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FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

We have identified a human gene encoding a 48-kDa protein that specifically interacts with the peptidyl prolyl isomerase FK506-binding protein 59 (FKBP59) and also with the well known FKBP12. FKBP59 and FKBP12 belong to the large family of immunophilins that bind the macrolide immunosuppressant drugs FK506 and rapamycin. The yeast two-hybrid system was used to isolate target proteins that interact with the immunosuppressant drug binding domain of the rabbit FKBP59. The cDNA for an as yet unidentified protein was isolated and cloned from a Jurkat cell library. The cDNA sequence of 1804 base pairs reveals an open reading frame of 417 amino acids. In vitro experiments suggest a direct interaction between FKBP59 and this new target protein. This specific association seems to be restricted to the FKBP family, since it also occurs both in vivo and in vitro with FKBP12 but not with cyclophilin 40. This novel protein was named FKBP-associated protein (FAP48). The formation of the complexes between FKBP59 or FKBP12 and FAP48 is prevented by FK506 and rapamycin in a dose-dependent manner. These results suggest that FAP48 shares or overlaps the macrolide binding site on FKBP59 as well as on FKBP12 and therefore may represent a natural common ligand of these immunosuppressant drug receptors.

FKBP59 is a protein usually associated with heat-shock protein hsp90 and steroid receptors. The N-terminal domain of the rabbit liver protein (149 amino acids) has a sequence homology with FKBP12, binds FK506 immunosuppressor, and has a peptidyl-prolyl cis-trans isomerase activity. The three-dimensional structure of this domain (FKBP59-I) was determined using homo- and heteronuclear multidimensional NMR spectroscopy, distance geometry, and molecular dynamics methods. Structure calculations used 1290 interproton distance restraints derived from nuclear Overhauser enhancement measurements, 29 dihedral phi angle restraints, and 92 hydrogen bond restraints. For the final 22 structures, the root mean square distance from the mean atomic coordinates, calculated for well-defined secondary structure fragments, is 0.47 +/- 0.05 and 1.26 +/- 0.15 A for backbone heavy atoms (N, C alpha, C') and for all non-hydrogen atoms, respectively. The global fold contains a twisted six-stranded antiparallel beta-sheet and a short alpha-helix packed on the hydrophobic side of the sheet. The 20 N-terminal and 12 C-terminal amino acids of the domain are disordered. The main-chain structure of FKBP59-I is globally similar to the NMR-derived and X-ray structures of unbound FKBP12. An unusual hydrogen bond interaction between the indole amino proton of Trp 89 and the aromatic cycle of Phe 129 was observed. This gives a large upfield shift (-4.8 ppm) and a significant exchange protection factor. The implications of the present structure determination on the ligand binding of FKBP59 are discussed.

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.

FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i.e. in microtubule depolymerizing conditions). Exposure of cells to 1 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS)

A protein of apparent molecular mass of approximately 59 kDa of the FK506-binding protein class (FKBP59) has been found associated with the heat shock protein hsp90 included in nontransformed steroid receptor complexes and termed FKBP59-HBI (HBI for Heat shock protein 90 Binding Immunophilin). Further data analysis has revealed that this immunophilin also belongs to the tetratricopeptide repeat family of proteins. In this work, we describe the hsp90-binding domain of FKBP59-HBI. Density gradient centrifugation, gel filtration, and immunoadsorption analyses failed to demonstrate a stable association between FKBP59-HBI and hsp90 in the rabbit reticulocyte lysate. Using a gel-retardation assay, we provide evidence for a specific ATP-independent interaction between highly purified wild-type rabbit FKBP59-HBI and human hsp90 beta. This interaction was not affected by the immunosuppressants FK506 and rapamycin. Examination of the behavior of several mutants led us to conclude that the tetratricopeptide motifs localized in the C-terminal part of FKBP59-HBI are necessary for hsp90 binding.

The effect of recombinant FKBP-59/HBI or of its first N-terminal domain FKBP-59/HBI-I on the phosphatase activity of calcineurin (a Ca(+2)-calmodulin dependent phosphatase) was tested in vitro in the presence or absence of the immunosuppressant drug FK506. Contrarily to the inhibition observed with the immunosuppressant complex FKBP-12-FK506, no significant inhibition was observed with FKBP-59/HBI or FKBP-59/HBI-I in the presence of FK506, even though FKBP-59/HBI-1 is nearly 55% homologous to the immunophilin FKBP-12. Inhibition was tested both with native calcineurin (calcineurin A: Mr 58-59 kDa) and with protease activated, calmodulin independent calcineurin (calcineurin A: Mr 45 kDa). There was no competitive effect of FKBP-59 on the inhibitory activity of the FKBP-12-FK506 complex, even when the molar concentration of FKBP-59/HBI was 100 times higher than that of FKBP-12. Clearly, although the first domain of FKBP-59/HBI displays several structural and functional features of FKBP-12, it does not interact with calcineurin.

In this study, the conservation of strong structural landmarks between all the members of two chaperone families (HSP60 and HSP70) was deduced from their sequences by hydrophobic cluster analysis. On this basis, we propose that the ATP-binding environment is maintained by a similar fold in both protein families. The observed similarities extend throughout the proteins, including both the ATPase domain and the C-terminal substrate-binding domain.

We have developed an in vivo system using coexpression of human glucocorticosteroid receptor (hGR) and chick hsp90 alpha (chsp90) in recombinant virus-infected Sf9 cells to study the formation of hetero-oligomeric complexes. We detected, in the cytosol, hGR complexes containing chsp90 as shown by the displacement of the [3H]triamcinolone acetonide bound hGR "8S" peak on glycerol/sucrose gradients by specific antibodies directed against chsp90 (BF4 and D7 alpha). We took advantage of this system and of the immunoadsorption of hGR containing complexes with anti-hGR antibody to analyze the effect of deletions introduced into the hsp90 molecule on the formation of complexes with the hGR. Deletion of the hydrophilic region "A", between amino-acids 221 and 290, abolished the formation of hGR/chsp90 complexes. Deletion of the hydrophilic region "B" (between amino-acids 530 and 581) or deletion of a leucine repeat region "Z" in the middle of the molecule (amino-acids 392 to 419) still allowed formation of hetero-oligomeric complexes detected by immunoadsorption but the hGR complexes formed with mutated chsp90s were devoid of steroid binding properties. These results are consistent with (1) the possible involvement of the "A" region in the interaction of hsp90 with steroid receptors and (2) a role of B and Z regions in the hsp90 structure for maintaining the steroid binding property of the hGR.

In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling.

p59/HBI, a Heat shock protein (hsp90)-Binding-Immunophilin of approximately 59 kDa, was originally detected in the heterooligomeric, non-DNA binding form of numerous mammalian steroid hormone receptors. P59/HBI belongs to the FKBP family since it binds the immunosuppressants FK506 and Rapamycin. Using immobilized FK506, we purified [3H]Org 2058-progesterone receptor (PR)-hsp90-complexes from chick oviduct cytosol in a species migrating at 9S in density gradient. This led to suppose that a protein present in these complexes is a FK506 binding protein. Following incubation of this FK506-affinity purified 9S-PR with BF4, a specific anti-chick hsp90 monoclonal antibody, a shift of the [3H]Org 2058-PR complexes from 9S to 11S has been observed, indicating the presence of hsp90, hsp70 also is included in the 9S-PR complexes as demonstrated by Western blotting and density gradient experiments. Two lines of evidence support the notion that an immunophilin of 50 kDa also associates to heterooligomeric 9S-PR complexes. Firstly, a shift at 11S of the FK506 eluted 9S-PR occurs in sucrose gradient after incubation with 419, a new polyclonal antibody raised against a peptide corresponding to the Hinge I region [Callebaut I., Renoir J. M., Lebeau M. C., Massol N., Burny A., Baulieu E. E., Mornon J. P. (1992) Proc. Natl. Acad. Sci. USA 89, 6270-6274] of the rabbit p59/HBI (amino acids Phe135 to Gly149). Secondly, in Western blotting experiments, both 419 and another anti-FKBP antibody, raised against purified Streptomyces Crysomallus FKBP12 [Pahl A. and Keller U. (1992) J. Bacteriol., 3, 325-333], allow detection of the same 50 kDa protein in the affinity purified PR.(ABSTRACT TRUNCATED AT 250 WORDS)

Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.

It has been previously proposed that the rabbit p59-HBI (Heat shock protein Binding Immunophilin) or rFKBP59 (FK506 Binding Protein), found associated with the 90 kDa heat shock protein in nontransformed steroid receptor complexes, has three domains structurally related to hFKBP12 (Callebaut, I., Renoir, J.M., Lebeau, M.C., Massol, N., Burny, A., Baulieu, E.E. and Mornon, J.P. (1992) Proc. Natl. Acad. Sci., USA 89, 6270-6274). Here we report the overexpression, as fusion proteins in E. coli, of the full length p59-HBI and a series of p59-HBI mutants delimiting these domains and their respective peptidyl prolyl cis trans isomerase (PPlase) activity. The PPlase activity of p59-HBI is comparable to that of hFKBP12 and is due to domain p59-HBI I which displays the highest homology with this immunophilin. The residual enzymatic activity found in domain p59-HBI II is discussed.

A protein of M(r) 59,000 (p59) was recently cloned and identified as a Heat shock protein Binding Immunophilin (p59/HBI). It participates to the heterooligomeric, non-DNA binding form of steroid receptors, in association with the heat shock protein of M(r) 90,000 (hsp90). It binds the immunosuppressants FK506 and rapamycin and possesses three FKBP-12 (FK506 binding protein of M(r) 12,000)--like domains (I to III), plus a tail containing a putative calmodulin binding site (domain IV). Following expression in E. Coli and purification on Glutathione-Sepharose of either the full-length recombinant p59/HBI, or the recombinant FKBP-like domains, we demonstrate by autoradiography of [alpha 32P]-8-azido ATP and of [alpha 32P]-8-azido GTP photoaffinity labeled complexes, that an ATP (GTP) binding site is located in the domain II. This nucleotide binding property is also found with the highly purified rabbit uterus p59/HBI. The latter, but not the recombinant protein, can be phosphorylated in vitro in the presence of Mn++ and/or of Ca++/Calmodulin in an ATP but not GTP dependent manner, suggesting copurification of a CaM kinase II-like enzyme. Thus it appears that p59/HBI is a multifunctional immunophilin which may be at the crossroad of the endocrine and immunological systems.

The heat-shock protein of 90 kDa (hsp90), constitutively expressed in most cells, is up-regulated by thermal stress and by developmental and mitogenic stimuli. When the serum-starved chicken hepatoma cell line DU249 is stimulated by serum, insulin, or growth factors acting via tyrosine kinase receptors, a transient induced expression of the hsp90 alpha gene takes place at both the messenger RNA and the protein synthesis level. This response is protein synthesis dependent and DNA synthesis independent. The maximum level of hsp90 alpha mRNA accumulation always precedes the maximum level of thymidine incorporation, suggesting a possible link between cell cycle and hsp90 alpha regulation (Jérôme, V., J. Léger, J. Devin, E.E. Baulieu, and M. G. Catelli. Growth Factors 4:317-327, 1991). Herein, we examine the subcellular distribution of hsp90 and the cell cycle-dependent regulation of the hsp90 alpha mRNA level. We show that, in contrast to hsp70, the 35S metabolically-labeled hsp90, which accumulates in the cytosoluble fraction 6 to 8 h after serum treatment, is not preferentially translocated to the nuclear compartment, although a small fraction is always present in the nucleus. We also demonstrated that serum- or insulin-induced accumulation of hsp90 alpha mRNA results from an activation of gene transcription and that hsp90 alpha promotor activity, which is low in quiescent DU249 cells, is induced approximately fivefold after serum stimulation. Finally, in cell culture synchronized by nocodazole or aphidicholin, hsp90 alpha mRNA accumulation seems an event specific to G1/S transition.

The cloning of chicken hsp90 beta, reported here, supports the hypothesis that the hsp90 alpha and beta genes are the result of a gene duplication event that occurred at the time of the emergence of vertebrates. Avian hsp90 beta mRNA is not inducible by thermal stress contrary to the mouse and human hsp90 alpha and beta mRNAs. Moreover, hsp90 beta mRNA level is not increased by mitogenic stimuli, like serum or insulin, which induce hsp90 alpha mRNA. Different functions and regulations of hsp90 alpha and beta will be probably more easily understood in the avian species than in higher vertebrates.

FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.

The M(r) 90,000 protein associated with steroid receptors in their non-transformed state has been identified as a heat shock protein (hsp90) but the relationship between hsp90 binding and receptor function is still poorly understood. In this work, we have obtained and characterized one monoclonal anti-rabbit hsp90 antibody (7C10), among more than 2000 wells plated. This antibody was able to complex both free and rabbit uterine progesterone receptor-associated hsp90 as demonstrated by sedimentation analysis on sucrose gradients. As assessed by ELISA, 7C10 displayed a high binding affinity for hsp90 (approximately 4 nM). A standardized and specific competitive binding assay was developed for accurate quantification of hsp90 in rabbit tissues including reticulocyte lysate. 7C10 also permitted immunolocalization of hsp90 in various rabbit tissues. In Western blot, the monoclonal antibody recognized a single polypeptide band of M(r) approximately 90,000 in crude or purified rabbit preparations but failed to cross-react with any other mammalian or avian hsp90. These findings suggest that hsp90, a highly conserved protein, is a weak immunogen and elicits a strict species specific immunological response. Owing to its high affinity and specificity for rabbit hsp90, the monoclonal antibody 7C10 was used for purification and total depletion of hsp90 from the reticulocyte lysate, an efficient system for in vitro receptor translation and reconstitution studies. Thus, 7C10 represents a new powerful tool to further investigate the importance of hsp90 in steroid hormone receptor function.

In the rabbit, a p59 protein included in the untransformed, non-DNA binding, "8-9S," steroid receptor complexes binds heat shock protein M(r) approximately 90,000 (hsp90). Sequence data [Lebeau, M. C., Massol, N., Herrick, J., Faber, L. E., Renoir, J. M., Radanyi, C. & Baulieu, E. E. (1992) J. Biol. Chem. 267, 4281-4284] and hydrophobic cluster analysis delineate, from the N terminus, two successive domains closely related to the immunosuppressant FK506 binding immunophilin FKBP (FK506 binding protein), consistent with recent purification of the human p56 immunophilin cognate protein by FK506 affinity chromatography [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A. & Deibel, M. R., Jr. (1992) J. Biol. Chem. 267, 2868-2871]. The first FKBP-like domain demonstrates all structural characteristics known to be necessary for immunosuppressant binding and for peptidylprolyl cis-trans isomerase (rotamase) activity. Hence, p59 is a "hsp binding immunophilin" (HBI). It is thus speculated that hsp binding immunophilin may help the assembly/disassembly mechanisms involved in steroid receptor trafficking and activity and participate in the poorly understood hsp90 function. ATP/GTP binding likely occurs within the second FKBP-like domain, near the FK506 binding site on the FKBP template. A third domain detected by the hydrophobic cluster analysis method is distantly structurally related to the two first FKBP-like domains and is followed by the C-terminal part of the protein, which contains a calmodulin binding consensus sequence. Hsp binding immunophilin may be involved in a number of immunological, endocrinological, and chaperone-mediated pathways.

The primary sequence of the rabbit liver cDNA coding for protein p59 has been determined. The protein binds to the 90-kDa heat shock protein (hsp 90) and is associated with it, including when hsp 90 participates in hetero-oligomeric complexes of untransformed mammalian steroid receptors that sediment at 8-10 S. The cloned cDNA codes for an open reading frame of 458 amino acids defining a yet unknown protein. However, 55% amino acid homology to peptidyl-prolyl isomerase is found between amino acids 41 and 137, suggesting rotamase activity for p59, which speculatively may apply to bound hsp 90 and thus be implied in the intracellular trafficking of hetero-oligomeric forms of steroid hormone receptors. A polyclonal antibody derived from the COOH-terminal peptide 441-458 demonstrates a good affinity for rabbit, rat, and human "p59" protein. It interacts with at least one epitope, available in 8-10 S untransformed steroid receptor complexes and different from that recognized by the monoclonal antibody KN382/EC-1.

We have previously reported that mineralocorticosteroid receptor (MR) is a 8-9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8-9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

In the absence of hormonal ligand, inactive, heterooligomeric, 8-10S steroid receptor complexes include a p59 protein (apparent M(r) approximately 59 kDa) bound to th heat shock protein hsp90 (apparent M(r) approximately 90 kDa), which itself binds to the ligand binding domain LBD of the receptor molecule, p59 is thus an hsp binding immunophilin HBI, which, through its interaction with a chaperone, may intervene in several cellular functions. We report that, in cell-free experiments at 0 degrees C, FK506 and rapamycin do not release p59 nor hsp90 from the 9.5S rabbit uterus progesterone receptor, suggesting that the binding of p59 to hsp90 does not interfere with the rotamase site of HBI. There is no "transformation/activation" of the receptor, but an up to 2 fold increase in progesterone agonist and antagonist binding to the receptor is observed. It is suggested that a functional interaction between HBI and receptor activity may be mediated by hsp90.

Previous work demonstrated that the ligand binding domain (LBD) was required to determine the formation of the cytosolic, untransformed, inactive, 8 S, heterooligomeric form of the human glucocorticosteroid receptor (hGR) which includes the 90-kDa heat shock protein (hsp90) (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguère, V., Cadepond, F.M., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). Truncations of hGR deleting all or almost all of the LBD give GR derivatives in the non-hsp90-interacting 4 S form able to stimulate transcription in a hormone-independent manner. To identify the LBD subregion(s) involved in 8 S formation, we analyzed the sedimentation behavior of hGR mutants with various LBD internal deletions and/or truncations transiently expressed in cells that contain hsp90 but very low levels of endogenous GR, and we correlated the results with their transcriptional activity. LBD has been divided into three subregions: two of them, LBD1 (between amino acids 551 and 626) and LBD2 (between amino acids 627 and 696), include amino acid sequences highly conserved in the steroid receptor superfamily, and LBD3 consists of the carboxyl-terminal part of the molecule (amino acids 697-777). Each of these subregions can be deleted without impeding 8 S heterooligomer formation, and the corresponding receptors do not have transcriptional activity in the absence as well as in the presence of hormone. When linked to hGR mutants truncated after amino acids 532 or 550, each subregion does separately promote 8 S heterooligomeric complex formation and repress the intrinsic constitutive transcriptional activity of the truncated receptors. These 8 S complexes contain hsp90. In a control experiment, the linkage of 1,017 amino acids of beta-galactosidase to the carboxyl-terminal of 1-532 hGR gave a hybrid receptor still constitutively transcriptionally active which did not bind hsp90. These results provide evidence that there is a strong correlation between the association with hsp90 and the loss of GR functional properties and that hsp90 may play a critical role in maintaining the receptor in a nonfunctional state.

Hsp90 is a heat-shock protein constitutively expressed in most cells. Besides regulation by thermal stress, the expression of hsp90 is also positively regulated by developmental and mitogenic stimuli. The effect of serum and insulin on protein and hsp90 alpha-mRNA levels has been studied in the chicken hepatoma cell line DU249. The culture of cells in serum-free medium resulted in a decrease of hsp90 alpha-mRNA level. A transient increase was observed at 6-9 h after serum restimulation. The expression of hsp90 gene was also increased by insulin alone in a dose-dependent manner and was maximum between 6 and 9 h treatment. The insulin induced increase of hsp90 alpha-mRNA was suppressed by cycloheximide (10 micrograms/ml) but not by an inhibitor of DNA synthesis, demonstrating that this induction requires protein neosynthesis. In serum starved cells, other growth factors (IGF1, EGF and bFGF) showed a positive effect on hsp90 alpha-mRNA level which took place before DNA synthesis with the same time-course as that of insulin. With PDGF, the induction of hsp90 alpha-mRNA occurred earlier. The time interval between the maximum of hsp90 alpha-mRNA induction and that of DNA synthesis was the same for all growth factors studied. From these results, we conclude that growth factors acting via tyrosine kinase receptors up-regulate hsp90 alpha-mRNA level in a DNA synthesis independent manner, possibly in late G1.

Several mutants of the human estrogen receptor (ER) were transiently expressed in Cos 7 cells in order to determine the regions involved in the formation of complexes with the heat shock protein Mr approximately 90,000 (hsp 90). The formation of the cytosol non-DNA binding 8-9 S complexes (8-9 S ER) was monitored by glycerol gradient ultracentrifugation. It was established that the N-terminal region of the receptor, including the two zinc fingers of the DNA binding domain (DBD), is not required for the formation of the 8-9 S ER complexes. Conversely, deletion of the entire ligand binding domain (LBD) produced truncated receptor mutants that are constitutive transcriptional activators and did not form 8-9 S ER complexes, confirming results obtained previously with the glucocorticosteroid receptor (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguerre, V., Cadepond, F., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). However, no limited subregion of the LBD was found to be uniquely involved in hsp 90 binding. A highly positively charged region situated at the C-terminal extremity of the DBD (between amino acids 251 and 271) also appeared to be implicated. Although not sufficient, this sequence is necessary for the formation of the 8-9 S ER; it also corresponds to the NL1 nuclear localization domain of steroid receptors. Taken together, these results suggest that the formation of complexes with hsp 90 involves several receptor regions, and they are consistent with the proposal that hsp 90 inhibits receptor function and can be released by hormone binding to the LBD.

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.

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.

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.

A monoclonal antibody (BF4) has been used to characterize and purify the heat-shock protein of Mr approximately 90,000 (hsp 90) present in the chick oviduct. In low salt cytosol, the sedimentation coefficient of hsp 90 is approximately 6.8 S, the Stokes radius approximately 7.1 nm, and the calculated Mr approximately 204,000, thus suggesting a dimeric structure. In 0.4 M KCl cytosol, only slightly smaller values were determined (approximately 6.5 S, approximately 6.8 nm, and approximately 187,000). Following purification by ion exchange and immunoaffinity chromatography, hsp 90 migrated as a single silver-stained band at Mr approximately 90,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient 6.2 S, the Stokes radius approximately 6.8 nm, and the Mr approximately 178,000 confirmed the dimeric structure. However, in both antigen or antibody excess conditions, only one molecule of monoclonal antibody could be bound to the hsp 90 dimer. Whether steric hindrance in a homodimer or the presence of two different 90-kDa proteins in a heterodimer explains this result cannot yet be decided. The dimer is not dissociated by high salt (1 M KCl) or the chaotropic agent (0.5 M NaSCN), but is disrupted by 4 M urea, suggesting a stabilization of the structure by hydrogen bonds. The molybdate-stabilized progesterone receptor hetero-oligomer form of approximately 8 S sedimentation coefficient was purified, and its hsp 90 component was then released by salt treatment. It was found to sediment at approximately 5.8 S and have a Stokes radius approximately 7.1 nm, giving Mr approximately 174,000. This observation is consistent with a previous report suggesting from specific activity determination, scanning of polyacrylamide gels, and cross-linking experiments that each purified nontransformed progesterone receptor molecule includes one progesterone binding unit per two 90-kDa protein molecules (Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023). This work brings direct evidence that both free hsp 90 and the non-hormone binding hsp 90 component released from the nontransformed steroid receptor in the cytosol are in a dimeric form.

Antisteroid hormones compete for hormone binding at the receptor level and prevent the hormonal response. A new concept is proposed for explaining the antiglucocorticosteroid activity of RU 486 in the chick oviduct system. It is based on the ability of the antisteroid to stabilize the hetero-oligomeric 8S-form of the glucocorticosteroid receptor (GR), which involves the interaction of the 94k-receptor and heat-shock protein MW 90,000 (hsp 90). It is proposed that hsp 90 caps the DNA binding site of the receptor, and this prevents it from binding to the DNA of hormone regulatory elements (HRE) and increasing transcription of regulated genes. This paper reviews other antiglucocorticosteroid and antiestrogen systems with reference to this hypothesis and also describes a four-step analysis of the molecular mechanism of antisteroid hormone action at the receptor level.

We report cDNA sequence, the complete derived aa sequence, and a predicted secondary structure of the chick hsp 90, a protein which has been found to form complexes with steroid hormone receptors. The modelling of the most negatively charged "region A" indicates that the alpha-helices of this portion of hsp 90 mimick DNA configuration. We propose that this region can, in absence of hormone, interact with and cap the positively charged DNA-binding domain of steroid receptors.

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.

The sequence of the entire chick 90 kDa heat shock protein (hsp90), the non hormone binding component of the heterooligomeric form of steroid receptors, is reported. A comparison of the amino acid sequence of the chick hsp90 to that of the homologous hsp90 from yeast to man, reveals 64-96% identity respectively, and even with E. coli hsp90 an identity of 44% is observed. Analysis of the sequence and a secondary structure prediction of chick hsp90 suggest that two hydrophilic regions A and B, predicted in alpha-helix may play a role in the interaction of hsp90 with other proteins such as steroid hormone receptors. While there are regions of the sequences completely conserved in all hsps90, the most negatively charged hydrophilic region (A) is absent in the E. coli protein.

We report the nucleotide sequence of a 2652 bp derived from a chicken 90-kDa heat shock protein (hsp 90) genomic clone. This fragment contains 890 bp of the 5' flanking region and 1762 bp of structural gene sequence encoding the first 85 amino acids of the protein. The start site of transcription was determined by primer extension and RNase mapping. Two introns have been identified. The first intron presents two features in common with the unique intron of the hsp 83 of drosophila: its location just before the ATG initiation codon and its length of approximately 1.3 Kb. The 5' flanking region contains a TATAA element, a CCAAT box and several putative cis-regulatory elements that might account for the basal level of expression and developmental regulation of the gene. Functional analyses show that hsp 90 gene expression is constitutive and heat inducible and that a full heat shock response requires the cooperativity of two distinct blocks of overlapping heat shock response elements.

Antisteroid hormones compete for hormone binding at the receptor level and prevent the hormonal response. A new concept is proposed for explaining the antiglucocorticosteroid activity of RU 486 in the chick oviduct system. It is based on the ability of the antisteroid to stabilize the hetero-oligomeric 8S-form of the glucocorticosteroid receptor, which involves the interaction of the 94,000-dalton receptor and the heat-shock protein MW 90,000 (hsp 90). It is proposed that hsp 90 caps the DNA-binding site of the receptor, and this prevents it from binding to the DNA of hormone regulatory elements and to increase transcription of regulated genes. This paper reviews other antiglucocorticosteroid and antiestrogen systems with reference to this hypothesis, and also describes a four-step analysis of the molecular mechanism of antisteroid hormone action at the receptor level.

Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.

Anti-steroid hormones compete for hormone binding at the receptor level and prevent the hormonal response. A new parameter is proposed for explaining both anti-progesterone and anti-glucocorticosteroid activities of RU486, a synthetic derivative of high receptor affinity. It is based on its ability to stabilize the heterooligomeric 8S form of the glucocorticosteroid (in the chick oviduct system) and progesterone (in the rabbit uterus system) receptors. These 8S complexes involve the interaction of the approximately 94,000 and approximately 120,000 mol. wt receptors with the heat shock protein of the mol. wt approximately 90,000 (hsp 90). It is proposed that hsp 90 caps the DNA binding site of the receptor and that this would prevent it from binding to the DNA of hormone regulatory elements (HRE) and modifying transcription of regulated genes. In contrast, hormone agonists induce the dissociation of the oligomer, thus unmasking the functional DNA binding domain of the receptor. Whether other differences between agonist- and antagonist-receptor complexes are involved in the expression of hormone and anti-hormone effects also is discussed in this paper.

1,10-Phenanthroline inhibited the DNA-cellulose binding of the transformed calf uterus estrogen receptor (homodimer of 66-kDa molecules: 5 S estrogen receptor) in a temperature- and concentration-dependent manner. This result appears related to the metal-chelating property of 1,10-phenanthroline, since the inhibition was decreased by addition of Zn2+ and Cd2+, but not by Ca2+, Ba2+, or Mg2+ for which the affinity of the chelator is low. Only a slight inhibition was observed in the presence of the 1,7-phenanthroline, a nonchelating analogue. After dialysis or filtration to remove free 1,10-phenanthroline, DNA binding of the 5 S estrogen receptor was still inhibited. Conversely, the chelator was unable to release prebound 5 S estrogen receptor from DNA-cellulose. The 5 S estrogen receptor DNA binding was inhibited when 1,10-phenanthroline was present during the transformation to activated receptor of the hetero-oligomeric nontransformed 9 S estrogen receptor, in which the hormone binding subunits are associated with heat shock protein, Mr 90,000 (hsp 90) molecules. In contrast, if 1,10-phenanthroline was removed before the transformation took place, only a slight inhibition was observed. Other experiments with EDTA indicated a similar inhibition of DNA-cellulose binding by the 5 S estradiol receptor, and all metal ions chelated by this agent prevented its inhibitory effect. The results indicate that 1,10-phenanthroline inhibited the DNA binding of the transformed 5 S estradiol receptor by chelating metal ion tightly bound to the receptor, which is not accessible to the chelator when the receptor is bound to DNA or to hsp 90. Therefore, they suggest that the metal ion may play a critical role in the interaction with DNA and hsp 90 by maintaining the structural integrity of the implicated receptor domain.