A comprehensive study on the immunological reactivity of the Hsp90 molecular chaperone

Periodontitis is a chronic infectious disease, Porphyromonas gingivalis being the most implicated pathogen. In the present study, we investigated the role of P. gingivalis HtpG (PgHtpG), a bacterial ortholog of mammalian Hsp90, in the growth of P. gingivalis and also assessed the immunological cross-reactivity of the members of the Hsp90 family. Antiserum against rat liver Hsp90 potently reacted with yeast Hsp90, called Hsc82, and also weakly with human Hsp90 (hHsp90) and human mitochondrial paralog Trap1, but did not react with PgHtpG, Escherichia coli HtpG, or human endoplasmic reticulum paralog Grp94. Moreover, among 19 monoclonal antibodies raised against hHsp90, nine cross-reacted with yeast Hsc82, and one with human Grp94, but none bound to PgHtpG or E. coli HtpG. Among them, three mAbs that strongly reacted with yeast Hsc82 recognized Asn(291)-Ile(304), a conserved region of the family protein. The polyclonal antibody raised against a peptide, Met(315)-Glu(328), of human Grp94, which corresponded to the conserved region of hHsp90, cross-reacted with hHsp90, but not with other Hsp90-family members. Thus, although mammalian Hsp90 shares some immunological reactivity with yeast Hsc82, human Grp94, and human Trap1, it is considerably distinct from its bacterial ortholog, HtpG. Disruption of the P. gingivalis htpG gene neither affected bacterial survival nor altered the sensitivity of P. gingivalis to various forms of stress.     

Hsp90 and co‐chaperones twist the functions of diverse client proteins

Hsp90 molecular chaperones are required for the stability and activity of a diverse range of client proteins that have critical roles in signal transduction, cellular trafficking, chromatin remodeling, cell growth, differentiation, and reproduction. Mammalian cells contain three types of Hsp90s: cytosolic Hsp90, mitochondrial Trap‐1, and Grp94 of the endoplasmic reticulum. Each of the Hsp90s, as well as the bacterial homolog, HtpG, hydrolyze ATP and undergo similar conformational changes. Unlike the other forms of Hsp90, cytosolic Hsp90 function is dependent on a battery of co‐chaperone proteins that regulate the ATPase activity of Hsp90 or direct Hsp90 to interact with specific client proteins. This review will summarize what is known about Hsp90's ability to mediate the folding and activation of diverse client proteins that contribute to human diseases, such as cancer and fungal and viral infections. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 211–217, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Evolutionary origins of Hsp90 chaperones and a deep paralogy in their bacterial ancestors

The 82-90 kD family of molecular chaperone proteins has homologs in eukaryotes (Hsp90) and many eubacteria (HtpG) but not in Archaebacteria. We used representatives of all four different eukaryotic paralogs (cytosolic, endoplasmic reticulum (ER), chloroplast, mitochondrial) together with numerous eubacterial HtpG proteins for phylogenetic analyses to investigate their evolutionary origins. Our trees confirm that none of the organellar Hsp90s derives from the endosymbionts of early eukaryotes. Contrary to previous suggestions of distant origins through lateral gene transfer (LGT) all eukaryote Hsp90s are related to Gram-positive eubacterial HtpG proteins. The nucleocytosolic, ER and chloroplast Hsp90 paralogs are clearly mutually related. The origin of mitochondrial Hsp90 is more obscure, as these sequences are deeply nested within eubacteria. Our trees also reveal a deep split within eubacteria into a group of mainly long-branching sequences (including the eukaryote mitochondrial Hsp90s) and another group comprising exclusively short-branching HtpG proteins, from which the cytosolic/ER versions probably arose. Both versions are present in several eubacterial phyla, suggesting gene duplication very early in eubacterial evolution and multiple independent losses thereafter. We identified one probable case of LGT within eubacteria. However, multiple losses can simply explain the evolutionary pattern of the eubacterial HtpG paralogs and predominate over LGT. We suggest that the actinobacterial ancestor of eukaryotes harbored genes for both eubacterial HtpG paralogs, as the actinobacterium Streptomyces coelicolor still does; one could have given rise to the mitochondrial Hsp90 and the other, following another duplication event in the ancestral eukaryote, to the cytosolic and ER Hsp90 homologs.     

Exploring the Functional Complementation between Grp94 and Hsp90

Grp94 and Hsp90 are the ER and cytoplasmic paralog members, respectively, of the hsp90 family of molecular chaperones. The structural and biochemical differences between Hsp90 and Grp94 that allow each paralog to efficiently chaperone its particular set of clients are poorly understood. The two paralogs exhibit a high degree of sequence similarity, yet also display significant differences in their quaternary conformations and ATPase activity. In order to identify the structural elements that distinguish Grp94 from Hsp90, we characterized the similarities and differences between the two proteins by testing the ability of Hsp90/Grp94 chimeras to functionally substitute for the wild-type chaperones in vivo. We show that the N-terminal domain or the combination of the second lobe of the Middle domain plus the C-terminal domain of Grp94 can functionally substitute for their yeast Hsp90 counterparts but that the equivalent Hsp90 domains cannot functionally replace their counterparts in Grp94. These results also identify the interface between the Middle and C-terminal domains as an important structural unit within the Hsp90 family.     

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 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.     

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.     

Hsp90 chaperones have an energetic hot‐spot for binding inhibitors

Although Hsp90‐family chaperones have been extensively targeted with ATP‐competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP‐binding pocket. A large‐scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen‐bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle‐specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen‐bond is pH‐dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked‐protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK_a associated with the D93 hydrogen‐bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK_a value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3–6 kcal/mol of stabilization (35–60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93?Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93?Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.     

Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotide

The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide‐dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species‐specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP‐bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X‐ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair‐like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs.     

Structural and energetic insight into the isoform-selective inhibitors of tumour marker Hsp90 against Grp94

The heat shock protein 90 (Hsp90) represents a new avenue for cancer therapy. A novel benzolactam inhibitor, compound 31, showed a great selectivity for Hsp90α and Hsp90β against Grp94. However, the structural basis for the great selectivity of compound 31 for Hsp90α/β versus Grp94 remains poorly understood. In this study, we carried out molecular docking, molecular dynamics simulations and binding free energy calculations (MM-GBSA) to address the isoform selective property. Molecular docking studies indicated the different binding modes of the Hsp90 and Grp94 with compound 31. The MM-GBSA calculations revealed that the hydrophobic interactions between compound 31 and proteins contributed the most to the binding affinity and the Grp94/compound 31 complex could result in a less energy-favourable complex compared with the Hsp90α/compound 31 and the Hsp90β/compound 31 complexes. This may render the weak binding of compound 31 to the Grp94. This study may be helpful for the future design of novel and selective Hsp90 inhibitors.     

The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.     

Molecular mechanism of bacterial Hsp90 pH‐dependent ATPase activity

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open‐to‐closed‐to‐open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH‐dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation‐specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti‐correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255‐ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255‐ATP salt‐bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH‐dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site‐specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

The overexpression of Hsp90B1 is associated with tumorigenesis of canine mammary glands

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics’ analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.     

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.     

Glucose-regulated Protein 94 Triage of Mutant Myocilin through Endoplasmic Reticulum-associated Degradation Subverts a More Efficient Autophagic Clearance Mechanism

Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that β-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.     

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.     

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.