Other PKR inhibitors that bind PKR,
such as the adenovirus VAI RNA(24) , function by competitively
inhibiting dsRNA binding to the kinase. We first examined whether P58
bound a PKR variant, which lacked the RBD-1 and which therefore failed
to bind detectable levels of dsRNA. Construction and characterization
of this histidine fusion PKR mutant has been extensively described
elsewhere (13, 32) . Despite the absence of RBD-1, the
PKR mutant can be efficiently autophosphorylated and radiolabeled in vitro in the presence of the polyanion
heparin(32) . Recombinant wild-type P58 (also expressed as a
histidine fusion protein; 40 pmol) was incubated with two
concentrations of P-labeled RBD-1 minus PKR (20 pmol, Fig. 5A, lanes 2 and 5; 40 pmol, Fig. 5A, lanes 3 and 6) followed by
immunoprecipitation either by the P58-specific monoclonal antibody,
2F8, or, as a control, normal mouse serum. The RBD-1 minus PKR was only
coprecipitated with P58 when the specific antibody was utilized (Fig. 5A, lanes 2 and 3).
Furthermore, coprecipitation of PKR depended on the presence of the
recombinant P58, demonstrating that there was no cross-reactivity
between PKR and the P58-specific antibody. Details of quantitation are
provided in the legend to Fig. 5.
Figure 5:
P58 complexes with a PKR mutant lacking
RBD-1 but not a PKR mutant containing only the amino terminus. Panel A, approximately 20 pmol (lanes 2 and 5) or 40 pmol (lanes 3 and 6) of PKR lacking
RBD-1 was radiolabeled in vitro in the presence of heparin and
incubated with purified histidine-tagged P58 (40 pmol). The mixture was
then immunoprecipitated with either P58-specific 2F8 mAb (lanes
1-3) or normal mouse serum (NMS) (lanes
4-6). As an additional control, precipitations were
performed with 20 pmol of radiolabeled PKR in the absence of P58 (lanes 1 and 4). The migration of delta RBD-1 PKR is
shown on the right of the panel. Phosphorimager
quantitation revealed that 8% of radiolabeled input PKR bound when 20
pmol was tested and 20% input radiolabeled PKR bound when 40 pmol was
tested. Quantitation also revealed that 12-18-fold more PKR bound
when the specific 2F8 antibody was utilized to coprecipitate PKR and
P58 compared with normal mouse serum. Panel B, left side
of panel, aliquots of [S]methionine-labeled in vitro translated full-length PKR (lane 1) and the
PKR mutant containing amino acids 1-242 (lane 2) were
analyzed by SDS-polyacrylamide gel electrophoresis. In vitro translated full-length PKR products were prepared from in
vitro transcribed RNA as described previously(33) . Right side of panel, increasing amounts of GST-P58 (20 and 50
µl, lanes 5 and 6 and lanes 9 and 10) or GST (20 and 50 µl, lanes 3 and 4 and lanes 7 and 8) containing lysates were mixed
with the in vitro translated full-length PKR (lanes
3-6) or mutant PKR (lanes 7-10) proteins.
Following selection on glutathione-agarose beads, samples were washed,
resuspended in SDS disruption buffer, and electrophoresed by
SDS-polyacrylamide gel electrophoresis (lanes
3-10).
The last experiment was
designed to directly test whether P58 bound a PKR mutant containing
only the amino half and therefore both dsRNA binding domains but no
catalytic domains(33) . Since such a PKR mutant was
catalytically inactive, it was necessary to develop another binding
assay and prepare in vitro translated wild-type and mutant PKR
radiolabeled with [S]methionine. Both
full-length PKR (as control) and PKR containing amino acids 1-242 (Fig. 5B, lanes 1 and 2,
respectively) were prepared as described in detail
previously(33) . Both the in vitro translated
full-length and mutant PKR proteins were previously found to
efficiently bind dsRNA(33) . The in vitro translated
proteins were then mixed with crude lysates prepared from E. coli overexpressing either the recombinant fusion protein, GST-P58, or,
as a control, GST alone. After incubation, GST-P58 and GST control
proteins were selected on glutathione beads and subjected to gel
electrophoresis. Coselected proteins were visualized by autoradiography (Fig. 5B, lanes 3-10). Radiolabeled
full-length PKR was coselected on agarose beads containing GST-P58 (lanes 5 and 6) but not GST (lanes 3 and 4). In contrast, the in vitro translated PKR mutant,
containing amino acids 1-242, was not coselected with GST-P58 and
thus did not bind P58 (lanes 9 and 10). These data,
taken together, suggest that PKR binding site(s) for P58 and dsRNA are
likely distinct and that P58 inhibits PKR by mechanisms independent of
dsRNA-mediated activation.
The current work was undertaken to
determine the molecular mechanisms underlying P58 action. Utilizing two
independent recombinant GST and histidine P58 fusion proteins,
glutathione-agarose coselection, and a variety of both monoclonal and
polyclonal antibodies, we determined that P58 forms a complex with PKR in vitro. The interaction between P58 and PKR is likely direct
and does not involve other cellular proteins, since complexes were
obtained utilizing highly purified proteins. Furthermore, this is
likely a high affinity interaction since complex formation occurred in
crude extracts and in the presence of 0.5% SDS and sodium deoxycholate.
Unlike the adenovirus-encoded PKR inhibitor, VAI RNA, P58 does not
function by binding to the dsRNA binding sites on PKR and can thus bind
to PKR variants, which lack the crucial first RNA binding domain but
not to a mutant containing only the RNA binding domains. It is
therefore likely that P58 binds to regions within the catalytic domains
of PKR. P58 is a member of the TPR family of proteins, characterized by
the presence of internal 34-amino acid repeats, motifs, which are
thought to form helix turn structures, each with a knob and hole,
acting as helix-associating domains. TPR proteins are reported to have
diverse functions; many play a role in mitosis and cell cycle
regulation, while others function in transcription, RNA splicing, and
protein import(36, 37) . It has been postulated that
TPR motifs may form amphipathic helices that could direct
protein-protein interactions, such as occurs between P58 and PKR. We
and others have found that not all TPR motifs are essential for
function (20, 38) nor as herein reported are they all
essential for P58 binding to PKR. For example, mutant 8-2, lacking TPR
motifs 8 and 9, and mutant 9-1, entirely lacking the first 3 motifs,
were still able to interact with PKR. The carboxyl terminus of P58,
containing the similarity to the DnaJ conserved J region is dispensable
both for P58 function and PKR binding in vitro, while the
central region of P58, containing the limited similarity to eIF-2
, appears to be essential for both. Although the sites of PKR that
interact with eIF-2
are currently unknown, it remains possible
that P58, based on its homology to the PKR substrate, interacts with a
similar site on PKR. However it should be stressed that P58 blocks the
autophosphorylation of PKR as well as eIF-2
phosphorylation,
indicating that P58 may need to interact with PKR at multiple sites to
regulate enzyme function.