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.     

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.     

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.     

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.     

Hsp90-binding immunophilins in plants: the protein movers

Studies of cytoplasmic-nuclear trafficking of the glucocorticoid receptor in mammalian cells suggest that the hsp90/hsp70-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in targeted movement of the receptor along microtubule tracts. Over the past few years, plant cells have been found to possess a similar multiprotein chaperone machinery. Plant cells also contain high molecular weight FKBPs that bind to plant hsp90 via a conserved protein interaction involving tetratricopeptide repeat domains. The hsp90/hsp70-based machinery and the plant FKBPs might be used to target the trafficking of signalling proteins in plants.     

Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus

We have previously shown that immunoadsorption of the FKBP52 immunophilin component of steroid receptor.hsp90 heterocomplexes is accompanied by coadsorption of cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. Coimmunoadsorption of dynein is competed by an expressed fragment of FKBP52 comprising its peptidylprolyl isomerase (PPIase) domain (Silverstein, A. M., Galigniana, M. D., Kanelakis, K. C., Radanyi, C., Renoir, J.-M., and Pratt, W. B. (1999) J. Biol. Chem. 52, 36980-36986). Here we show that cotransfection of 3T3 cells with the FKBP52 PPIase domain and a green fluorescent protein (GFP) glucocorticoid receptor (GR) chimera inhibits dexamethasone-dependent movement of the GFP-GR from the cytoplasm to the nucleus. Cotransfection with FKBP12 does not affect GFP-GR movement. Inhibition of movement by the FKBP52 PPIase domain is abrogated in cells treated with colcemid to eliminate microtubules prior to steroid addition. After withdrawal of colcemid, microtubules reform, and PPIase inhibition of GFP-GR movement is restored. These observations are consistent with the notion that FKBP52 targets retrograde movement of the GFP-GR along microtubules by linking the receptor to the dynein motor. Here, we also show that native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR.hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.     

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.     

The 70-kDa protein bound to hsp90 in wheat germ lysate is a plant homologue of animal Hop

The hsp90-based chaperone machinery is implicated in numerous cellular processes including signal transduction, genomic silencing, and protein degradation. Hop is a component of the animal hsp90 multichaperone complex, whose function is to link the two chaperones, hsp90 and hsp70. Currently there exists little information on a plant Hop homologue. Herein it is reported that a 70-kDa protein in wheat germ lysate is associated with hsp90 and hsp70 and that this protein is a wheat homologue of Hop. It is also shown that, in addition to being detected in complexes, the wheat Hop as well as the previously identified immunophilin FKBP73, can bind directly to purified plant hsp90. In the steroid receptor folding assay, the wheat Hop was not detected in receptor complexes, but the wheat immunophilin FKBP73 could be detected when mammalian p23 was added to the plant lysate. The present results identify two hsp90-binding proteins and provide a useful framework on which to further investigate their functions.     

Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR).hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR.hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of approximately 0.03, and its elimination by immunoadsorption did not affect GR folding and GR. hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR.hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR.hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.     

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

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.