All of the protein interactions that link steroid receptor.hsp90.immunophilin heterocomplexes to cytoplasmic dynein are common to plant and animal cells

Both plant and animal cells contain high molecular weight immunophilins that bind via tetratricopeptide repeat (TPR) domains to a TPR acceptor site on the ubiquitous and essential protein chaperone hsp90. These hsp90-binding immunophilins possess the signature peptidylprolyl isomerase (PPIase) domain, but no role for their PPIase activity in protein folding has been demonstrated. From the study of glucocorticoid receptor (GR).hsp90.immunophilin complexes in mammalian cells, there is considerable evidence that both hsp90 and the FK506-binding immunophilin FKBP52 play a role in receptor movement from the cytoplasm to the nucleus. The role of FKBP52 is to target the GR.hsp90 complex to the nucleus by binding via its PPIase domain to cytoplasmic dynein, the motor protein responsible for retrograde movement along microtubules. Here, we use rabbit cytoplasmic dynein as a surrogate for the plant homologue to show that two hsp90-binding immunophilins of wheat, wFKBP73 and wFKBP77, bind to dynein. Binding to dynein is blocked by competition with a purified FKBP52 fragment comprising its PPIase domain but is not affected by the immunosuppressant drug FK506, suggesting that the PPIase domain but not PPIase activity is involved in dynein binding. The hsp90/hsp70-based chaperone system of wheat germ lysate assembles complexes between mouse GR and wheat hsp90. These receptor heterocomplexes contain wheat FKBPs, and they bind rabbit cytoplasmic dynein in a PPIase domain-specific manner. Retention by plants of the entire heterocomplex assembly machinery for linking the GR to dynein implies a fundamental role for this process in the biology of the eukaryotic cell.     

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.     

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.     

Cyclophilin-A is bound through its peptidylprolyl isomerase domain to the cytoplasmic dynein motor protein complex

Although cyclophilin A (CyP-A) is a relatively abundant small immunophilin present in the cytoplasm of all mammalian cells, its general function(s) in the absence of the immunosuppressant drug cyclosporin A is not known. In contrast, the high molecular weight hsp90-binding immunophilins appear to play a role in protein trafficking in that they have been shown to link glucocorticoid receptor-hsp90 and p53.hsp90 complexes to the dynein motor protein for retrograde movement along microtubules. These immunophilins link to cytoplasmic dynein indirectly through the association of the immunophilin peptidylprolyl isomerase (PPIase) domain with dynamitin, a component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Here, we show that CyP-A exists in native heterocomplexes containing cytoplasmic dynein that can be formed in cell-free systems. Prolyl isomerase activity is not required for forming the dynein complex, but the PPIase domain fragment of FKBP52 blocks complex formation and CyP-A binds to dynamitin in a PPIase domain-dependent manner. CyP-A heterocomplexes containing tubulin and dynein can be formed in cytosol prepared under microtubule-stabilizing conditions, and CyP-A colocalizes in mouse fibroblasts with microtubules. Colocalization with microtubules is disrupted by overexpression of the PPIase domain fragment. Thus, we conclude that CyP-A associates in vitro and in vivo with the dynein/dynactin motor protein complex and we suggest that CyP-A may perform a general function related to the binding of cargo for retrograde movement along microtubules.     

Evidence for glucocorticoid receptor transport on microtubules by dynein

Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR.hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR.hsp90.immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR(-) L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.     

Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.     

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.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.     

hsp70 interacting protein Hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1

Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes. Using purified proteins, we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly-hsp90 and hsp70-and three proteins that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly. Despite the fact that Hip does not affect assembly, it is recovered in GR.hsp90 heterocomplexes assembled by both systems. In the five-protein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1.     

Visualization and mechanism of assembly of a glucocorticoid receptor.Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft

A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR.hsp70 complex that primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR.hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size approximately 1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR.hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/554C, and it is the subsequent assembly step with hsp90 that is defective.     

Nucleotide binding states of hsp70 and hsp90 during sequential steps in the process of glucocorticoid receptor.hsp90 heterocomplex assembly

A minimal system of five purified proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR, which primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23 (Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., and Pratt, W. B. (2000) J. Biol. Chem. 275, 18054-18060). Here we have examined the nucleotide-bound states of the two essential chaperones in each step. We show that it is the ATP-bound state of hsp70 that interacts initially with the GR. After rapid priming and washing, the primed GR.hsp70 complex rapidly binds hsp90 in the second step reaction in a nucleotide-independent manner. The rate-limiting step is the ATP-dependent opening of the steroid binding cleft after hsp90 binding. This activating step requires the N-terminal ATP-binding site of hsp90, but we cannot establish any role for a C-terminal ATP-binding site in steroid binding cleft opening. The reported specific inhibitors of the C-terminal ATP site on hsp90 inhibit the generation of steroid binding, but they have other effects in this multiprotein system that could explain the inhibition.     

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.