The Cellular Inhibitor of the PKR Protein Kinase, P58IPK, Is an Influenza Virus-activated Co-chaperone That Modulates Heat Shock Protein 70 Activity*

Mark W. MelvilleDagger §, Seng-Lai TanDagger , Marlene WambachDagger , Jaewhan Songparallel **, Richard I. Morimotoparallel **, and Michael G. KatzeDagger Dagger Dagger §§

From the Dagger  Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, parallel  Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, and Dagger Dagger  Regional Primate Research Center, University of Washington, Seattle, Washington 98195

    ABSTRACT
Top
Abstract
Introduction
References

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 alpha -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

Viral infection and the stress response have been linked because DnaJ and DnaK were first characterized as accessory factors for lambda  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.

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 alpha -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 alpha -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.

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.

    MATERIALS AND METHODS

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::GAL1right-arrowlacZ) was cotransformed with BD-1-386, BD-P53 or pGBT9, and with AD-P58IPK, AD-hSRP1, or pGAD424. MATalpha strainY187 (ura3-52, his3, ade2-101, trp1-901, leu2-3, -112, gal4, gal80, +URA3::GAL4right-arrowlacZ) 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 beta -galactosidase assay as described by CLONTECH Laboratories. Yeast cell extracts were prepared by a glass bead method as described previously (46).

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.

    RESULTS

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.


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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.

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.


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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.

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.


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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.

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).


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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.

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 beta -galactosidase reporter gene under control of the GAL4 promoter, and the strength of the hybrid protein interaction is measured by beta -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 beta -galactosidase assay. The chart in panel A shows beta -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.

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.


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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. [alpha -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. (bullet ), negative control (no P58IPK or chaperones); (open circle ), 1-386 alone; (times ), hsp40; (black-square), P58IPK + 1-386; (), P58IPK + hsp40 + 1-386.


    DISCUSSION

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.


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Fig. 7.   Model of chaperone-regulated P58IPK-PKR pathway. See text for details.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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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Abstract
Introduction
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