C-terminal sequences outside the tetratricopeptide repeat domain of FKBP51 and FKBP52 cause differential binding to Hsp90

Hsp90 assembles with steroid receptors and other client proteins in association with one or more Hsp90-binding cochaperones, some of which contain a common tetratricopeptide repeat (TPR) domain. Included in the TPR cochaperones are the Hsp70-Hsp90-organizing protein Hop, the FK506-binding immunophilins FKBP52 and FKBP51, the cyclosporin A-binding immunophilin CyP40, and protein phosphatase PP5. The TPR domains from these proteins have similar x-ray crystallographic structures and target cochaperone binding to the MEEVD sequence that terminates Hsp90. However, despite these similarities, the TPR cochaperones have distinctive properties for binding Hsp90 and assembling with Hsp90.steroid receptor complexes. To identify structural features that differentiate binding of FKBP51 and FKBP52 to Hsp90, we generated an assortment of truncation mutants and chimeras that were compared for coimmunoprecipitation with Hsp90. Although the core TPR domain (approximately amino acids 260-400) of FKBP51 and FKBP52 is required for Hsp90 binding, the C-terminal 60 amino acids (approximately 400-end) also influence Hsp90 binding. More specifically, we find that amino acids 400-420 play a critical role for Hsp90 binding by either FKBP. Within this 20-amino acid region, we have identified a consensus sequence motif that is also present in some other TPR cochaperones. Additionally, the final 30 amino acids of FKBP51 enhance binding to Hsp90, whereas the corresponding region of FKBP52 moderates binding to Hsp90. Taking into account the x-ray crystal structure for FKBP51, we conclude that the C-terminal regions of FKBP51 and FKBP52 outside the core TPR domains are likely to assume alternative conformations that significantly impact Hsp90 binding.     

Control of glucocorticoid and progesterone receptor subcellular localization by the ligand-binding domain is mediated by distinct interactions with tetratricopeptide repeat proteins

The TPR proteins FKBP52, FKBP51, Cyp40, and PP5 are found in steroid receptor (SR) complexes, but their receptor-specific preferences and roles remain unresolved. We have undertaken a systematic approach to this problem by examining the contribution of all four TPRs to the localization properties of glucocorticoid (GR) and progesterone (PR) receptors. The GR of L929 cells was found in the cytoplasm in a complex containing PP5 and FKBP51, while the GR of WCL2 cells was nuclear and contained PP5 and FKBP52. Cyp40 did not interact with the GR in either cell line. To test whether FKBP interaction determined localization, we overexpressed Flag-tagged FKBP51 in WCL2 cells and Flag-FKBP52 in L929 cells. In WCL2 cells, the GR exhibited a shift to greater cytoplasmic localization that correlated with recruitment of Flag-FKBP51. In contrast, Flag-FKBP52 was not recruited to the GR of L929 cells, and no change in localization was observed, suggesting that both cell-type-specific mechanisms and TPR abundance contribute to the SR-TPR interaction. As a further test, GR-GFP and PR-GFP constructs were expressed in COS cells. The GR-GFP construct localized to the cytoplasm, while the PR-GFP construct was predominantly nuclear. Similar to L929 cells, the GR in COS interacted with PP5 and FKBP51, while PR interacted with FKBP52. Analysis of GR-PR chimeric constructs revealed that the ligand-binding domain of each receptor determines both TPR specificity and localization. Lastly, we analyzed GR and PR localization in cells completely lacking TPR. PR in FKBP52 KO cells showed a complete shift to the cytoplasm, while GR in FKBP51 KO and PP5 KO cells showed a moderate shift to the nucleus, indicating that both TPRs contribute to GR localization. Our results demonstrate that SRs have distinct preferences for TPR proteins, a property that resides in the LBD and which can now explain long-standing differences in receptor subcellular localization.     

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.     

The 90-kDa heat-shock protein (Hsp90)-binding immunophilin FKBP51 is a mitochondrial protein that translocates to the nucleus to protect cells against oxidative stress

Confocal microscopy images revealed that the tetratricopeptide repeat motif (TPR) domain immunophilin FKBP51 shows colocalization with the specific mitochondrial marker MitoTracker. Signal specificity was tested with different antibodies and by FKBP51 knockdown. This unexpected subcellular localization of FKBP51 was confirmed by colocalization studies with other mitochondrial proteins, biochemical fractionation, and electron microscopy imaging. Interestingly, FKBP51 forms complexes in mitochondria with the glucocorticoid receptor and the Hsp90/Hsp70-based chaperone heterocomplex. Although Hsp90 inhibitors favor FKBP51 translocation from mitochondria to the nucleus in a reversible manner, TPR domain-deficient mutants of FKBP51 are constitutively nuclear and fully excluded from mitochondria, suggesting that a functional TPR domain is required for its mitochondrial localization. FKBP51 overexpression protects cells against oxidative stress, whereas FKBP51 knockdown makes them more sensitive to injury. In summary, this is the first demonstration that FKBP51 is a major mitochondrial factor that undergoes nuclear-mitochondrial shuttling, an observation that may be related to antiapoptotic mechanisms triggered during the stress response.     

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.     

A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins.

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-Hsp90-FKBP52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport.     

Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90

The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity.     

Analysis of Hsp90 cochaperone interactions reveals a novel mechanism for TPR protein recognition

The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.     

The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain

Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.     

Binding of hsp90-associated immunophilins to cytoplasmic dynein: direct binding and in vivo evidence that the peptidylprolyl isomerase domain is a dynein interaction domain

FKBP52 is a steroid receptor-associated immunophilin that binds via a tetratricopeptide repeat (TPR) domain to hsp90. FKBP52 has also been shown to interact either directly or indirectly via its peptidylprolyl isomerase (PPIase) domain with cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. The functional role for the PPIase domain in receptor movement was demonstrated by showing that expression of the PPIase domain fragment of FKBP52 in 3T3 cells inhibits dexamethasone-dependent nuclear translocation of a green fluorescent protein-glucocorticoid receptor chimera. Here, we show that cytoplasmic dynein is co-immunoadsorbed with two other TPR domain proteins that bind hsp90 (the cyclophilin CyP-40 and the protein phosphatase PP5). Both proteins possess PPIase homology domains, and co-immunoadsorption of cytoplasmic dynein with each is blocked by the PPIase domain fragment of FKBP52. Using purified proteins, we show that FKBP52, PP5, and the PPIase domain fragment bind directly to the intermediate chain of cytoplasmic dynein. PP5 colocalizes with both cytoplasmic dynein and microtubules, and expression of the PPIase domain fragment of FKBP52 in 3T3 cells disrupts its cytoskeletal localization. We conclude that the PPIase domains of the hsp90-binding immunophilins interact directly with cytoplasmic dynein and that this interaction with the motor protein is responsible for the microtubular localization of PP5 in vivo.     

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.     

The Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically

Cdc37 associates with the heat-shock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 co-chaperones. Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases. It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or -independent functions or both. Sti1 and Cpr7 are non-essential Hsp90 co-chaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR). We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37. Similarly, the endogenous proteins are also found in a complex. Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90. Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be co-purified with exogenously expressed Cpr7 fused to glutathione-S-transferase. The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37. Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Interaction of Heat Shock Protein 90 and the Co-chaperone Cpr6 with Ura2, a Bifunctional Enzyme Required for Pyrimidine Biosynthesis

The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6-dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones.     

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.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.