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.     

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.     

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.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

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.     

Corresponding functional dynamics across the Hsp90 Chaperone family: insights from a multiscale analysis of MD simulations

Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones.     

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.     

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 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.     

Structures of the N-terminal and middle domains of E. coli Hsp90 and conformation changes upon ADP binding

Hsp90 is an abundant molecular chaperone involved in many biological systems. We report here the crystal structures of the unliganded and ADP bound fragments containing the N-terminal and middle domains of HtpG, an E. coli Hsp90. These domains are not connected through a flexible linker, as often portrayed in models, but are intimately associated with one another. The individual HtpG domains have similar folding to those of DNA gyrase B but assemble differently, suggesting somewhat different mechanisms for the ATPase superfamily. ADP binds to a subpocket of a large site that is jointly formed by the N-terminal and middle domains and induces conformational changes of the N-terminal domain. We speculate that this large pocket serves as a putative site for binding of client proteins/cochaperones. Modeling shows that ATP is not exposed to the molecular surface, thus implying that ATP activation of hsp90 chaperone activities is accomplished via conformational changes.     

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     

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 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.     

Interaction of Hsp90 with the nascent form of the mutant epidermal growth factor receptor EGFRvIII

EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.     

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.     

Structural basis for topoisomerase VI inhibition by the anti-Hsp90 drug radicicol

Members of the GHL ATPase superfamily, including type II topoisomerases, Hsp90-class chaperones, and MutL, all share a common GHKL-type ATP-binding fold and act as nucleotide-controlled 'molecular clamps'. These enzymes' ATP-binding sites have proven to be rich drug targets, and certain inhibitors of type II topoisomerases and Hsp90 bind to this region and competitively inhibit these enzymes. Recently, it was found that radicicol, a drug known to block Hsp90 function, also inhibits the archaeal type IIB topoisomerase topo VI. Here, we use X-ray crystallography to show that despite low sequence identity ( approximately 10-12%) between topo VI and Hsp90, radicicol binds to the ATPase sites of these two enzymes in an equivalent manner. We further demonstrate that radicicol inhibits both the dimerization of the topo VI ATPase domains and ATP hydrolysis, two critical steps in the enzyme's strand passage reaction. This work contributes to a growing set of structures detailing the interactions between GHL-family proteins and various drugs, and reveals radicicol as a versatile scaffold for targeting distantly related GHL enzymes.     

The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s

Both the Grp170 and Hsp110 families represent relatively conserved and distinct sets of stress proteins, within a more diverse category that also includes the Hsp70s. All of these families are found in a wide variety of organisms from yeasts to humans. Although Hsp110s or Grp170s are not Hsp70s any more than Hsp70s are Hsp110s or Grp170s, it is still reasonable to refer to this combination of related families as the Hsp70 superfamily based on arguments discussed above and since no obvious prokaryotic Hsp110 or Grp170 has yet been identified. These proteins are related to their counterparts in the Hsp70/Grp78 family of eukaryotic stress proteins but are characterized by significantly larger molecular weights. The members of the Grp170 family are characterized by C-terminal ER retention sequences and are ER localized in yeasts and mammals. As a Grp, Grp170 is recognized to be coregulated with other major Grps by a well-known set of stress conditions, sometimes referred to as the unfolded protein response (Kozutsumi et al 1988; Nakaki et al 1989). The Hsp110 family members are localized in the nucleus and cytoplasm and, with other major Hsps, are also coregulated by a specific set of stress conditions, most notably including hyperthermic exposures. Hsp110 is sometimes called Hsp105, although it would be preferable to have a uniform term. The large Hsp70-like proteins are structurally similar to the Hsp70s but differ from them in important ways. In both the Grp170 and Hspl10 families, there is a long loop structure that is interposed between the peptide-binding ,-domain and the alpha-helical lid. In the Hsp110 family and Grp170, there are differing degrees of expansion in the alpha-helical domain and the addition of a C-terminal loop. This gives the appearance of much larger lid domains for Hsp110 and Grp170 compared with Hsp70. Both Hsp110 and Grp170 families have relatively conserved short sequences in the alpha-helical domain in the lid, which are conserved motifs in numerous proteins (we termed these motifs Magic and TedWylee as discussed earlier). The structural differences detailed in this review result in functional differences between the large (Grp170 and Hspl10) members of the Hsp70 superfamily, the most distinctive being an increased ability of these proteins to bind (hold) denatured polypeptides compared with Hsc70, perhaps related to the enlarged C-terminal helical domain. However, there is also a major difference between these large stress proteins; Hsp110 does not bind ATP in vitro, whereas Grp170 binds ATP avidly. The role of the Grp170 and Hsp110 stress proteins in cellular physiology is not well understood. Overexpression of Hsp110 in cultured mammalian cells increases thermal tolerance. Grp170 binds to secreted proteins in the ER and may be cooperatively involved in folding these proteins appropriately. These roles are similar to those of the Hsp70 family members, and, therefore, the question arises as to the differential roles played by the larger members of the superfamily. We have discussed evidence that the large members of the superfamily cooperate with members of the Hsp70 family, and these chaperones probably interact with a large number of chaperones and cochaperones in their functional activities. The fundamental point is that Hsp110 is found in conjunction with Hsp70 in the cytoplasm (and nucleus) and Grp170 is found in conjunction with78 in tha ER in every eucaryotic cell examined from yeast to humans. This would strongly argue that Hsp110 Grp170 exhibit functions in eucaryotes not effectively performed by Hsp70s or Grp78, respectively. Of interest in this respect is the observation that all Hsp110s loss of function or deletion mutants listed in the Drosophila deletion project database are lethal. The important task for the future is to determine the roles these conserved molecular chaperones play in normal and physiologically stressed cells.     

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.