William B. Pratt

Regulation of Signaling Protein Function and Trafficking by the hsp90/hsp70-Based Chaperone Machinery

Nearly 100 proteins are known to be regulated by hsp90. Most of these substrates or “client proteins” are involved in signal transduction, and they are brought into complex with hsp90 by a multlprotein hsp90/hsp70-based chaperone machinery. In addition to binding substrate proteins at the chaperone site(s), hsp90 binds cofactors at other sites that are part of the heterocomplex assembly machinery as well as immunophllins that connect assembled substrate·hsp90 complexes to protein-trafficking systems. In the 5 years since we last reviewed this subject, much has been learned about hsp90 structure, nucleotide-binding, and cochaperone interactions; the most important concept is that ATP hydrolysis by an intrinsic ATPase activity results in a conformational change in hsp90 that is required to induce conformational change in a substrate protein. The conformational change induced in steroid receptors is an opening of the steroid-binding cleft so that it can be accessed by steroid. We have now developed a minimal system of five purified proteins—hsp90, hsp70, Hop, hsp40, and p23—that assembles stable receptor·hsp90 heterocomplexes. An hsp90·Hop·hsp70·hsp40 complex opens the cleft in an ATP-dependent process to produce a receptor·hsp90 heterocomplex with hsp90 in its ATP-bound conformation, and p23 then interacts with the hsp90 to stabilize the complex. Stepwise assembly experiments have shown that hsp70 and hsp40 first interact with the receptor in an ATP-dependent reaction to produce a receptor·hsp70·hsp40 complex that is “primed” to be activated to the steroid-binding state in a second ATP-dependent step with hsp90, Hop, and p23. Successful use of the five-protein system with other substrates Indicates that it can assemble signal protein-hsp90 heterocomplexes whether the substrate is a receptor, a protein kinase, or a transcription factor. This purified system should facilitate understanding of how eukaryotlc hsp70 and hsp90 work together as essential components of a process that alters the conformations of substrate proteins to states that respond in signal transduction.

The hsp90-based Chaperone System: Involvement in Signal Transduction from a Variety of Hormone and Growth Factor Receptors

Abstract A variety of transcription factors and protein kinases involved in signal transduction are recovered from cells in heterocomplexes containing the abundant protein chaperone hsp90. Genetic studies in yeast have demonstrated that binding of steroid receptors, the dioxin receptor, and some protein kinases to hsp90 is critical for their signal transducing function in vivo. These heterocomplexes are formed by a multiprotein chaperone machinery consisting of at least four ubiquitous proteins—hsp90, hsp70, p60 and p23. Four high-molecular-weight immunophilins have been discovered as components of steroid receptor or other transcription factor complexes with hsp90. The immunophilins, protein chaperones with prolyl isomerase activity, bind the immunosuppressant drugs FK506 or CyP-40. These immunophilins all bind via tetratricopeptide repeat (TPR) domains to a single TPR binding site on each hsp90 dimer, and multiple heterocomplexes exist for each protein chaperoned by hsp90 according to the immunophilin that is bound to this TPR binding site at any time. Three components of the MAP kinase signalling system (Src, Raf, and Mek) exist in complexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein cdc37. The p50cdc37 binds to hsp90 at a site that is close to but different from the TPR binding site of the immunophilins, and like the immunophilins, p50cdc37 is thought to be involved in targeting and trafficking of the protein kinases. The recent introduction of the benzoquinone antibiotic geldanamycin has facilitated the identification of proteins that are chaperoned by the hsp90-based system. Geldanamycin binds to members of the hsp90 protein family, blocking assembly of hsp90 heterocomplexes and destabilizing preformed heterocomplexes. In the presence of geldanamycin, the function of hsp90-chaperoned proteins is disrupted, and the proteins undergo rapid degradation by an ubiquitin-dependent proteasomal mechanism. It is becoming clear that hsp90 chaperoning is not only essential to a variety of signal transduction pathways, but is critical for proper folding, stabilization, and trafficking of an expanding list of proteins.

Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315–32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR·hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ∼35% contains PP5. Only a small (but undetermined) fraction of the native GR·hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

Folding of the Glucocorticoid Receptor by the Heat Shock Protein (hsp) 90-based Chaperone Machinery THE ROLE OF p23 IS TO STABILIZE RECEPTOR·hsp90 HETEROCOMPLEXES FORMED BY hsp90·p60·hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor·hsp90 heterocomplexes. We have reconstituted a minimal receptor·hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR·hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997)J. Biol. Chem. 272, 13047–13054). In this work, we show that addition of p23 to native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR·hsp90 heterocomplexes prepared with the minimal (hsp90·p60·hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR·hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90·p60·hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR·hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90·p60·hsp70 forms a GR·hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex to inactivation and disassembly.

The Tetratricopeptide Repeat Domain of Protein Phosphatase 5 Mediates Binding to Glucocorticoid Receptor Heterocomplexes and Acts as a Dominant Negative Mutant

We previously identified a protein-serine phosphatase designated PP5, based on the binding of its tetratricopeptide repeat (TPR) domain to the atrial natriuretic peptide receptor (Chinkers, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079). We have now identified another protein complex to which PP5 is targeted through its TPR domain. A 90-kDa protein, identified as heat shock protein 90 (hsp90) by immunoblotting, specifically co-immunoprecipitated from COS-7 cell lysates with the FLAG-tagged TPR domain of PP5. hsp90 also co-immunoprecipitated with full-length FLAG-tagged PP5 overexpressed in COS-7 cells and with endogenous PP5 from untransfected COS-7 cells or rat brain. During gel filtration, PP5 and hsp90 comigrated in a high molecular weight complex. Since glucocorticoid receptors (GR) exist as large heterocomplexes containing hsp90 bound to TPR proteins, we hypothesized that PP5 might be associated with these complexes. Consistent with this hypothesis, PP5 specifically co-immunoprecipitated with GR from mouse L cell lysates. To test the functional importance of this TPR-mediated association in living cells, we used a dominant negative PP5 mutant consisting only of its TPR domain. The mutant inhibited GR-mediated transactivation by approximately 70% in transfected CV-1 cells. This is the first evidence that the TPR proteins in steroid receptor heterocomplexes may be required for signaling in vivo.

The hsp90-binding Antibiotic Geldanamycin Decreases Raf Levels and Epidermal Growth Factor Signaling without Disrupting Formation of Signaling Complexes or Reducing the Specific Enzymatic Activity of Raf Kinase

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras·Raf, Ras·Src, Raf·Mek, and Raf·Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf·hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor·hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery THE INITIAL hsp90·p60·hsp70-DEPENDENT STEP IS SUFFICIENT FOR CREATING THE STEROID BINDING CONFORMATION

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles steroid receptors into a complex with hsp90. The glucocorticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding conformation. Recently, we have reconstituted a receptor·hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). In this work we show that when the GR is incubated with hsp90, hsp70, and p60, steroid binding sites are generated despite the absence of p23. In this minimal reconstituted system, the GR is incubated with the chaperones in the presence of [3H]triamcinolone acetonide ([3H]TA), which binds to the receptor as GR·hsp90 complexes are formed. When molybdate or p23 is also present during the incubation with chaperones at 30 °C, the formation of steroid binding sites can be assayed by incubating the washed GR with [3H]TA after heterocomplex assembly at 30 °C. However, in the absence of p23 or molybdate, rapid disassembly of GR·hsp90 complexes apparently occurs simultaneously with assembly, such that [3H]TA must be present during the assembly process to trap evidence of conversion of the GR HBD from a non-steroid binding to a steroid binding conformation. Mixture of purified rabbit hsp90 and hsp70 with bacterial lysate containing human p60 results in spontaneous formation of an hsp90·p60·hsp70 complex that can be adsorbed with anti-p60 antibody, and the resulting immune complex converts the GR HBD to a steroid binding state in an ATP-dependent and K+-dependent manner. When the GR is incubated with hsp90, hsp70, and p60 in the presence of the hsp90-binding antibiotic geldanamycin, GR·hsp90·p60· hsp70 complexes are formed, but they have no steroid binding activity. Our data suggest that hsp90, hsp70, and p60 work together as a chaperone complex that possesses all of the folding/unfolding activity necessary to generate the high affinity steroid binding conformation of the receptor.

A Model of Protein Targeting Mediated by Immunophilins and Other Proteins That Bind to hsp90 via Tetratricopeptide Repeat Domains

We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR·hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src·hsp90 and Raf·hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor·hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.

Proposal for a role of the Hsp90/Hsp70-based chaperone machinery in making triage decisions when proteins undergo oxidative and toxic damage

The Hsp90/Hsp70-based chaperone machinery plays a well-established role in signaling protein function, trafficking and turnover. A number of recent observations also support the notion that Hsp90 and Hsp70 play key roles in the triage of damaged and aberrant proteins for degradation via the ubiquitin-proteasome pathway. In the mid-1990s, it was discovered that Hsp70 is required for ubiquitin-dependent degradation of short-lived and abnormal proteins, and it became clear that inhibition of Hsp90 uniformly leads to the proteasomal degradation of Hsp90 client proteins. Subsequently, CHIP and parkin were shown to be Hsp70-binding ubiquitin E3 ligases that direct ubiquitin-charged E2 enzymes to the Hsp70-bound client protein. Stabilization by Hsp90 reflects the interaction of the chaperone with the ligand binding cleft of the client protein. These hydrophobic clefts must be open to allow passage of ligands to binding sites in the protein interior, and they are inherent sites of conformational instability. Hsp90 stabilizes the open state of the cleft and prevents Hsp70-dependent ubiquitination. In the model we propose here, progressive oxidative events result in cleft opening as the initial step in protein unfolding, and as long as Hsp90 can interact to stabilize the cleft, it will buffer the effect of oxidative damage. When cleft opening is such that Hsp90 can no longer interact, Hsp70-dependent ubiquitination occurs. We summarize evidence that Hsp90 interacts very dynamically with a variety of proteins that are not classic Hsp90 clients, and we show that this dynamic cycling of Hsp90 with nitric oxide synthase protects against CHIP-mediated ubiquitination. Scientific interest to date has focused on stringent regulation of the classic client proteins, which have metastable clefts and are inherently short lived. But, the recognition that Hsp90 cycles dynamically with longer lived proteins with more stable clefts may permit extension of the triage model to the quality control of damaged proteins in general.