Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines.

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1 h, 42 degree C). In IGROV-1 cells, a low concentration (15 microg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27(Kip1) and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.

RU49953: a non-hormonal steroid derivative that potently inhibits P-glycoprotein and reverts cellular multidrug resistance.

Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux. However, their hormonal properties limit their potential for clinical trials. The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486. Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine. It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486. RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.

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

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

beta-Amyloid efflux mediated by p-glycoprotein.

A large body of evidence suggests that an increase in the brain beta-amyloid (Abeta) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Abeta, however, less is known regarding its secretion from cells. We now report that p-glycoprotein (p-gp), an ATP-binding cassette (ABC) transporter, is an Abeta efflux pump. Pharmacological blockade of p-gp rapidly decrease extracellular levels of Abeta secretion. In vitro binding studies showed that addition of synthetic human Abeta1-40 and Abeta1-42 peptides to hamster mdr1-enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Abeta peptides across the plasma membranes of p-gp enriched vesicles, and showed that this phenomenon was both ATP- and p-gp-dependent. Taken together, our study suggests a novel mechanism of Abeta detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.

Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein.

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor.hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Dexamethasone and triamcinolone acetonide accumulation in mouse fibroblasts is differently modulated by the immunosuppressants cyclosporin A, FK506, rapamycin and their analogues, as well as by other P-glycoprotein ligands.

In mouse fibroblasts (LMCAT cells) stably transfected with the reporter gene chloramphenicol acetyl transferase under the control of the mouse mammary tumor virus promoter (MMTV-CAT), cyclosporin A (CsA), FK506, and rapamycin (Rap) at micromolar concentrations potentiate dexamethasone- (Dex) induced CAT gene activity in a dose-dependent way (Renoir J.-M., Mercier-Bodard C., Hoffmann K., Le Bihan S., Ning Y. M., Sanchez E. R., Handschumacher R. E. and Baulieu E. E., Proc. Natl. Acad. Sci. U.S.A., 92, 1995, 4977-4981). In this work, we used LMCAT and 1471.1 cells, another mouse fibroblast cell line stably transfected with the MMTV-CAT construct, and found that exposure to immunosuppressants affected steroid-induced transcription differently. Indeed, all immunosuppressants, including inactive analogues, potentiated not only Dex- but also TA-induced CAT gene expression in LMCAT cells. The extent of this potentiation was 3 times lower for TA than for Dex. These immunosuppressants have no effect in 1471.1 cells. In addition, no difference of glucocorticosteroid affinity for the GR was observed in 1471.1 cells, in contrast to LMCAT cells. In both cell lines, the drugs tested increased [3H] Dex and [3H] TA (although to a lesser extent) accumulation. Since it is known that immunosuppressants can reverse the membrane Phospho-glycoprotein (P-gp) activity responsible for an active efflux of small hydrophobic molecules from numerous cell types, we therefore measured the relative efficiency of other P-gp ligands (including vinca alkaloids and the inactive CsA analogue, PSC833), on [3H] Dex and [3H] TA accumulation. In both cell lines, and depending on the drugs, reversal of Dex export was more pronounced than that of TA export (approximately 11 times in LMCAT and approximately 2 times in 1471.1 cells). However, the antiprogestin/antiglucocorticosteroid RU 38 486 and its 17beta derivatives RU 49 953 which does not bind to GR, both identified as strong reversal molecules of P-gp activity, had respectively, no and a strong inhibiting effect on steroid accumulation in both cell lines. These results suggest that a mechanism resembling but different from P-gp can modulate steroid entry into these mouse fibroblasts. This is confirmed by the failure to demonstrate the presence of P-gp by immunoprecipitation and Western blot experiments in membrane preparations from both cell lines. From these data, we conclude: (i) that the two synthetic GR ligands do not accumulate similarly in mouse fibroblasts, (ii) that RU 49 953 increases steroid efflux, in contrast to other agents known to reverse P-gp activity (iii) that cellular entry and export of Dex and TA can be modulated by membrane efflux mechanism(s), different from P-gp, and (iiii) that immunosuppressant potentiation of Dex- and TA-induced CAT activity involves such a mechanism in LMCAT cells. In 1471.1 cells, the lack of any enhancing effect upon steroid-induced transcription of all the drugs tested, although they all increase steroid accumulation, suggests involvement of immunosuppressant-influenced factor(s) acting downstream from steroid entry, in the hormone receptor-mediated transcription pathway(s).

Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells.

The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR species extracted from cells treated with progestin only. The nuclear 9S-[3H]Org2058-PR resulting from cells exposed to Rap, contained, in addition to the heat shock proteins of 90 kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52 but not FKBP51, although the latter was part of unliganded PR heterocomplex associated with hsp90. These results suggest that Rap and KN62 act upon the PR by distinct mechanisms, with only Rap impeding progestin-induced PR transformation. FKBP51 appeared to dissociate from the receptor heterocomplex, but not from hsp90, after hormone binding to PR in vitro and in vivo, whether in the presence or not of Rap and KN62. Immunoprecipitation experiments distinguished two PR- and glucocorticosteroid (GR)-associated molecular chaperone complexes, containing hsp90 and hsp70 and FKBP52 or FKBP51. Another complex identified in T47D cytosol contained hsp90 and the cyclosporin A-binding cyclophilin of 40 kDa, CYP40, but not hsp70, PR, or GR. These observations support the concept that FKBP51 and FKBP52 can act as regulators of Rap and FK506 activity upon PR and GR-mediated transcription, a mechanism that could be also regulated by type II and/or type IV CaMKs.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52.

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.

Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.

We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus.

The FK506-binding immunophilin hsp56 (FKBP52) is one of several chaperone proteins associated with untrasformed steroid receptors in a multiprotein heterocomplex. The function of heat shock protein 56 (hsp56) with respect to receptor action is unknown. hsp56 is not required for glucocorticoid receptor heterocomplex assembly or for proper folding of the receptor hormone-binding domain into a high affinity steroid-binding conformation. In intact cells, the majority of the hsp56 is located in the nucleus, with a minority colocalizing with microtubules in the cytoplasm. hsp56 contains a conserved negatively charged domain that we speculate might serve as a nuclear localization signal recognition sequence. Here we show that injection of an antibody raised against this negative sequence into intact L cells impedes subsequent dexamethasone-mediated shift of the glucocorticoid receptor into the nucleus. Nonimmune rabbit serum and an antibody raised against another site on hsp56 do not affect receptor movement. Inhibition of receptor movement by the 419 antibody against the negative sequence is blocked by preincubation with purified hsp56, but not by preincubation with purified hsp90, hsp70, or BSA. These observations are consistent with the possibility that hsp56 is involved in receptor trafficking to the nucleus, possibly functioning as the nuclear localization signal recognition protein. Receptor trafficking to the nucleus is not affected by FK506, indicating that the peptidylprolyl isomerase activity of hsp56 is not involved.

Cyclosporin A potentiates the dexamethasone-induced mouse mammary tumor virus-chloramphenicol acetyltransferase activity in LMCAT cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression.

As previously observed for FK506, we report here that cyclosporin A (CsA) treatment of mouse fibroblast cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid (LMCAT cells) results in potentiation of dexamethasone (Dex)-induced CAT gene expression. Potentiation by CsA is observed in cells treated with 10-100 nM Dex but not in cells treated with 1 microM Dex, a concentration of hormone which results in maximum CAT activity. At 10 nM Dex, 1-5 microM CsA provokes an approximately 50-fold increase in CAT gene transcription, compared with transcription induced by Dex alone. No induction of CAT gene expression is observed in cells treated with CsA or FK506 in the absence of Dex. The antisteroid RU 486 abolishes effects obtained in the presence of Dex. Using a series of CsA, as well as FK506, analogs, including some devoid of calcineurin phosphatase inhibition activity, we conclude that the potentiation effects of these drugs on Dex-induced CAT gene expression in LMCAT cells do not occur through a calcineurin-mediated pathway. Western-blotting experiments following immunoprecipitation of glucocorticosteroid receptor (GR) complexes resulted in coprecipitation of GR, heat shock protein hsp90 and two immunophilins: the FK506-binding protein FKBP59 and the CsA-binding protein cyclophilin 40 (CYP40). Two separate immunophilin-hsp90 complexes are present in LMCAT cells: one containing CYP40-hsp90, the other FKBP59-hsp90. Thus, both FKBP59 and CYP40 can be classified as hsp-binding immunophilins, and their possible involvement as targets of immunosuppressants potentiating the GR-mediated transcriptional activity is discussed.

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.

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)

Effects of immunosuppressants FK506 and rapamycin on the heterooligomeric form of the progesterone receptor.

The non-DNA binding form of the rabbit uterus cytosol progesterone receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M(r) 90kDa (hsp90), a Heat shock protein Binding Immunophilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosuppressants FK506 and Rapamycin (RAP) and belongs to the FK506 binding protein family. A recombinant p59/HBI-glutathione-S-transferase fusion protein, purified by Sephadex LH-20 filtration of tritiated drug-p59/HBI complexes, binds FK506 and RAP with apparent Kd values of 75 +/- 40 and 40 +/- 15 nM, respectively. Immunopurification from cytosol of [3H]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded "9S"-PR species in which hsp90, hsp70 and p59/HBI were present. In the absence of tungstate ions, only the 4-6S PR was eluted, and Western blot analysis demonstrated the absence of hsps and p59/HBI. In contrast 30 to 50% of the original 9S-PR species containing hsps and p59/HBI, was eluted in the absence of tungstate ions but after exposure of cytosol to 5 microM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25 degrees C with 0.05 to 10 microM FK506 or RAP, then with [3H]steroids (the agonist [3H]Org 2058 or the anti-progestin [3H]RU486), contains greater amounts of 9S-PR species than that detected in non-immunosuppressant exposed control cytosol. Scatchard analysis showed an up to 2-fold decrease of the Kd value for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on immobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59/HBI associated to PR units. Altogether, these results suggest that binding of immunosuppressants to p59/HBI does not promote hsps dissociation from the receptor and, as a consequence, that inhibition of peptidyl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumption that hsp90 binds to receptor and that p59/HBI binds hsp90 but not directly to receptor, immunosuppressants affect hormone binding by an unknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the EC1 antibody specific for several mammalian p59/HBI, also binds to FK506 columns and can be eluted by exchange with either FK506 or RAP, suggesting that there is an avian HBI homolog.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunological identification of a 50 kDa Mr FK506-binding immunophilin as a component of the non-DNA binding, hsp90 and hsp70 containing, heterooligomeric form of the chick oviduct progesterone receptor.

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)

The mammalian heat shock protein binding immunophilin (p59/HBI) is an ATP and GTP binding protein.

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.

A novel monoclonal anti-rabbit hsp90 antibody: usefulness for studies on hsp90-steroid receptor interaction.

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.

Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein.

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.

An immunophilin that binds M(r) 90,000 heat shock protein: main structural features of a mammalian p59 protein.

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.

P59, an hsp 90-binding protein. Cloning and sequencing of its cDNA and preparation of a peptide-directed polyclonal antibody.

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.

[Effect of immunosuppressants FK506 and rapamycin on the function of progesterone receptor: protein "p59-HBI", intersection between immunology and endocrinology?]. / Un effet des immunosuppresseurs FK506 et rapamycine sur le fonctionnement du récepteur de la progestérone: la protéine "p59-HBI", un carrefour de l'immunologie et de l'endocrinologie?

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.