The 59 kDa FK506-binding protein, a 90 kDa heat shock...

2037Journal of Cell Science 108, 2037-2051 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding

immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton

and mitotic apparatus

Martine Perrot-Applanat1,*, Christian Cibert2, Gérard Géraud2, Jack-Michel Renoir3

and Etienne-Emile Baulieu3

1INSERM U135 Hormones et Reproduction, Faculté de Médecine de Bicêtre, 78 rue du Général Leclerc, 94275 Kremlin-BicêtreCedex, France2CNRS UMR 9922, Institut Jacques Monod, T43, 2 place Jussieu, 75005 Paris, France3INSERM U33 Communications Hormonales, Faculté de Médecine de Bicêtre, 80 rue du Général Leclerc, 94276 Kremlin-BicêtreCedex, France

*Author for correspondence at present address: INSERM U 344, Endocrinologie Moléculaire, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard,75730 Paris Cedex 15, France

FKBP59-HBI, a 59 kDa FK506 binding protein whichbinds the 90 kDa heat shock protein hsp90 and thus is aheat shock protein binding immunophilin (HBI), was orig-inally discovered in association with unliganded steroidreceptors in their heat shock protein containing het-erooligomer form. It belongs to a growing family includingother FKBPs which bind the immunosuppressants FK506and rapamycin, and cyclophilins which bind cyclosporin A,all having rotamase (peptidyl-prolyl cis-trans isomerase)activity which may be involved in protein folding. Targetsfor drug-immunophilin complexes have been mostlystudied in vivo in T lymphocytes; however, immunophilinsare present in all cell types, where their role and distribu-tion are still unknown.

Here we report the localization of FKBP59-HBI invarious non lymphoid cells (mouse fibroblasts (L-929),monkey kidney cells (Cos-7), Madin-Darby canine kidneyepithelial cells (MDCK), and mouse neuronal cells (GT1)).Two polyclonal antipeptide antibodies directed against theC-terminal end (amino acids 441-458) (Ab 173) or thesequence 182-201 (Ab 790) of the FKBP59-HBI were usedin light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphasecells. Specific immunofluorescence was much stronger inthe cytoplasm than in the nucleus when using Ab 173, andstronger in the nucleus than in the cytoplasm with Ab 790.Detailed observations of L-cells, which have a particularlyflat morphology, showed a punctate as well as a fibrouscytoskeletal staining in the cytoplasm using antibody 173,a result which suggests interactions of FKBP59-HBI withan organized network. Colocalization experiments (usingantibodies against tubulin, vimentin or actin) and use ofcytoskeletal-disrupting drugs revealed partial association

of FKBP59-HBI with the microtubules. Western blotexperiments confirmed that the protein was present in thesubcellular fractions containing either ‘soluble’ proteinsreleased from cells exposed to NP40 detergent, or proteinsreleased from the cytoskeleton exposed to calcium ions (i.e.in microtubule depolymerizing conditions). Exposure ofcells to 1 µM FK506 and rapamycin for 1 hour did notmodify significantly the staining, although rapamycintreatment rendered the network stained by 173 clearlyvisible. Interestingly, during mitosis FKBP59-HBI segre-gated from the region of the chromosomes; it mainlylocalized with the mitotic apparatus (centrosome, spindleand interzone separating the chromosomes), the cleavagefurrow and the midbodies during cytokinesis. It appearedagain as a fibrous network in the cytoplasm of the twodaughters cells. In conclusion, the present immunocyto-chemical analysis documents the presence of FKBP59-HBIin different cell compartments, where the differentiallabelling with Ab 173 and Ab 790 suggests different con-formations of the protein or different interactions withother proteins which need to be clarified using biochemicalmethods. The role of FKBP59-HBI, as well as the roles ofother FKBPs present in the same cells, needs further inves-tigation. Preliminary immunofluorescence observationsshowed that FKBP12, a FK506 binding protein of 12 kDahomologous to the N-terminal region of FKBP59 (59%identity with amino acids 18-134), was only present in thecytoplasm associated with elements of the cytoskeleton, notconcentrated into the mitotic apparatus.

Key words: immunophilin, immunocytochemistry, cytoskeleton,mitosis

SUMMARY

2038 M. Perrot-Applanat and others

INTRODUCTION

A 59 kDa protein, p59, was originally detected in associationwith the progesterone receptor (PR) isolated from rabbit uterus(Nakao et al., 1985; Tai et al., 1986). It was later identified asa common component of non transformed progesterone,androgen, estrogen and glucocorticoid receptors from a varietyof sources (Renoir et al., 1990a; Sanchez et al., 1990; Rexin etal., 1992; Smith et al., 1993), as well as dioxin receptor(Perdew, 1988). It forms complexes with two chaperoneproteins involved in protein folding, the heat shock proteinshsp90 and hsp70 (Joab et al., 1984; Catelli et al., 1985; and seeBaulieu et al., 1989; Pratt et al., 1992; Perdew and Whitelaw,1991, for reviews). However, p59 does not bind to the receptoritself (Renoir et al., 1990a; Sanchez et al., 1990). Themechanism by which p59 may cooperate with two heat shockproteins to regulate steroid receptors is unknown. It has beenproposed that the association of p59, hsp90 and hsp70 withsteroid receptors maintains the receptors in an inactive statewhile binding of steroid dissociates the receptors from hsp90(see Baulieu et al., 1989, and Pratt et al., 1992, for reviews).

Recently, the cloning and sequencing of the rabbit uterinep59 (Lebeau et al., 1992; Callebaut et al., 1992) and biochem-ical experiments (Tai et al., 1992; Yem et al., 1992) have estab-lished that it is an immunophilin, binding both FK506 andrapamycin (Tai et al., 1992; Wiederrecht et al., 1992; Yem etal., 1992; Renoir et al., 1994). Immunophilins are ubiquitous,abundant and well conserved proteins; they have been identi-fied in bacteria, yeast and a variety of tissues (Gething andSambrook, 1992; Schreiber, 1992; Heitman et al., 1992, 1993).These proteins bind immunosuppressive drugs and may beclassified as cyclophilins, which bind to cyclosporin A, andFKBPs (FK506 binding proteins), which bind FK506 andrapamycin. These drugs are inhibitors of specific signal trans-duction patways that lead to T lymphocyte activation (seeRosen and Schreiber, 1992, for a review). In addition to havingimmunosuppressant binding properties, all immunophilinspossess a rotamase (peptidyl-prolyl cis-trans isomerase)activity, whose inhibition by binding to immunosuppressantsdoes not explain immunosuppressive activity (Rosen andSchreiber, 1992). It has also been speculated thatimmunophilins play a role in protein folding (Rosen andSchreiber, 1992; Schreiber, 1992) as well as in protein traf-ficking in the cell (Gething and Sambrook, 1992). Since thep59 protein binds immunosuppressants (FK506 andrapamycin) and hsp90, it has also been named p59/HBI (i.e.p59 heat shock protein binding immunophilin) or FKBP59-HBI. Indeed, the deduced amino acid sequences from rabbitand human p59/HBI cDNA (Lebeau et al., 1992; Peattie et al.,1992) revealed the existence of four domains, the first at the Nterminus of the protein bearing close homology to FKBP12(Callebaut et al., 1992), a smaller FK506-binding protein.FKBP59-HBI also binds at its C-terminal end to calmodulin(CaM) an intracellular receptor for Ca2+, which modulates theactivity of CaM-binding proteins (Massol et al., 1992), as wellas to ATP and GTP (Le Bihan et al., 1993).

The ubiquitous tissue distribution among mammalianspecies as well as the association of immunophilin FKBP59-HBI with heat shock proteins and steroid receptors promptedus to study the cellular localization of FKBP59-HBI in non-lymphoid cells. Preliminary immunohistochemical analysis

localized FKBP59-HBI to the cytoplasmic and/or the nuclearcompartment in T lymphocytes (Sanchez et al., 1990), rabbituterus (Gasc et al., 1990) or kidney cells (Ruff et al., 1992)using monoclonal or polyclonal antibodies (Nakao et al., 1985;Ruff et al., 1992). In the present study, we used two differentrabbit anti-peptide antibodies (173 and 790) directed againstthe N- and C-terminal ends of the protein for the detection ofFKBP59-HBI in fibroblasts (mouse L-929 and monkey Cos-7cells), epithelial (Madin-Darby canine kidney, MDCK) andneuronal (GT1) cells. Using conventional light microscopy andconfocal laser scaning microscopy, we showed that FKBP59-HBI was localized throughout the nucleus of interphase cells,with a punctate and fibrous cytoskeletal staining clearly visiblein the cytoplasm, using antibody 173. In mouse L929 cells,which have a particularly flat morphology (Perrot-Applanat etal., 1992), we have been able to compare the distribution ofFKBP59-HBI with those of cytoskeletal elements in variousconditions, including colocalization experiments, use ofcytoskeleton-disrupting drugs and western blot analysis. Wedetected a partial association of FKBP59-HBI with the micro-tubules using antibody 173. Interestingly, during mitosis theFKBP59-HBI labeling patterns showed an association with themitotic apparatus and the midbody during cytokinesis inaddition to the cytoplasmic region, in all the cell lines that werestudied. These various labeling patterns probably reflectdifferent conformations of this protein. Finally, since FKBP59includes in its N-terminal region the sequence of FKBP12, anabundant and well characterized member of the FKBP family,we have briefly compared the distributions of the two proteinsin the same cells.

MATERIALS AND METHODS

Cell cultureMouse L929 fibroblasts, obtained from the American Type CultureCollection (Bethesda, MD) were grown in Dulbecco’s modifiedEagle’s medium (DMEM) containing 10% fetal calf serum, asdescribed (Perrot-Applanat et al., 1992). Monkey Cos-7 fibroblastsand Madin-Darby canine kidney epithelial cells (MDCK) were grownin DMEM supplemented with 10% fetal calf serum. Mouse neuronalcells (hypothalamus, GT1) were obtained from Dr Weiner (Univer-sity of California Medical School, San Francisco, CA).

AntibodiesTwo different peptides corresponding to amino acids Lys441 toAla458 (COOH-terminal peptide), and to Leu182 to Pro201 (N-terminal peptide, within domain II; Callebaut et al., 1992; Renoir etal., 1993) of FKBP59-HBI were synthesized (Neosystem, Strasbourg,France) and coupled to KLH (keyhole limpet hemocyanin). Thesequence of the two peptides revealed no homology with other knownprotein sequences when the Genpro, Swissprot, NBRF and EMBLbanks were screened with the help of BLASTP and FASTA programs.Several multi injections (250 µg total coupled peptides) in Freund’scomplete adjuvant were performed subcutaneously on the back andin the lymphoid nodes of immature male New Zealand rabbits.Boosters were given 6 weeks after the previous injection withidentical amount of coupled peptides (in incomplete Freund’sadjuvant). Blood was collected from the marginal ear vein 10-12 daysfollowing the 2nd booster, and the IgG fraction was purified byammonium sulfate precipitation and DEAE-Sephacryl (Pharmacia)chromatography. The purified IgG containing either antibody 173 orantibody 790 was stored frozen at −70°C, in buffer (25 mM Tris-HCl,

2039Immunocytochemical localization of FKBP59

150 mM NaCl, pH 7.4). Antibody 173 was previously shown to cross-react with rat and mouse FKBP59-HBI, which is included in thehetero-oligomeric form of several steroid hormone receptors (Lebeauet al., 1992). Monoclonal antibody against recombinant humanFKBP12 (Kobayashi et al., 1993) was a gift from Dr Kobayashi(Fujisawa, Osaka, Japan).

Immunofluorescence studiesIndirect immunofluorescence staining was performed on fixed cells aspreviously described (Perrot-Applanat et al., 1992). In most experi-ments, cells were fixed in methanol at −20°C for 10 minutes. Fixationin 4% (w/v) paraformaldehyde in PBS, pH 7.4, at room temperature,followed by permeabilization with 0.2% Triton X-100 (v/v) (SigmaChemical) for 5 minutes, was also used. Fixed cells were preincubatedin goat serum diluted 1:40 (v/v) in PBS, then incubated with rabbitanti-FKBP59-HBI antibody 173 (125 µg/ml total IgG in PBS) orantibody 790 (80 µg/ml total IgG) for 12 hours at 4°C, and rinsedthree times over a period of 15 minutes. The cells were then incubatedfor 60 minutes with fluorescein isothiocyanate (FITC)-conjugatedsecondary antibody (goat anti-rabbit IgG), washed three times andmounted in PBS/glycerol. Controls were carried out with an identicalprocedure, except that the primary antibody was replaced with a nonimmune serum or PBS. Controls also included the incubation of cellswith antibodies 173 and 790 which had been previously saturated withpeptides 182-201, 441-458, or recombinant domains I-II of FKBP59-HBI (amino acids 1-269, 300 µg/ml). This recombinant protein wasproduced in Escherichia coli as a fusion protein with glutathione S-transferase (GST; Chambraud et al., 1993). Control experiments werealso performed in the presence of 1% KLH carrier protein.

Double immunofluorescence studies of FKBP59-HBI and compo-nents of the cytoskeleton were performed as previously described(Perrot-Applanat et al., 1992), using a monoclonal mouse anti-chickenbrain tubulin (dilution 1:300, Amersham), a polyclonal goat anti-vimentin (dilution 1:40, Miles Laboratories Inc, Naperville, IL).Microfilaments and stress fibers were visualized using either anti-actinantibodies (dilution 1:10, Biomedical Technologies, Stoughton, MA)or phalloidin (Molecular Probes Inc, Oregon). Second antibodiesincluded FITC-conjugated goat anti-mouse IgG and rhodamine-con-jugated goat anti-rabbit IgG (dilution 1:40; Nordic).

Confocal microscopyConfocal laser scanning microscopy was performed using a Bio-RadMRC-600 (Bio-Rad Laboratories, Palo Alto, CA), mounted on anOptiphot II Nikon microscope equipped with a 60× objective (planapochromatic, NA 1.4). An argon ion laser adjusted at 488 nm wave-length was used for the analysis of fluorescein, and a helium-neon ionlaser adjusted at 543 nm was used for the analysis of rhodamine, aspreviously described (Perrot-Applanat et al., 1992). The electronictrain of the microscope was adjusted to obtain the maximum dynamicrange (0-255) from each of the samples that we have observed.

Drug treatmentsFK506 and rapamycin were gifts from Drs K. Murato (Fujisawa,Osaka, Japan) and Sehgal (Wyeth-Ayerth, Princeton, NJ), respec-tively. The cell cultures were treated on the second to third day ofsubculture with either 10−6 M FK506 or 10−6 M rapamycin for 1 hour.Cells were then fixed and analyzed by indirect immunofluorescence.Cell cultures incubated in the absence of immunosuppressive drugswere used in control experiments. In a second set of experiments, cellswere incubated in the presence of 5 µM demecolcine (N-deacetyl-N-methyl-colchicine, colcemid) for 1 to 6 hours. This treatment resultsin the disappearance of the microtubules and the collapse of the inter-mediate filaments in these cells (Perrot-Applanat et al., 1992).

Cytosol preparation and western blot analysisFor cytosol preparation, 75×106 cells were homogenized with aglass/glass homogenizer in 500 µl of 10 mM Hepes, pH 7.6, con-

taining 10% glycerol, 2 mM DTT and 20 mM Na2WO4 (buffer A)plus a cocktail of protease inhibitors: PMSF (phenylmethylsulfonylfluoride; 0.3 mM), leupeptin (Sigma; 20 µM), aprotinin from bovinelung (Sigma; 0.5 i.u./ml), pepstatin A (Sigma; 0.25 µM), bacitracin(Sigma; 10 µg/ml) and benzamidine (Sigma; 2.5 µM). Ultracentrifu-gation (45 minutes at 45,000 rpm at 2°C) in a Ti-45 rotor allowedrecovery of the cytosoluble protein fraction (cytosol).

Electrophoresis of cytosols from mouse L929 fibroblasts, neuronalGT1, monkey Cos-7 fibroblasts and canine epithelial MDCK cellswas performed in 10% acrylamide gels according to Laemmli (1970).Controls included the purified rabbit recombinant FKBP59-HBI,expressed in E. coli and cleaved from GST with thrombin(Chambraud et al., 1993). Proteins were transferred to nitrocellulosemembranes (Schleicher & Schuell) with a horizontal X-Blot apparatus(Ceralabo, France). Non-specific sites were blocked with 10% non fatdried milk in PBS; membranes were incubated with specific anti-bodies 173 and 790, used at 10 µg purified IgG per ml PBS. TheFKBP59-HBI antibody complexes were revealed with the anti-rabbitVectastain kit (Biosys, Burlingame, CA) according to the manufac-turer’s instructions. Proteins determination was performed asindicated by Renoir et al. (1990a).

Immunopurification of β-tubulin and FKBP59 from L929cellsPurified IgGs of antibody 173 or the IgG fraction of the β-tubulin anti-bodies were coupled to Protein A-Sepharose (Pharmacia, Upsalla,Sweden, 50 µl of packed gel) at 1 mg/ml according to the method ofShneider et al. (1982). Control immunoadsorbents with rabbit and ratnon-immune IgGs were also synthesized. Portions of cytosol fromL929 cells (500 µl) were added to the immunoadsorbents (50 µl) andthe suspensions were rotated for 4 hours at 4°C. The gels were rinsedwith buffer A (5 ml) and buffer A containing 0.3 M NaCl (5 ml), andthen reequilibrated in buffer A; bound proteins were eluted with 50mM diethylamine, pH 10.5 (3×50 µl), for 1 hour at 4°C, and theeluates were immediately neutralized before western blot analysis aspreviously described.

Preparation of cytoskeletal fractions from detergent-extracted and Ca-treated cellsCytoskeletal fractions were prepared by extracting cells withdetergent under conditions which stabilize cytoskeleton, essentiallyaccording to the procedure of Salomon et al. (1979). L-cells (70.106)were washed twice with PBS, scraped with a rubber policeman andcentrifuged at 800 rpm for 10 minutes. The cell pellet was resus-pended for 10 minutes in extraction buffer A (0.1 M PIPES, 1 mMMgSO4, 2 mM EGTA, 2 M glycerol, pH 6.8) containing 0.2% NP40and the cocktail of protease inhibitors mentioned above. The nucleiand cytoskeleton (P1) were pelleted by centrifugation at 1500 g for10 minutes and the resulting supernatant of ‘soluble’ proteins (orcytoplasmic fraction, S1) was collected, boiled in Laemmli samplebuffer and stored at −20°C. The nuclear-cytoskeletal fraction (P1) waswashed twice by repeating the extraction procedure, then treated for10 minutes with buffer B (0.1 M PIPES, 1 mM MgSO4, 2 mM EGTA,2 M glycerol, pH 6) containing 0.2% NP40 and 12 mM CaCl2. Theresidual cytoskeleton fraction (P6, which lacks microtubules) waspelleted by centrifugation at 1500 g for 10 minutes, and the super-natant, S4, was collected. The pellet was washed twice with thecalcium containing extraction cocktail, using 100 vol per wash, andthen boiled in Laemmli sample buffer. Aliquots of S1, S4 and P6 wereanalyzed by SDS-PAGE.

L-cells were also extracted in monolayer as follows. Subconfluentcells were incubated for three sequential 30 second intervals in extrac-tion buffer A containing protease inhibitors and 0.2% NP40, and thenwashed with the same buffer. Soluble proteins were released duringextraction. These cells were further extracted under microtubuledepolymerizing conditions by adding 12 mM CaCl2 to the extractionbuffer, and then treated for immunofluorescence studies.


RESULTS

Specificity of the FKBP59-HBI antibodiesFig. 1 indicates that both 173 and 790 antibodies recognize aprotein migrating at ~55-56 kDa in L 929 cell cytosol (lanes2). Antibody 173 recognizes the same protein in canine MDCKcells and in GT1 mouse neuronal cells (lanes 4 and 5, respec-tively). With monkey kidney COS-7 cells, a doublet wasdetected with antibody 173, as has already been observed inthis species (Renoir et al., 1990a), and in IM-9 human lym-phocytes (Sanchez et al., 1990), with the mouse monoclonalEC1 obtained against purified rabbit FKBP59 (Nakao et al.,1985; Tai et al., 1986); both signals represent isoforms ofFKBP59. Antibody 790 reacts with a 55-56 kDa protein inMDCK cells as in L-cells (lanes 2 and 4), but it does notrecognize any protein at the FKBP59 position in the cytosolfrom COS-7 or from GT1 cells (Fig. 1B). However, thisantibody reveals FKBP59 in the eluate fraction obtained fromFK506-affinity-purification of COS-7 cell cytosol and of GT1cytosol (Renoir, unpublished results). Purified recombinantFKBP59-HBI was used as an internal standard (lanes 1).

As the protein recognized by antibody 173 has a Mr close tothat of tubulin and a distribution which we show below to berelated to the cytoskeleton and the mitotic spindle, it wasimportant to check the absence of cross-reactivity of theantibody with tubulin. Cytosol from L-cells was immunopuri-

Fig. 1. Antigenic specificities of antibodies 173 and 790. Aliquots(containing 20 µg total protein) of cytosol from L929 cells (lanes 2),COS7 cells (lanes 3), MDCK cells (lanes 4), and neuronal mouseGT1 cells (lanes 5) were boiled in denaturing Laemmli sample bufferand loaded on top of 10% SDS-PAGE gels. After electrophoresis andwestern blotting as indicated in Materials and Methods, specificproteins were revealed with the antibodies 173 (A) used at 10 µgpurified IgG/ml, and 790 (B) used at 6 µg purified IgG/ml. Theantigen/antibody complexes were revealed with the Vectastain Kit.Position of standards (Rainbow Stds, Amersham) of knownmolecular mass are indicated on the left of the figure. In lanes 1, 800ng of purified FKBP59-HBI expressed in E. coli as a fusion proteinwith GST and cleaved with thrombin (see Materials and Methods),was loaded. As already published (Chambraud et al., 1993), differentFKBP/GST products were obtained that corresponded to C-terminalproteolysis; this explains the presence of one or two signalsrecognized by Ab 173 and 790, respectively.

fied with antibody 173, and the eluate was analyzed on westernblot using either antibody 173 or monoclonal anti-β-tubulin.As shown in Fig. 2, the protein immunopurified by antibody173 has an apparent Mr that is similar to that of β-tubulin butit is not recognized by anti-β-tubulin antibodies. Similarly, theβ-tubulin purified from anti-tubulin immunoadsorbent is notrecognized by antibody 173. Thus, the anti-FKBP59-HBI 173does not cross react with tubulin. Similar conclusions wereobtained with antibody 790 (not shown).

Localization of FKBP59-HBI in L-cellsIndirect immunofluorescence, with both light microscopy andconfocal laser microscopy, and various fixation conditions (seeMaterials and Methods) were used. These studies wereperformed first in mouse L929 fibroblasts, since they have aparticularly flat morphology, and the nucleus and cytoplasm

Fig. 2. Lack of cross reactivity between Ab 173 and anti-tubulinantibodies. Portions of cytosol (500 µl) from L929 cells (75 106)were incubated (4 hours at 4°C) with 50 µl of differentimmunoadsorbents (Ab173 Protein A-Sepharose; anti-β-tubulin/Protein A-Sepharose; control non-immune rabbit IgG/ProteinA-Sepharose and control non-immune rat IgG/Protein A-Sepharose).After washing, proteins were eluted from the gels (in 180 µl totalvolume), as described in Materials and Methods. The eluates of eachgel were boiled in denaturing Laemmli sample buffer. Aliquots ofeluates were analyzed by western blotting using antibody 173 (10µg/ml) (A) or anti-β-tubulin antibody (B). (A) Lane 1, 50 µl (0.3 µgprotein) from control rabbit non-immune IgG-immunoadsorbent;lane 2, 30 µl (0.9 µg protein) from anti-β-tubulin immunoadsorbent;lane 3, 20 µl (0.3 µg protein) from anti-173 immunoadsorbent; lane4, 50 µl (0.83 µg protein) from anti-173 immunoadsorbent plus 30 µlfrom anti-β-tubulin immunoadsorbent. (B) Lane 1, 30 µl from anti-β-tubulin immunoadsorbent; lane 2, 50 µl from anti-173immunoadsorbent; lane 3, 30 µl from anti-β-tubulin plus 50 µl fromanti-173 immunogel and lane 4, 150 µl from (0.5 µg protein) controlanti-rat immunogel. The positions of the standards are indicated onthe left.


are quite distinct. Immunofluorescence was observed in inter-phase cells and mitotic cells, using antibodies 173 and 790.

ControlsThe specificity of the localization was ensured by the followingcontrols: fluorescence was absent if cells were incubated withpreimmune IgG, with second antibody alone or with theFKBP59-HBI-specific IgG (60-80 µg/ml), preincubated withpurified FKBP59-HBI or homologous peptide (10−6 to 5×10−6 M) (compare Fig. 3a and b, e and g). Fluores-cence was not modified if cells were incubated with antibodies173 or 790 in the presence of heterologous peptide (Fig. 3a andc, e and f) or in the presence of the carrier protein KLH (notshown). In addition, when the concentration of antibody 173decreased from 250 to 12 µg/ml, immunofluorescence succes-sively disappeared from the different compartments, asdescribed below (cytoplasmic network, 60 µg/ml; nuclei andmitotic apparatus, 40 µg/ml; and finally diffuse clusters, below20 µg/ml), suggesting either different affinities of the antibodyfor different pools of FKBP59-HBI or different concentrationsof the antigen in these pools.

Interphase cellsPositive staining was found in both the cytoplasm and nucleus.With antibody 173 (Fig. 4a,c-d), cytoplasmic staining wasclearly visible and was fibrous in nature, suggesting acytoskeletal localization of FKBP59-HBI. Punctate immuno-

Fig. 3. Specificity of the immunofluorescence localization of FKBP59-Hantibodies directed against peptides 441-458 or 182-201, respectively; (bwith peptide 441-458 (b), peptide 182-201 (c) and domain I+II (delineat790 antibodies (80 µg/ml) presaturated with peptide 441-458 (f), peptideperformed with an excess of 100-fold peptide (10−6 to 5 10−6 M); these ppeptide (compare a with b, and e with g) but not with the heterologous p

fluorescence was also observed, depending on the cell (Fig.4d). The network was more clearly visible in well spread cells(Fig. 4c). Confocal laser microscopy producing 0.5 µm thickoptical sections of the cell confirmed these observations anddemonstrated that the network was preferentially distributedclose to the substratum, independent of the perinuclearclusters (Fig. 5a-b). The distribution of antibody 790-directedimmunofluorescence, as shown in Fig. 3b, was nuclear andcytoplasmic without any obvious cytoskeletal localization.The nuclei were stained by the two antibodies 173 and 790;however, staining was more intense with antibody 790. Nostaining was observed in nucleoli. Confocal analysis demon-strated that the staining was present in all planes of thenucleus.

Although much is known about the function of FK506-binding proteins in T-cell activation, FK506 and rapamycinhave also recently been shown to be active in L-cells, wherethey potentiate the dexamethasone-induced transcription of areporter gene (Ning and Sanchez, 1993). Incubation of cellswith 10−6 M FK506 or rapamycin for 1 hour did not modifythe intensity of fluorescence in the cytoplasm or in the nucleus.In addition, FK506 treatment did not modify the punctate andfibrous cytoskeletal staining (Fig. 5c-d). Incubation of cellswith rapamycin resulted in changes of cell morphology andFKBP59-HBI staining in the cytoplasm: cells were very wellspread, and staining of the network observed using antibody173 was more clearly distinguishable (Fig. 5e-f).

BI in L cells. (a and e) Cells incubated with 173 (a) or 790 (e),c and d) cells incubated with 173 antibodies (60 µg/ml) presaturated

ed between amino acids 1 and 269) (d); f, g and h, cells incubated with 182-201 (g) and domain I + II (h). Pretreatment of antibodies wasretreatments completely prevented staining with the homologouseptide (compare a with c and e with f). Methanol fixation. ×400.


Mitotic cellsAs shown in Fig. 6, FKBP59-HBI specific fluorescence wasexcluded from the region of chromosome segregation inmitotic cells and it was also punctate as previously mentionedfor interphase cells. An intense immunofluorescence was asso-ciated with the mitotic apparatus during all steps of mitosis,using both antibodies 173 and 790. Using antibody 173,staining first appeared near the centrosomes as cells enteredprophase (Fig. 6a), whilst at metaphase staining was concen-trated in the pericentriolar regions, with strands of fluorescenceprojecting towards the chromosomes (Fig. 6b); and duringanaphase and telophase, staining was present between the polesand chromosomes (half spindles) and in the region betweenseparating chromosomes (interzone) (Fig. 6c-e). Throughoutcytokinesis, immunofluorescence was present in the cleavagefurrow (Fig. 6f), and in the two new daughter cells stainingwas found throughout the midbody and as a fibrous networkin the cytoplasm (Fig. 6g,h). A similar distribution of fluores-cence outside the chromosome segregation region wasobserved with antibody 790 (Fig. 6i-k). Fluorescence waspresent at the centrosome and the mitotic pole, but was not con-centrated at the interzone, the cleavage furrow and themidbody compared to that seen with antibody 173. Doubleimmunofluorescence analysis using anti-β-tubulin antibodiesby confocal microscopy reflects a distribution of staining byantibody 173 close to that of tubulin in mitotic cells (Fig. 7).FKBP59-HBI-specific immunofluorescence of the mitoticspindle persisted upon treatment of cells at 4°C, as is the casefor cold-stable microtubules (not shown).

Immunofluorescence staining of FKBP59-HBI in thecytoplasm of interphase L-cells: relation to thecytoskeleton The pattern of distribution of FKBP59-HBI in the L-cell

Fig. 4. Immunofluorescencelocalization of FKBP59-HBI ininterphase L-cells. Cells werefixed in methanol and stained asdescribed under Materials andMethods using antibody 173 (a,c-d) or 790 (b). Note thepredominant cytoplasmic stainingin a and the predominant nuclearand perinuclear staining in b.×400. In c and d, highermagnification (×800) of wellspread cells incubated withantibody 173 shows a filamentousnetwork (c) and punctateimmunostaining (d). The networkwas more clearly visible inmethanol-fixed cells than afterparaformaldehyde fixation (notshown).

cytoplasm using antibody 173 showed clear filamentous struc-tures and suggested the possibility that FKBP59-HBImolecules were interacting with an organized network (Fig. 8aand b). Various attempts were made to identify the nature ofthis network: colocalization experiments using antibodiesagainst components of the cytoskeleton (microtubules, micro-filaments and vimentin-intermediary filaments), use ofcytoskeleton-disrupting drugs and subcellular fractionation.

FKBP59-HBI (as shown in Figs 3 and 8) gave patternsclearly different from the typical staining of actin representedby stress fibers, subcortical actin and ruffles, as observed withanti-actin antibodies or with phalloidin (Fig. 8c). This obser-vation led us to suggest that FKBP59-HBI did not colocalizewith these forms of actin. In contrast, double-labeling experi-ments performed using anti-FKBP59-HBI and anti-tubulinantibodies showed coincidence in the fibrous staining patternsin approximately 50% of the cells (Fig. 8e-f). Incubation ofcells with 5 µM demecolcine for 5 hours, which resulted in acomplete disappearance of microtubules (Fig. 8g), also showedthe disappearance of the fibrous staining of FKBP59-HBI inmost cells (Fig. 8h). However, a few treated cells (less than10%) showed residual filamentous structures with antibody173 that were characterized by intensive perinuclear stainingwith coil-like fluorescence (Fig. 8i). Similarly, the vimentincontaining filaments appeared clumped and took up coiledforms (Fig. 8j).

The distribution of FKBP59-HBI staining was also studiedin L-cells extracted in monolayer with 0.2% NP40 in thepresence or absence of calcium ions, which depolymerizemicrotubules (Fig. 9C). When cells were extracted with NP40in the absence of calcium, diffuse immunofluorescence dis-appeared; the remaining immunofluorescence was distributedpreferentially in the perinuclear region and appeared fibroustowards the less dense edge of the cells. In mitotic cells,


immunofluorescence was still observed on the mitoticapparatus and midbodies. When cells were extracted in thepresence of calcium ions, the immunofluorescent networkappeared inconspicuous.

We use selective sequential extraction of L-cells in suspen-sion to corroborate these morphological observations. Threefractions were generated sequentially: (1) the soluble proteinsreleased from cells exposed to 0.2% NP40 under conditionswhich stabilize cytoskeleton (S1); (2) proteins released from

Fig. 5. Localization of FKBP59-HBI in L cells in the presence orabsence of immunosuppresivedrugs. Confocal laser microscopywas used. Cells were treated withFK506 (c-d) or rapamycin (e-f) (1µM for 1 hour), then fixed inmethanol and stained as describedin Materials and Methods, usingantibody 173. Control cellswithout treatment are shown in aand b. For each situation, thefigure shows two optical 0.5µmthick sections at 4 µmintervals, near the middle of thecell (a,c,e) or close to thesubstratum (b,d,f). Thismicrograph was created usingPhotoshop.

stabilized cytoskeleton exposed to calcium ions (S4, i.e. inmicrotubule depolymerizing conditions); and (3) proteinsremaining in the residual cytoskeleton after exposure tocalcium ions (P6, depleted of microtubules). Tubulin wasincluded in the first and second fractions (S1 and S4). The thirdfraction contained vimentin and actin (not shown). Westernblot analysis of these three fractions with antibody 173 (Fig.9A) revealed the presence of the antigen mainly in the fractionof soluble proteins and the fraction of proteins which were


released from the cytoskeleton when exposed to calcium ions.The third fraction of residual cytoskeleton which was depletedof microtubules (P6) contained FKBP59-HBI to a lesser extent.

Fig. 6. Immunofluorescence localization of FKBP59-HBI in mitotic L-cetreated with methanol and stained with antibody 173 (a-h) or 790 (i-p). (atelophase; (f,g,o) the two daughter cells are connected by a midbody. ×80(arrow) and p (×5,000). Note also the stained centrosome in i and j (arrow

From these results, we conclude that, in the cytoplasm of L-cells, FKBP59-HBI is partly soluble, and partly associatedwith the cytoskeleton. It is associated most probably with

lls, using confocal laser microscopy. Asynchronous cultures were,i,j) Prophase; (b,k) metaphase; (c,d and l,m) anaphase; (e,n)0. The midbody shown in g can be seen at higher magnification in hs).


microtubules, and to a lesser extent with other filamentousstructures, which appear to be resistant to colcemid (such asintermediary filaments).

FKBP59-HBI in other cellsWe also used antibodies 173 and 790 in immunofluorescenceand western blot experiments to examine the distribution andrelative expression of FKBP59-HBI in other cells. As seen inFig. 1, mouse GT1-I neuronal cells, canine kidney epithelialcells (MDCK) and monkey kidney fibroblasts (COS-7), inaddition to the L mouse fibroblasts discussed above, allcontained a ~55-56 kDa protein which was recognized byantibody 173. Immunofluorescence (Fig. 10) was alwaysobserved during mitosis in the mitotic apparatus. Usingantibody 173, an immunofluorescent fibrous network was alsoshown to be present in interphase cells, as is the case in L-cells.

Association of FKBP12 with the cytoskeleton in L-cellsThe distribution of FKBP12, another FK506-binding protein(12kDa) which is homologous to the N-terminal region ofFKBP59, was also analyzed by immunofluorescence in L-cells(Fig. 11). Cytoplasmic staining, with a clear fibrous networkwas observed. Confocal microscopy confirmed the absence ofFKBP12 in the nucleus. In mitotic cells, immunofluorescencewas punctate and no staining was present in the mitotic spindle,the interzone or the midbody, in contrast to the FKBP59-HBIimmunofluorescence observed with antibody 173 (comparewith Fig. 6).

Fig. 7. Colocalization of FKBP59-HBI and tubulin inmitotic cells, using confocal microscopy. Doubleimmunofluorescence with 173 (a and c) and anti-tubulin (b and d) was performed on the same cells, asdescribed in Materials and Methods. (a,b and c,d)Metaphase and anaphase cells, respectively. Themitotic apparatus was stained by both antibodies;additional staining in the perichromosomal region withantibody173 (arrow) was visible . Each micrographrepresents one optical 0.5 µm thick section without anytreatment. Bar, 5 µm.

DISCUSSION

The amino acid sequence of rabbit FKBP59-HBI defines threesuccessive domains related to FKBP12 (the first described andbest known FK506-binding protein of 12 kDa), as well as aputative calmodulin binding site located at the C-terminal endof the protein (Callebaut et al., 1992; Massol et al., 1992). TheN-terminal first FKBP12-like domain displays FK506 bindingand catalytic (prolyl cis-trans isomerase) activity (Callebaut etal., 1992; Lebeau et al., 1992; Peattie et al., 1992; Schreiber,1992; Chambraud et al., 1993). Here, we report for the firsttime the detailed immunocytochemical distribution ofFKBP59-HBI and a brief comparison with the localization ofFKBP12.

Most studies which focused on the molecular recognition ofimmunosuppressants by immunophilins and the biologicalconsequences of their interactions have been performed inlymphoid cells (Schreiber, 1992). The paucity of cytoplasm inT lymphocytes, which are pharmacological targets forimmunosuppressants, makes it difficult to explore in detailinteractions of FKBP59-HBI with structural cell componentssuch as the cytoskeleton. Since FKBP59 and other FKBPs suchas FKBP12 are ubiquitous and not limited to the immunesystem, we have studied the localization of these proteins innon lymphoid cells

Localization in L-cells and other cellsFKBP59-HBI localization was studied in L929 cells and COS-7 cells (fibroblasts), MDCK cells (kidney epithelial cells) andGT1 (neuronal cells). We used two antibodies (173 and 790)


directed against two different epitopes located in the C-terminal (amino acids 441-458), or in the N-terminal (aminoacids 181-201) domain of the protein, respectively. The speci-ficity of the anti-peptide antibody 173 has been previouslydescribed (Lebeau et al., 1992); it was extensively confirmedin this study, and that of 790 was established, by both westernblot analysis, using pure protein and crude cytosol, and in situsaturation experiments, using either the recombinant protein orvarious peptide fragments. Light microscopic examinationrevealed that FKBP59-HBI was present in the cytoplasm (aspunctate and fibrous cytoskeletal staining) and nucleus of all

Fig. 8. Cytoplasmic distribution of FKBP59-HBI in L cells: association phase-contrast of the same cell; (c) actin pattern; (d) vimentin pattern; (e(e) and anti-β-tubulin (f) antibodies; (g-i) immunofluorescence localizatiintermediate filament networks. L cells were treated with demecolcine (1(g), antibody 173 (h,i), or anti-vimentin antibodies (j). All morphologicawith demecolcine, were reversible after moving the cells to a normal me

cell types and species studied. Results obtained with twodifferent anti-peptide antibodies applied to the same cells inidentical experimental conditions revealed differential local-ization, depending on the antibody used. This observation wasconfirmed in several cell types.

The nuclear staining was preferentially observed withantibody 790 directed against an epitope located in domain II,close to but distinct (Le Bihan et al., 1993; Chambraud et al.,1993) from that of the monoclonal EC1 (Nakao et al., 1985;Tai et al., 1986) and similar to one of the epitopes of the poly-clonal antibody UPJ 56 used by Ruff et al. (1992). EC1 and

with microtubules. (a and b) Immunofluorescence of p59/HBI and and f) double-labeling immunofluorescence with anti-FKBP59-HBIon of FKBP59-HBI in the presence of drugs disrupting microtubules or0 µM, 5 hours), then fixed and stained with anti-β-tubulin antibodiesl changes, as well as FKBP59-HBI staining induced by the treatmentdium for 2 hours. ×400, except for e and f, ×650.


UPJ56 antibodies were also used in immunohistochemistry inorder to localize FKBP59-HBI (named hsp56 by Sanchez,1990), and several reports mentioned a dualcytoplasmic/nuclear distribution of the antigen, depending onthe cells (Sanchez et al., 1990; Gasc et al., 1990; Ruff et al.,

Fig. 10. Localization of FKBP59-HBI in various cell types. GT1neuronal cells from mouse hypothalamus (a), canine kidneyepithelial cells (MDCK) (b) and monkey fibroblasts Cos-7 (c) werefixed in methanol and incubated as described under Materials andMethods, using antibody 173. Note the presence ofimmunofluorescence in the mitotic apparatus (arrow) in a and b, andin the fibrous network of two interphase cells in c. ×570.CaCl2

CaCl2

1992). In the cytoplasm of L-cells, using cold methanolfixation, a fibrillar staining was preferentially observed withantibody 173, a distribution also previously observed with theUPJ 56 antibody (Ruff et al., 1992). Since the epitope locatedin the 441-458 domain is preferentially accessible in thecytoplasm and the epitope located in the 182-201 domain ispreferentially accessible within the nucleus, it is possible thatthere are different conformations of FKBP59-HBI in differentcell compartments, and/or interactions of the FKBP59-HBIwith different proteins in the cytoplasm and the nucleus.

Localization of FKBP59-HBI in the cytoplasmDetailed immunofluorescence of L-cells was performed usingphase-contrast confocal analysis, double-labeling immunoflu-

Fig. 9. Subcellular distribution of FKBP59-HBI during extraction inL 929 cells. (A and B) Western blot analysis. Aliquots (30 µg totalproteins) of soluble proteins obtained after extraction of L-cells withNP40 (S1) (see Materials and Methods), as well as of Ca2+-extractedproteins from the nuclear-cytoskeleton fraction (S4) and of residualcytoskeleton fraction (P6) were loaded on 10% SDS-PAGE gels.Immunoblotting was performed as described in Materials andMethods with anti-β-tubulin antibody (B) and antibody 173 (10 µgpurified IgG/ml; A). The positions of the molecular mass standardsare indicated on the left of the figure. (C) FKBP59-HBIimmunostaining of cells extracted in monolayer. Top, NP40-extracted cells (0.2% for 90 seconds); bottom, cells extracted withNP40 and CaCl2 (12 mM). Methanol fixation. ×400.


Fig. 11. Immunofluorescence localization of FKBP12 in L-cells.Cells were fixed in methanol and stained using a monoclonal anti-FKBP12 antibody (gift from Dr Kobayashi) and FITC-conjugatedanti-mouse IgG. Interphase (a) and mitotic cells (b,c) were analyzedby light microscopy. Confocal laser microscopy (d) confirmed theexclusive cytoplasmic localization of FKBP12.

orescence and western blotting in various conditions, includingthe use of detergents, high Ca2+ levels and cytoskeleton-dis-rupting drugs. A substantial proportion of the protein wasfound in a particulate form (vesicles) in interphase and mitoticcells, not associated with the fibrous network stained byantibody 173. In addition, if soluble components wereextracted by detergent lysis, most of the FKBP59-HBI stainingof the vesicles remained in interphase cells, with a distributiondifferent from that of the fibrous network. FKBP59-HBI wasfound to colocalize with microtubules and possibly othercytoskeletal elements not clearly identified. The significance ofsuch a localization of FKBP59 is unknown. hsp90, which canbind to FKBP59, may itself be associated with elements of thecytoskeleton (Koyasu et al., 1986; Sanchez et al., 1988;Redmond et al., 1989). As shown in this study, FKBP12staining gives a predominant fibrous network in L-cells. Cal-cineurin, a calmodulin Ca2+ dependent phosphatase whoseactivity is inhibited by FK506/FKBP12 complexes in T lym-phocytes (Liu et al., 1991), has also been described to be partlyassociated with the cytoskeleton (C. Klee, personal communi-cation), especially with microtubules of dendrites (Wood et al.,1980). Association of FKBP59-HBI with cytoskeletalelement(s) also occurs in the presence of FK506, as previouslymentioned by Ruff et al. (1992). Incubation of L cells with

rapamycin, an immunosuppressive drug which blocks cellcycle progression of lymphocytes in G1 (Morice et al., 1993)and inhibits the p70 S6 kinase (Chung et al., 1992; Price et al.,1992), results in cell spreading and a more clearly distinguablecytoskeletal network. However, whether this result indicates aclose interaction of rapamycin binding to FKBP59-HBI withthe cytoskeleton or depends on an independent effect ofrapamycin on the cell cycle remains unclear.

FKBP59-HBI as a nuclear proteinConfocal microscopy demonstrated that FKBP59-HBI, but notFKBP12, is located throughout the nucleus with the exceptionof the nucleoli. The presence of FKBP59-HBI has been docu-mented in the nuclei of both non lymphoid cells (Gasc et al.,1990) and Jurkat cells (Ruff et al., 1992). Unliganded proges-terone, estradiol and glucocorticosteroid receptors are presentin the nucleus of steroid target cells (King and Green, 1984;Welshons et al., 1984; Perrot-Applanat et al., 1985, 1992; Gascet al., 1984, 1990; Martins et al., 1991). The presence ofFKBP59-HBI in the nucleus of steroid target cells, like that ofhsp90 (Renoir et al., 1990b; Ruff et al., 1992; Kwang et al.,1994), which is a molecular chaperone shown to be part ofsteroid receptor heterocomplexes, suggests that FKBP59-HBImay play an active role in steroid receptor function. Recently,potentiation by FK506 of dexamethasone-induced glucocor-ticoid receptor mediated transcription has been reported inimmortalized L-cells LMCAT (Ning and Sanchez, 1993). Thisis consistent with the finding that immunophilin drugs, FK506and cyclosporin, inhibit the activity of nuclear transcriptionfactors for IL-2 gene activation (NFAT, AP3, NFIL, andNFkB) in T lymphocytes (Emmel et al., 1989; Matila et al.1990; Azhderian et al., 1993). It is thus suggested that immuno-suppressants may affect the function of important nuclearsignals not only in T lymphocytes but also in other cell types.

FKBP59-HBI and mitosisA striking observation of FKBP59-HBI localization is its con-centration in the mitotic apparatus (MA) throughout thevarious stages of mitosis and in the centrosome (pericentriolarregion) of interphase cells. Located in the spindle early inmitosis, it is concentrated at the interzone at anaphase andfinally localized to the midbody after cytokinesis. The speci-ficity of the antibodies has been discussed above. Results ofpresaturation analysis of antibodies with purified proteins andwith antigenic peptides (homologous or heterologous) excludethe possibility that this observation results in non specificantibody binding. Finally, the possibility of cross-reactivity ofthe polyclonal antibody with other proteins is also unlikely,from the absence of sequence homology with other knownpeptide sequence(s). Co-localization experiments of FKBP59-HBI and tubulin during mitosis show that both proteins arecomponents of the mitotic apparatus, with a close, but nottotally similar, distribution. FKBP59-HBI (which representsless than 0.1% of proteins) is a calmodulin (CaM)/Ca2+ bindingprotein (Massol et al., 1992) and could be phosphorylated invitro by calmodulin/Ca2+ kinase II (Le Bihan et al., 1993), anenzyme with broad substrate specificity (MAP-2, tau,vimentin, synapsin I) (Schulman, 1993). During mitosis, CaMkinase II and CaM are dynamic components of the mitoticapparatus (Ohta et al., 1990; Welsh et al., 1979). CaM kinaseII was found at the level of centrosomes and between the


spindle poles in metaphase and anaphase; during telophase,CaM kinase II was present at the midzone of the intercellularbridge between two daughter cells, a site where FKBP59-HBI,but not β-tubulin or CaM, was also found. CaM was also con-centrated within the mitotic spindle, with a preferential asso-ciation with kinetochore-to-pole microtubules (Welsh et al.,1979). In contrast to FKBP59, FKBP12, which has no CaM-binding site, does not associate with the mitotic apparatus. Theassociation of FKBP59-HBI with the mitotic apparatussuggests that it may play a role in the cell cycle progression ofmammalian cells, even if microinjections of Ab173 into inter-phase cells do not block mitosis in preliminary experiments.

A large number of FKBPs, all possessing peptidyl prolylisomerase activity, exist in eukaryotes including yeast, and inbacteria. FKBP13 is present in the endoplasmic reticulum (Jinet al., 1991; Nielsen et al., 1992; Partaledis et al., 1992).FKBP25 is found in the nucleus, associated with casein kinaseII and nucleolin (Jin and Burakoff, 1993) and in yeast, theNPI46 gene (also named FPR3; Manning-Krieg et al., 1994)encodes for a prolyl cis-trans isomerase within the nucleolus(Shan et al., 1994). FKBP33, recently cloned from Strepto-myces chrysomallus, was localized to cell membranes (Pahland Keller, 1994). These different cellular localizations ofFKBPs, in addition to the localization of FKBP59-HBI indifferent compartments including the cytoskeleton and thenucleus, may reflect functions of proline isomerase involved atthe cross-roads of protein folding, cell compartimentalization,signal transduction and immunosuppression (Gething andSambrook, 1992; Heitman et al., 1992).

In conclusion, we report the wide cellular distribution ofFKBP59-HBI. Clearly, further investigation is needed for anundestanding of the cellular function(s) of FKBP59-HBI. Inaddition, to having a possible important role in steroid receptorassembly and activity, it may be involved in several importantcellular functions, via association with proteins such as CaMor hsp90. The recent cloning and sequencing of Cyp40, a 40kDa cyclophilin-related protein (Kieffer et al., 1993; Ratajczaket al., 1993), revealed the homology of a 150 amino acid C-terminal segment in this protein with one in FKBP59-HBI,including three tetratrico-peptide repeats (TPR) terminated bya potential site for calmodulin interaction. Cyp40 is acomponent of the unactivated estrogen complex (Ratajczak etal., 1993). Interestingly, similar TPR units have recently beenidentified in a number of proteins involved in cell division, theheat shock response, protein transport and neurogenesis (seeKieffer et al., 1993). FKBP59-HBI and perhaps Cyp40 may beheat shock protein (hsp 90)-binding member(s) of a large TPRsuperfamily of proteins.

We thank Dr Kobayashi (Fujisawa, Osaka, Japan) and M.C. Lebeau(INSERM U33) for the gift of anti-FKBP12 antibodies, and BernardMaro (Institut Jacques Monod, Paris) for his comments on the man-uscript. This work was supported by the Institut National de la Santéet de la Recherche Médicale, the Centre National de la Recherche Sci-entifique and the Association pour la recherche sur le cancer (to J.-M.R., no. 6510).

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(Received 18 July 1994 - Accepted 11 January 1995)

The 59 kDa FK506-binding protein, a 90 kDa heat shock...

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