From the Department of Microbiology, School of
Medicine, University of Washington, Seattle, Washington 98195,
Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University, Evanston, Illinois 60208, and
Regional Primate Research Center,
University of Washington, Seattle, Washington 98195
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ABSTRACT |
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P58IPK, a member of the
tetratricopeptide repeat and J-domain protein families, was first
recognized for its ability to inhibit the double-stranded RNA-activated
protein kinase, PKR. PKR is part of the interferon-induced host defense
against viral infection, and down-regulates translation initiation via
phosphorylation of eukaryotic initiation factor 2 on the Viral infection and the stress response have been linked because
DnaJ and DnaK were first characterized as accessory factors for The group of proteins known as molecular chaperones work together to
promote nascent chain folding of proteins during translation, transport, and secretion (reviewed in Ref. 8). Originally characterized as heat shock proteins, they constitute a critical part of the general
cellular response to stress (9, 10). The bacterial DnaJ and DnaK
protein families and their eukaryotic counterparts, hsp40 and
hsp/Hsc70, protect cells by refolding proteins that have been denatured
as a result of stress (11). However, recent studies have extended the
role of molecular chaperones beyond nascent peptide chain folding and
protection from cellular stress. For example, chaperones are required
for assembly of steroid receptor complexes (12, 13), and are involved
in apoptosis (14) and kinase maturation (15). In many cases, molecular
chaperones are assisted by a number of proteins, referred to as
co-chaperones, which interact in a specific manner with the chaperones
to modify their activities (16).
Our current study suggests that the cellular protein P58IPK
may also be a co-chaperone. P58IPK was originally
characterized as an influenza virus-activated protein that inhibits the
protein kinase PKR (17, 18). The inhibition of PKR by
P58IPK requires direct protein-protein interaction, which
is mediated by one of P58IPK's nine tandemly arranged
1TPR domains (19, 20). P58IPK also contains a
J-domain homology region to DnaJ at its C terminus. Importantly, the
J-domain is required for the in vivo inhibition of PKR (20).
PKR is a serine/threonine kinase that is activated upon binding
double-stranded RNA (21). The kinase has roles in signal transduction
(22, 23), cell growth and differentiation (24, 25), tumor suppression
(26-28), and apoptosis (29, 30). The best understood role of PKR,
however, is its ability to down-regulate mRNA translation via
phosphorylation of the In this report, we further define the molecular details and regulation
of the hsp40-P58IPK interaction, and for the first time
describe a role for hsp/Hsc70 in this increasing complex regulatory
pathway. By establishing a role for these molecular chaperones in the
regulation of the kinase, we provide a mechanism by which cellular
stress can regulate the PKR pathway. Moreover, these findings highlight
the molecular interplay between the pathways that regulate the cellular
stress response, viral defense, and growth control.
Cells and Bacterial Strains--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, 100 units/ml
penicillin G, 100 µg/ml streptomycin sulfate in monolayer cultures at
37 °C in a CO2 incubator. Cell lysates were prepared from 150-cm2 flasks in 400 µl Buffer A (50 mM
HEPES, 50 mM KCl, 2 mM MgCl2, 5%
Triton X-100, 1 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). The WSN strain of
influenza virus was grown and titered as described previously (40).
Interferon-treated extracts were prepared as described (41). For heat
shock experiments, the medium for HeLa cells was replaced with fresh
medium at 42 °C. The cultures were incubated at 42 °C for 30 min
and then returned to 37 °C. Plasmids expressing glutathione
S-transferase (GST)-P58IPK or GST alone were
carried in the Escherichia coli strain BL-21 as described
(19).
Purified Proteins and Antibodies--
GST-tagged
P58IPK and GST alone were prepared as described earlier
(19). Human hsp40 was expressed as full-length protein in E. coli and purified as described (42). The stress-inducible form of
hsp70 (hsp72) was also expressed in E. coli, and purified as
described (43). The ATPase domain of the constitutive form of hsp70
(Hsc70), referred to as 1-386, was purified as described (43).
Polyclonal antiserum to hsp40 was kindly provided by W. Welch
(University of California, San Francisco, CA). The anti-hsp70 mouse
monoclonal antibody, N27F3-4, was purchased from StressGen (Canada),
and used to detect full-length hsp70 (recognizes both hsp70 and Hsc70).
The anti-hsp70 mouse monoclonal antibody, 5A5, was purchased from
Affinity BioReagents (Golden, CO), and used to detect the 1-386 protein
fragment. Mouse monoclonal antisera to the Gal4 activation domain and
Gal4 binding domain were purchased from CLONTECH
(Palo Alto, CA). Mouse monoclonal antiserum to actin was purchased from
ICN (Costa Mesa, CA).
GST-pulldown Assay--
We used two different GST-pulldown
assays for these experiments. The first was used to co-purify hsp40 and
hsp70 from whole cell extract with GST-P58IPK or GST as
binding substrate. For these experiments, 100 µl of E. coli extract prepared from cells expressing GST-P58IPK
or GST alone was incubated with 50 µl of glutathione agarose beads
for 1 h at 4 °C. The beads were washed twice in
phosphate-buffered saline containing 1% Triton X-100, 2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and once in Buffer
A (25 mM HEPES, pH 7.5, 5 mM MgCl2,
50 mM KCl, 10 mM dithiothreitol). 100-250 µg
of HeLa cell extract prepared from untreated, influenza virus-infected, or heat-stressed cells was added. The reactions were incubated at room
temperature for 1 h with agitation. Beads were washed twice in
Buffer A and 1 mM ATP. Elution of bound proteins was achieved by addition of 30 µl of 50 mM Tris/HCl, pH 8.8, and 50 mM free glutathione. Eluted proteins were separated
by 10% SDS-PAGE and transferred to a nitrocellulose membrane,
according to the procedure of Towbin et al. (44). The filter
was blocked in 5% nonfat dry milk, and hsp40 and hsp70 were detected
by Western blot analysis as described earlier (38). Autoradiograms of
the Western analyses were scanned using an Epson scanner, and the signal was quantified using ImageQuant software.
The second variation of the GST-pulldown used purified proteins, rather
than cell extracts, as a source for hsp40, hsp70, and 1-386. Reactions
were carried out in 20 µl of Buffer A with proteins at the following
concentrations: 2.5 µM GST or GST-P58IPK,
1.75 µM hsp40, and 0.75 µM hsp70 or 1-386. Some reactions also contained ATP at a concentration of 1 mM. Reactions were incubated for 15 min at 30 °C.
Glutathione-agarose beads were added (20 µl), and reactions were
incubated for 30 min at 4 °C. Beads were washed three times in
Buffer A ± 1 mM ATP, resuspended in 20 µl elution
buffer, and incubated for 10 min at room temperature. Eluted proteins
were separated and analyzed as above.
Yeast Two-hybrid Plus Assay--
The hsp40 yeast expression
plasmid was generated by inserting the whole hsp40 coding sequence from
an EcoRI digest of hsp40 in pBluescript (Stratagene) into
the EcoRI site of pYX222 (Novagen). The ATPase domain of
Hsc70 was expressed as a Gal4 binding domain fusion protein in the
pGBT10 vector (BD-1-386) (45). P58IPK was expressed as a
Gal4 activation domain fusion protein as described previously (46). To
create pYX-PP1C, an EcoRI -SalI
fragment containing the whole PP1C coding sequence was
released from pYES2-PP12 and cloned
into the corresponding sites of pYX222 (Novagen). hSRP1 was
expressed as a Gal4 activation domain fusion in the pGAD424 vector
(47). The two-hybrid plasmids pGBT9 and pGAD424 (CLONTECH) were used for Gal4 DNA binding domain
(BD) and Gal4 transcriptional activation domain (AD) fusions,
respectively. pGBT9 and pGAD424 contain the selectable auxotrophic
markers, TRP1 and LEU2, respectively. BD-P53 was
purchased from CLONTECH Laboratories.
To develop a yeast two-hybrid plus assay, we modified the yeast
two-hybrid system by introducing a third protein into the system using
the plasmid pYX222. pYX222 contains an HIS3+
selectable marker and is compatible with the two-hybrid plasmids, pGBT10 and pGAD424. Yeast strains SFY526 (CLONTECH)
and Y187 (obtained from Dr. S. J. Elledge, Baylor College of
Medicine) were used as hosts. MATa strain SFY526 (ura3-52,
his3-200, ade2-101, lys2-801, trp1-901, leu2-3,-112,
canr, gal4-542, gal80-538,
+URA3::GAL1 ATPase Assay--
ATPase assays were performed as described
earlier (43). GST-P58IPK and GST were prepared as above,
except they were dialyzed against 25 mM Tris/HCl, pH 7.5 before use. 1-386 was used at a final concentration of 0.45 µM, hsp40 was used at 0.9 µM, and GST and
GST-P58IPK were used at 3.2 µM.
P58IPK Is Activated in Influenza Virus-infected Cells
by Dissociation from hsp40--
Previously, we found that
P58IPK was present in an inactive form in uninfected cells,
but was activated upon infection with influenza virus (17, 18). We
recently identified the molecular chaperone, hsp40, as a negative
regulator of P58IPK, and found that these proteins stably
interacted in rabbit reticulocyte lysate (38). We hypothesized that
influenza virus activates P58IPK by dissociating it from
hsp40, allowing P58IPK to inhibit the antiviral activity of
PKR. We therefore evaluated the ability of P58IPK to
bind to hsp40 in cell extracts prepared from influenza virus-infected or mock-infected HeLa cells. Using a GST-pulldown approach, we analyzed
the binding of recombinant GST-tagged P58IPK to endogenous hsp40.
HeLa cells were mock infected, or infected with influenza virus, and
cell extracts were prepared at various time points postinfection. Glutathione-agarose beads, precoated with GST-P58IPK, were
then added to each extract. The beads were washed, and bound proteins
were eluted with free glutathione and subjected to SDS-PAGE. hsp40 was
detected by Western blotting, and the signal was quantified. We found
that hsp40 present in untreated or mock-infected cell extract
co-purified with GST-P58IPK (Fig.
1). In a parallel experiment using
agarose beads precoated with GST alone, hsp40 was not detected (data
not shown). In contrast, when GST-P58IPK was reacted with
influenza virus-infected extracts there was a transient increase in the
amount of hsp40 binding to P58IPK, followed by a dramatic
reduction 5 h postinfection. Western blot analysis of the input
cell extract confirmed that the loss of hsp40 binding was not because
of a reduction in hsp40 protein level (Fig. 1). This finding correlates
with our earlier work, which showed that P58IPK was
activated in influenza virus-infected cells at 4-5 h postinfection (18). Furthermore, these results strongly support our hypothesis that
P58IPK is activated by influenza virus infection through
the dissociation of hsp40.
Heat Shock Mimics Influenza Virus Infection:
P58IPK-hsp40 Binding Is Disrupted--
Because there is
evidence for an association between viral infection and the stress
response, we next examined the effects of heat shock on the ability of
P58IPK to bind to hsp40. We used the GST-pulldown approach
to examine the ability of P58IPK to bind to hsp40 in
extracts prepared from cells subjected to heat shock. Extracts were
prepared from HeLa cells that were subjected to a mild heat shock (30 min at 42 °C) and allowed to recover at 37 °C for 0-5 h, or from
untreated cells as a control. GST-pulldowns and quantification of bound
hsp40 were carried out as described above. We found that the amount of
hsp40 bound to P58IPK remained unchanged until 5 h
into the recovery period (Fig. 2). At
5 h, there was little hsp40 bound to GST-P58IPK.
Again, we confirmed that the changes in hsp40-P58IPK
binding were not because of a loss of hsp40 present in the extract (Fig. 2). These results support the idea that the
PKR-P58IPK pathway is regulated similarly in heat-stressed
cells and cells infected with influenza virus. Furthermore, these
experiments provide evidence that the stress-regulated functions of PKR
may be controlled in part by P58IPK.
P58IPK Forms a Stable Complex with hsp40 and the ATPase
Domain of Hsc70--
The experiments described above demonstrated that
P58IPK and hsp40 bind, but did not rule out the possibility
that other factors were mediating the association. The simplest
approach to answer this question was to examine purified hsp40 and
purified GST-P58IPK, in the absence of other proteins, for
the ability to interact. Using the GST-pulldown approach, we found that
hsp40 was retained on beads with GST-P58IPK, but not GST or
buffer alone (Fig. 3). This evidence
strongly suggested that hsp40 and P58IPK are able to
interact directly. P58IPK shares structural similarities
with hsp70-interactive proteins, such as the co-chaperones Hip, Hop,
and Cyp40, all of which contain TPR domains that are required for
stable interaction with hsp70 (reviewed in Ref. 48). We reasoned that
because of these similarities with hsp70-interactive proteins,
P58IPK might also interact with hsp70. We therefore
examined whether P58IPK was able to form a stable complex
with hsp70, in the absence or presence of hsp40. Because ATP is known
to affect the ability of hsp70 to stably interact with other proteins,
we also examined its effect in this assay.
GST-P58IPK was incubated with purified recombinant hsp70
(the inducible form) and hsp40, separately or in combination, and the proteins were purified and examined. Western blot analysis demonstrated that in the absence of ATP, there was a weak interaction between hsp70
and P58IPK, which was neither enhanced nor diminished with
the addition of hsp40 (Fig.
4A). In the presence of both
hsp40 and ATP, however, significantly more hsp70 was bound by
GST-P58IPK. In addition, hsp40 also co-purified in the
complex. Control experiments confirmed that hsp40 and hsp70 were not
similarly purified on GST alone (data not shown). As an initial step to determine whether the P58IPK-hsp70 interaction was limited
to the C-terminal refolding domain of hsp70, we repeated the binding
experiments using the amino-terminal 44-kDa ATPase domain of Hsc70
(referred to as 1-386). We found that P58IPK also formed a
stable complex with 1-386 (Fig. 4B) and that this interaction was enhanced by ATP and dependent upon hsp40. The ability
of ATP to stabilize the complex between 1-386, hsp40, and
GST-P58IPK is analogous to the TPR protein Hip, where ATP
also enhanced Hip-hsp40-1-386 binding (49). Similarly, ATP stimulated
binding between auxilin and Hsc70 (50).
As an independent confirmation of our in vitro binding
results, we examined the formation of this trimeric complex in an
in vivo environment using the yeast two-hybrid plus system.
This is a method to analyze the interaction of three proteins in
vivo and quantitatively compares different binding conditions. We
expressed P58IPK as a fusion protein with the GAL4
activation domain (AD-P58IPK) and hsp40 as a full-length
protein on a different plasmid (pYX-hsp40). The yeast strain SFY526 was
co-transformed with both plasmids, and grown on selective medium. The
ATPase domain of Hsc70 was expressed as a GAL4 binding domain fusion
(BD-1-386) in the yeast strain Y187. The yeast were mated, and progeny
were grown on selective medium for maintenance of the three plasmids.
These yeast contain a P58IPK Stimulates the ATPase Activity of
hsp70--
Our results, which show that P58IPK interacted
with the ATPase domain of Hsc70, suggested that the interaction might
have a functional consequence. As is evident with most J-domain
proteins that interact with the ATPase domain of Hsc70 to regulate
ATPase activity. This has been demonstrated for hsp40, Hdj-2, Hsj-1,
auxilin, and most other J-domain proteins (reviewed in Ref. 51). We
thus hypothesized that P58IPK could also stimulate the
ATPase activity of Hsc70. For these experiments, we used the ATPase
fragment of Hsc70 alone (1-386), which retains ATPase activity even in
the absence of the C-terminal refolding domain (43). P58IPK
stimulated this activity in a dose-dependent manner, and to
the same degree as observed for hsp40 (Fig.
6). Furthermore, when combined in the
same reaction, hsp40 and P58IPK displayed an additive
effect, which suggested that the proteins do not work synergistically.
Control experiments using GST alone confirmed that the stimulation was
not because of contaminating E. coli proteins present in the
purified preparation (data not shown). As a control, we verified that
the GST-P58IPK preparation contained no significant
endogenous ATPase activity (data not shown). These experiments
demonstrate that P58IPK, like other J-domain proteins, is
able to specifically interact with and stimulate the ATPase activity of
Hsc70.
P58IPK Is a Co-chaperone Protein--
Co-chaperones
are defined on the basis of their ability to interact with molecular
chaperones and to modify their activity or provide specificity. Thus,
the ability of P58IPK to bind to Hsc70 and stimulate the
ATPase activity indicates that P58IPK is a novel member of
the co-chaperones. Although we have not yet determined whether
P58IPK's ability to regulate Hsc70 directly plays a role
in the inhibition of PKR, recent studies of two other proteins provide
examples of J-domain co-chaperones that recruit Hsc70 to specific
protein targets. First is the clathrin-uncoating factor, auxilin, which contains a C-terminal J-domain homology region. The J-domain is required for the clathrin-uncoating activity of auxilin, as well as
interaction with Hsc70 (50, 52). The current hypothesis is that auxilin
targets Hsc70 to specific sites on the clathrin basket and stimulates
the chaperone to refold the clathrin molecule, and thus uncoat the
vesicle (53). Indeed, deletion of the J-domain of auxilin abrogates
clathrin uncoating (54). Furthermore, the clathrin-uncoating activity
can be blocked by other J-domain proteins, such as yeast Ydj-1 and
human Hsj-1 (52, 55). Presumably, this is because these general
chaperones do not contain a clathrin-binding domain, and thus compete
with auxilin for Hsc70.
The second co-chaperone is the tumor antigen (T antigen) of the
polyomavirus, simian virus 40, which has a J-domain at its NH2 terminus. Work by Kelley and Georgopoulos (56) showed
that the T antigen J-domain could functionally substitute for the
J-domain in DnaJ of E. coli. More recently, a
reciprocal experiment found that the J-domain from Hsj-1 could
functionally substitute for the T antigen J-domain (57). The T antigen
also contains a binding site for the retinoblastoma tumor suppressor
protein (pRb), which the T antigen may inactivate through direct
protein-protein interaction. However, recent reports demonstrate that
the J-domain of the T antigen is critical for the inactivation of pRb
and malignant transformation (58). Srinivasan et al. (59)
proposed that the T antigen targets Hsc70 to pRb and stimulates the
chaperone to alter the conformation of pRb. This tenet is supported by
a report showing that a functional J-domain in the T antigen is critical for the accumulation of free E2F and activation of E2F promoter sequences (58). Thus, the T antigen and auxilin systems highlight the potential of J-domain co-chaperone proteins to target specific proteins for refolding by hsp70.
Model for P58IPK-PKR Pathway--
We showed in this
study that hsp40 and P58IPK associate in extracts prepared
from HeLa cells and that binding was disrupted by infection with
influenza virus (Fig. 1). We are not certain of the significance of the
increased hsp40 binding to GST-P58IPK immediately upon
influenza virus infection. Perhaps hsp40 is released from other
regulatory pathways, thus increasing the pool of hsp40 available to
bind to GST-P58IPK. The interaction between hsp40 and
P58IPK also was abrogated during recovery from heat shock,
suggesting P58IPK may have roles in the general stress
response (Fig. 2). We observed no increase in hsp40 binding to
P58IPK immediately following heat shock compared with
influenza virus-infected cells. However, the same conclusion could be
drawn: hsp40 was released from other regulatory pathways, but in
heat-shocked cells hsp40 was recruited by hsp70 to bind to denatured
proteins. The interaction between P58IPK and hsp40 was
direct, requiring no other binding partners (Fig. 3).
P58IPK binding to hsp70, however, required both ATP and
hsp40. Significantly, the interaction was specific to the ATPase domain
of Hsc70, indicating that the complex was not merely an intermediate
step in a refolding reaction (Fig. 4).
Therefore, we propose in our revised model that P58IPK is
inactive before heat shock or influenza virus infection because it is
bound to its own inhibitor, hsp40 (Fig.
7). In response to a stress event, such
as heat shock or virus infection, P58IPK then binds to
hsp/Hsc70. Our in vitro binding results indicate that hsp40
may be required for the association of hsp/Hsc70, and there might exist
a trimeric hsp40-P58IPK-hsp/Hsc70 complex (Fig. 4).
However, given our earlier results showing that hsp40 impairs the
ability of P58IPK to inhibit PKR, hsp40 likely dissociates
before P58IPK binds to PKR. The mechanism of the release of
P58IPK by hsp40 is unclear. It is possible that the
cellular stress response triggered by heat shock or influenza virus
infection recruits hsp40, along with the other stress response
proteins, to participate in the general protection of the cell, and
thus free sufficient P58IPK to down-regulate PKR.
Alternatively, the chaperone is modified in some way, so that it is no
longer able to bind to P58IPK. Regardless of the cause of
the release of P58IPK, the final step in the PKR pathway is
the inhibition of PKR. We speculate that P58IPK brings
hsp/Hsc70 to PKR, and stimulates the chaperone to refold, and thus
inactivate the kinase. This hypothesis is supported by the observation
that P58IPK stimulates the ATPase activity of Hsc70. The
inhibition of PKR ensures that translation will not be impaired in the
case of influenza virus infection, or that it will be restored in the
case of recovery from heat stress. We acknowledge the speculative
nature of the model and emphasize that we present it as merely a
hypothesis and a guideline for future studies.
Previously, we have shown that P58IPK inhibited PKR in a
stoichiometric manner by direct protein-protein interaction. Our
studies also showed that the 6th TPR domain was required for direct
interaction with PKR, and inhibition of the kinase. Furthermore, Tan
et al. (60) demonstrated that P58IPK binding to
PKR prevents dimerization of the kinase, which is a required step for
full catalytic activity. Interestingly, monomerization of the E. coli P1 replication initiator RepA requires a functional DnaK-DnaJ
system (61, 62), thus implicating another J-domain protein in the
disruption of protein multimers. Just as most J-domain proteins require
their hsp70/Hsc70 counterpart, the requirement of P58IPK
for its J-domain to inhibit PKR in vivo supports the notion
that hsp/Hsc70 may be involved in P58IPK activity. As
indicated in Fig. 7, we have identified a second negative regulator of
P58IPK, called P52rIPK, which also binds to and
inhibits P58IPK (39). This is particularly interesting in
the context of molecular chaperones, because P52rIPK
contains a region of homology to hsp90. The conditions under which
P52rIPK and P58IPK interact within the cell,
however, and the consequences of this interaction, remain to be determined.
These findings are significant for the study of P58IPK
because they demonstrate the likely mechanism by which influenza virus activates P58IPK, namely by disrupting the complex between
P58IPK and its negative regulator, hsp40. Furthermore, this
report is the first evidence of a role for P58IPK in the
absence of influenza virus infection, as evidenced by the liberation of
P58IPK during recovery from heat shock, and the
co-chaperone activity of P58IPK. We conclude that the cell
may regulate the ability of PKR to mediate antiviral activity, growth
suppression, signal transduction, and translational control by
balancing the activities of hsp40, hsp/Hsc70, P58IPK, and
possibly P52rIPK.
-subunit.
P58IPK is activated in response to infection by influenza
virus, and inhibits PKR through direct protein-protein interaction.
Previously, we demonstrated that the molecular chaperone heat shock
protein 40 (hsp40) was a negative regulator of P58IPK. We
could now report that influenza virus activates the P58IPK
pathway by promoting the dissociation of hsp40 from P58IPK
during infection. We also found that the P58IPK-hsp40
association was disrupted during recovery from heat shock, which
suggested a regulatory role for P58IPK in the absence of
virus infection. The PKR pathway is even more complex as we show in
this report that the molecular chaperone, hsp/Hsc70, was a component of
a trimeric complex with hsp40 and P58IPK. Moreover, like
other J-domain proteins, P58IPK stimulated the ATPase
activity of Hsc70. Taken together, our data suggest that
P58IPK is a co-chaperone, possibly directing hsp/Hsc70 to
refold, and thus inhibit kinase function.
INTRODUCTION
Top
Abstract
Introduction
References
phage replication in Escherichia coli (1). In many cases, viral infection elicits a cellular stress response, similar to heat
shock or chemical treatment. This has been demonstrated for vaccinia
virus (2) and Sindbis virus (3). Many viruses also use specific
molecular chaperones to aid in viral replication and assembly. For
example, hepatitis B virus recruits hsp90 for efficient replication (4,
5). In addition, vaccinia virus (2), adenovirus (6), and rabies virus
(7) enlist the aid of the molecular chaperone,
hsp1/Hsc70, during viral assembly.
-subunit of eukaryotic initiation factor 2 (31). This has been characterized most extensively as a host defense
against viral infection, but recent studies revealed that the
-subunit of eukaryotic initiation factor 2 of phosphorylation by PKR
may also be a general response to cellular stress (32). Indeed, PKR was
activated by heat stress (33, 34), as well as chemical stressors, such
as sodium arsenite (35) or calcium ionophore (36, 37). Work from our
laboratory also supported a connection between PKR and the stress
response, because we have found that P58IPK directly
interacted with hsp40, and was negatively regulated by the molecular
chaperone (38). Furthermore, we recently identified a novel
hsp90-related protein, P52rIPK, which is also a
negative regulator of P58IPK, suggesting that
P58IPK is probably regulated in response to different
stress signals (39). Therefore, we reasoned that P58IPK
activity might also be modulated by stress, perhaps to down-regulate PKR activity once the stress was relieved, and cells begin to recover.
MATERIALS AND METHODS
lacZ) was cotransformed with BD-1-386,
BD-P53 or pGBT9, and with AD-P58IPK, AD-hSRP1, or pGAD424.
MAT
strainY187 (ura3-52, his3, ade2-101, trp1-901, leu2-3,
-112, gal4, gal80, +URA3::GAL4
lacZ) was transformed with pYX-hsp40, pYX-PP1C, or pYX222. A mating procedure was
used to combine all three plasmids, and the diploids were streaked on
synthetic defined plates (Biolol, Vista, CA) lacking Trp, Leu, and His
for plasmid selection. The lacZ reporters integrated in the
genomes of both strains were used to assess protein-protein interactions based on a
-galactosidase assay as described by CLONTECH Laboratories. Yeast cell extracts were
prepared by a glass bead method as described previously (46).
RESULTS
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Fig. 1.
P58IPK binding to hsp40 disrupted
in influenza virus-infected cells. Glutathione-agarose beads (50 µl) precoated with GST-P58IPK were mixed with 300 µg of
HeLa cell extract from uninfected, mock-infected, and cells infected
with influenza virus for 1, 3, or 5 h. Bound proteins were
separated by SDS-PAGE as described under "Materials and Methods."
hsp40 was detected by Western blotting. The Western blot signal was
quantified using ImageQuant software, background binding was
subtracted, and the signals were expressed as arbitrary binding units.
At the bottom is a Western blot analysis showing the input level of
hsp40 in each extract.
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Fig. 2.
P58IPKhsp40 binding blocked
during recovery from heat shock. HeLa cells were subjected
to a mild heat stress as described under "Materials and Methods,"
and allowed to recover for the indicated times. GST-P58IPK
pulldowns were performed as shown in Fig. 1 and described under
"Materials and Methods," and the Western blot signal of hsp40 was
quantified using ImageQuant software.
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Fig. 3.
P58IPK binds to purified
hsp40. Purified hsp40 was mixed with buffer alone (lane
1), GST-P58IPK (lane 2), or GST (lane
3). Following a brief incubation, glutathione-agarose beads were
added, incubated, and then washed with binding buffer. Proteins were
eluted in free glutathione, separated by SDS-PAGE, and hsp40 was
detected by Western blot analysis.
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Fig. 4.
ATP and hsp40 required for P58IPK
binding to hsp70. Purified GST alone, GST-P58IPK, or
binding buffer was used as a binding substrate. In all panels, the
first three lanes were reactions without ATP, and the last three were
plus ATP. Panel A, full-length hsp70 was mixed
with GST or GST-P58IPK, with or without hsp40, and purified
on glutathione-agarose beads. hsp40 and hsp70 were detected by Western
blotting. Panel B is a similar experiment, using the 1-386 fragment of hsp70.
-galactosidase reporter gene under control of
the GAL4 promoter, and the strength of the hybrid protein interaction
is measured by
-galactosidase activity. Western blotting analysis of
each yeast transformant confirmed that each protein was expressed at similar levels (Fig. 5B). We
found that the P58IPK-1-386 interaction was stimulated
approximately 6-fold in the presence of hsp40 (compare lanes
1 and 4). Moreover, the interaction was specific to the
ATPase domain of Hsc70, which supports the results of our in
vitro binding experiments. It is interesting to note that the
amount of P58IPK-1-386 binding in the absence of hsp40 was
the same as background (compare lane 4 to lanes
2, 3, and 5). This suggests that the endogenous yeast DnaJ protein, Ydj-1 did not substitute for hsp40 in
this interaction, further corroborating the specificity of the
P58IPK-hsp40-1-386 interaction.
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Fig. 5.
P58IPK-Hsc70 interaction specific
to the ATPase domain of hsp70. Yeast expressing
AD-P58IPK with pYX-hsp40 or vector alone were mated with
yeast expressing BD-1-386. The resulting yeast were grown on
triple-selective medium, and the P58IPK-1-386 interaction
measured by -galactosidase assay. The chart in panel A
shows
-galactosidase units from yeast expressing:
AD-P58IPK, pYX-hsp40, and BD-1-386 (lane 1);
AD-P58IPK, pYX-hsp40, and BD-P53 (lane 2);
AD-hSRP1, pYX-hsp40, and BD-1-386 (lane 3);
AD-P58IPK, pYX-PPIc, and BD-1-386 (lane 4); and
pGBT9, pYX223, and pGAD424 (lane 5). Western blotting was
performed to confirm expression of the proteins (panel
B). The actin probe, shown at the bottom, confirms that each
lane contains approximately the same amount of protein.
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Fig. 6.
P58IPK stimulates ATPase activity
of hsp70. Purified GST-P58IPK was mixed with purified
44-kDa ATPase domain of Hsc70 (1-386), in the absence or presence of
hsp40. [ -32P]ATP was added as a substrate for 1-386 ATPase activity, and reactions were carried out for 1 h, as
described under "Materials and Methods." The amount of ADP was
calculated and normalized to the amount of 1-386 present in the assay.
(
), negative control (no P58IPK or chaperones); (
),
1-386 alone; (
), hsp40; (
), P58IPK + 1-386; (
),
P58IPK + hsp40 + 1-386.
DISCUSSION
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Fig. 7.
Model of chaperone-regulated
P58IPK-PKR pathway. See text for details.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Höhfeld (Heidelberg) for the gift of the BD-1-386 construct, Dr. T. Enomoto for pGAD424-hSRP1, and Dr. J. Potter for guidance with the ATPase assay. We also thank Dr. M. Korth for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI-22646, RR-00166, and AI-41629 (to M. G. K.).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 the Public Health Service, National Research Service Award T32 GM07270. Current address: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305.
¶ Supported by the Gustavus & Louise Pfeiffer Research Foundation.
** Supported by National Institutes of Health Grant GM38109.
§§ To whom correspondence should be addressed: Dept. of Microbiology, School of Medicine, Box 357242, University of Washington, Seattle, WA 98195. Tel.: 206-543-8837; Fax: 206-685-0305; E-mail: honey{at}u.washington.edu.
The abbreviations used are: hsp, heat shock protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; BD, binding domain; AD, activation domain; T antigen, tumor antigen; pRb, retinoblastoma tumor suppressor protein.
2 S.-L. Tan and M. G. Katze, unpublished results.
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REFERENCES |
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