Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding

X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.     

Adenosine nucleotides and the regulation of GRP94-client protein interactions

The molecular chaperone heat shock protein 90 (Hsp90) serves essential roles in the regulation of signaling protein function, trafficking, and turnover. Hsp90 function is intimately linked to intrinsic ATP binding and hydrolysis activities, the latter of which is under the regulatory control of accessory factors. Glucose-regulated protein of 94 kDa (GRP94), the endoplasmic reticulum Hsp90, is highly homologous to cytosolic Hsp90. However, neither accessory factors nor adenosine nucleotides have been clearly implicated in the regulation of GRP94-client protein interactions. In the current study, the structural and regulatory consequences of adenosine nucleotide binding to GRP94 were investigated. We report that apo-GRP94 undergoes a time- and temperature-dependent tertiary conformational change that exposes a site(s) of protein-protein interaction; ATP, ADP, and radicicol markedly suppress this conformational change. In concert with these findings, ATP and ADP act identically to suppress GRP94 homooligomerization, as well as both local and global conformational activity. To identify a role(s) for ATP or ADP in the regulation of GRP94-client protein interactions, immunoglobulin (Ig) heavy chain folding intermediates containing bound GRP94 and immunoglobulin binding protein (BiP) were isolated from myeloma cells, and the effects of adenosine nucleotides on chaperone-Ig heavy chain interactions were examined. Whereas ATP elicited efficient release of BiP from both wild-type and mutant Ig heavy chain intermediates, GRP94 remained in stable association with Ig heavy chains in the presence of ATP or ADP. On the basis of these data, we propose that structural maturation of the client protein substrate, rather than ATP binding or hydrolysis, serves as the primary signal for dissociation of GRP94-client protein complexes.     

Radicicol-sensitive peptide binding to the N-terminal portion of GRP94

GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on- and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.     

The ATPase cycle of the endoplasmic chaperone Grp94

Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a k(cat) of 0.36 min(-1) at 25 degrees C. Kinetic and equilibrium constants of the partial reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared with other Hsp90s investigated, with dissociation constants (K(D)) of approximately 4 microm for ADP, ATP, and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, 5'-N-ethylcarboxamidoadenosine) studied here bind with similar rate constants for association (0.2-0.3 x 10(6) M(-1) s(-1)). Furthermore, there is a marked difference from cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide-binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.     

Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.     

Ligand-induced conformational shift in the N-terminal domain of GRP94, an Hsp90 chaperone

GRP94 is the endoplasmic reticulum paralog of cytoplasmic Hsp90. Models of Hsp90 action posit an ATP-dependent conformational switch in the N-terminal ligand regulatory domain of the chaperone. However, crystal structures of the isolated N-domain of Hsp90 in complex with a variety of ligands have yet to demonstrate such a conformational change. We have determined the structure of the N-domain of GRP94 in complex with ATP, ADP, and AMP. Compared with the N-ethylcarboxamidoadenosine and radicicol-bound forms, these structures reveal a large conformational rearrangement in the protein. The nucleotide-bound form exposes new surfaces that interact to form a biochemically plausible dimer that is reminiscent of those seen in structures of MutL and DNA gyrase. Weak ATP binding and a conformational change in response to ligand identity are distinctive mechanistic features of GRP94 and suggest a model for how GRP94 functions in the absence of co-chaperones and ATP hydrolysis.     

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.     

Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basis for nucleotide-induced conformational change

GRP94, the endoplasmic reticulum paralog of Hsp90, is regulated by adenosine nucleotides that bind to its N-terminal regulatory domain. Because of its weak affinity for nucleotides, the functionally relevant transition in GRP94 is likely to be between the unliganded and nucleotide-bound states. We have determined the structure of the unliganded GRP94 N-domain. The helix 1-4-5 subdomain of the unliganded protein adopts the closed conformation seen in the structure of the protein in complex with inhibitors. This conformation is distinct from the open conformation of the subdomain seen when the protein is bound to ATP or ADP. ADP soaked into crystals of the unliganded protein reveals an intermediate conformation midway between the open and closed states and demonstrates that in GRP94 the conversion between the open and closed states is driven by ligand binding. The direction of the observed movement in GRP94 shows that nucleotides act to open the subdomain elements rather than close them, which is contrary to the motion proposed for Hsp90. These observations support a model where ATP binding dictates the conformation of the N-domain and regulates its ability to form quaternary structural interactions.     

Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones

GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 A crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a "twisted V" conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.     

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.     

Basolateral expression of GRP94 in parietal cells of gastric mucosa

GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na₂CO₃ treatment dissociates part of it from the membrane. However, 100 mM Na₂CO₃ treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-lig- and binding experiments. Both the K _D value obtained in saturation experiments with N-ethylcarboxamido-[³H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.     

The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain

The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652-678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met(658)-Met(662)) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.     

A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket.

In vivo function of the molecular chaperone Hsp90 is ATP-dependent and requires the full-length protein. Our earlier studies predicted a second C-terminal ATP-binding site in Hsp90. By applying direct biochemical approaches, we mapped two ATP-binding sites and unveiled the C-terminal ATP-binding site as the first example of a cryptic chaperone nucleotide-binding site, which is opened by occupancy of the N-terminal site. We identified an N-terminal gamma-phosphate-binding motif in the middle domain of Hsp90 similar to other GHKL family members. This motif is adjacent to the phosphate-binding region of the C-terminal ATP-binding site. Whereas novobiocin disrupts both C- and N-terminal nucleotide binding, we found a selective C-terminal nucleotide competitor, cisplatin, that strengthens the Hsp90-Hsp70 complex leaving the Hsp90-p23 complex intact. Cisplatin may provide a pharmacological tool to dissect C- and N-terminal nucleotide binding of Hsp90. A model is proposed on the interactions of the two nucleotide-binding domains and the charged region of Hsp90.     

GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.     

Identification of the N-terminal peptide binding site of glucose-regulated protein 94

Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the beta sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys117 within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His125, which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the beta sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone.     

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

The Hsp90 chaperone complex is both a facilitator and a repressor of the dsRNA-dependent kinase PKR.

PKR, a member of the eukaryotic initiation-factor 2alpha (eIF-2alpha) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co-chaperone p23 bind to PKR through its N-terminal double-stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF-2alpha. A short-term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf-MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates.