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In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ERalpha polyubiquitination, increases the level of ERalpha protein, and enhances ERalpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERalpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERalpha, but eliminates the effects of RAP80 on ERalpha polyubiquitination, the level of ERalpha protein, and ERalpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERalpha as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERalpha activity.

The structurally related natural products rapamycin and FK506 bind to the same intracellular receptor, FKBP12, yet the resulting complexes interfere with distinct signalling pathways. FKBP12-rapamycin inhibits progression through the G1 phase of the cell cycle in osteosarcoma, liver and T cells as well as in yeast, and interferes with mitogenic signalling pathways that are involved in G1 progression, namely with activation of the protein p70S6k (refs 5, 11-13) and cyclin-dependent kinases. Here we isolate a mammalian FKBP-rapamycin-associated protein (FRAP) whose binding to structural variants of rapamycin complexed to FKBP12 correlates with the ability of these ligands to inhibit cell-cycle progression. Peptide sequences from purified bovine FRAP were used to isolate a human cDNA clone that is highly related to the DRR1/TOR1 and DRR2/TOR2 gene products from Saccharomyces cerevisiae. Although it has not been previously demonstrated that either of the DRR/TOR gene products can bind the FKBP-rapamycin complex directly, these yeast genes have been genetically linked to a rapamycin-sensitive pathway and are thought to encode lipid kinases.

In addition to their application in organ transplantation, immunosuppressive drugs are valuable tools for studying signal transduction in eukaryotic cells. Using affinity chromatography, we have purified immunosuppressive drug receptors (immunophilins) from fava bean. Proteins belonging to both major classes of the immunophilin family identified from animal sources [FK506- and rapamycin-binding proteins (FKBPs) and cyclophilins] were present in this higher plant. FKBP13, the most abundant FKBP family member in leaf tissues, was not detected in root tissues, whereas other FKBPs were present in both tissues. While the abundance of cyclophilin A in leaves was similar to that in roots, cyclophilin B/C was expressed at a much higher level in leaf tissues than in root tissues. Subcellular localization of immunophilins in mesophyll cells showed that chloroplasts contained FKBP13 and cyclophilin B/C but not other members, which explains the preferential expression of these two proteins in leaves over roots. The abundance of chloroplast-localized immunophilins, FKBP13 and cyclophilin B/C, was regulated by light. Although etiolated leaves produced detectable levels of cyclophilin B/C, they did not express FKBP13. Illumination of etiolated plants dramatically increased the expression of both FKBP13 and cyclophilin B/C. The light-induced expression of FKBP13 is closely correlated with the accumulation of chlorophyll in the leaf tissue. Our findings suggest that FKBP13 and cyclophilin B/C may play a specific role in chloroplasts.

Avian progesterone receptor complexes contain five major co-purifying proteins, including hsp90, hsp70, and a 23-kDa protein. The other receptor-associated proteins, p50 and p54, share amino acid sequence similarities with a 52-59-kDa component (p59, hsp56, or FKBP52) of mammalian steroid receptor complexes that is also a member of the FK506-binding proteins (FKBP) family of immunophilins. We show here that p50, but not p54, cross-reacts with a rabbit antiserum prepared against human FKBP52. Both p50 and p54 bind an FK506 affinity resin at 0.5 M KCl, but only p50 binds efficiently in low salt conditions. Glycerol density gradient analyses show that both p50 and p54 exist predominantly in oligomeric complexes at low ionic strength. The poor retention of p54 on FK506 resin at low ionic strength compared with the high retention of p50 suggests that these proteins may largely exist in separate complexes and may interact with other proteins, such as progesterone receptor, in distinctive manners. In HeLa cell extracts, a 55-kDa FK506-binding protein, distinct from FKBP52, cross-reacts with anti-p54 antibody FF1. We conclude that p50 is avian FKBP52, whereas p54 and the 55-kDa human FF1 antigen are FKBP54, a novel immunophilin.

Upon entering a cell the natural product rapamycin, like the structurally related immunosuppressant FK506, associates with members of the FKBP family of proteins. One or more of the resulting FKBP-rapamycin complexes blocks signaling pathways emanating from some growth factor receptors. Recently, the addition of rapamycin was shown to inhibit the phosphorylation and activation of a 70-kDa ribosomal S6 protein kinase, which normally occurs minutes after the activation of certain cytokine and growth factor receptors. We now report that rapamycin can be added 4 to 6 h after the addition of serum growth factors to quiescent human osteosarcoma cells and still arrest these cells in G1. This window of action correlates with the inducible appearance of a cyclin-dependent kinase (cdk) activity, and the induction of this activity is inhibited by the addition of rapamycin. Furthermore, p36cyclin D1 associates with this cdk protein complex in lysates of untreated cells, but does not associate with this cdk protein complex in lysates of rapamycin-treated cells. Together, these studies demonstrate that FKBP-rapamycin can modulate a cyclin-dependent kinase activity and a cyclin D1-cdk association during early G1 in MG-63 human osteosarcoma cells.

P59 [also known as FK506 binding protein 59 (FKBP59) or heat shock protein 56 (hsp56)] and heat shock proteins 90 and 70 (hsp90 and hsp70) associate with steroid receptors and are believed to maintain the receptors in an inactive state. Recently, we showed that p59 purified from human lymphocytes is an immunophilin (FKBP59) which binds both FK506 and rapamycin. It was also demonstrated that immunosuppressant-FKBP59 complexes associate with hsp90, hsp70, and the glucocorticoid receptor [Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. Here we provide evidence that rabbit uterine p59 also binds FK506 and rapamycin and that p59 or its homologue is associated with nontransformed progesterone receptors of rabbit uterus and chicken oviduct. This suggests that the immunophilin-heat shock protein-steroid receptor interaction is ubiquitous and not limited to immune systems. A FKBP59 homologue complexed with hsp90-hsp70 was also detected in yeast, which suggests that the immunophilin-heat shock protein association has been evolutionarily conserved. In addition, we found that the FKBP59-hsp complexes are more complicated than previously thought, involving other proteins such as actin and a 63-kDa protein, p63. The association of p63 to the p59 complex was inhibited by FK506 and rapamycin, suggesting that p63 could be a potential target for the immunosuppressive actions of these two drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.

Immunophilins, a family of proteins that exhibit rotamase (peptidyl-prolyl cis-trans isomerase) activity in vitro, are expressed in many organisms and most tissues. Although some immunophilins can mediate the immunosuppressive actions of FK506, rapamycin, and cyclosporin A, the physiological role of the unligated proteins is not known. A 59-kilodalton member of the FK506- and rapamycin-binding class was found to associate in the absence of these drugs with two heat shock proteins (hsp90 and hsp70) and the glucocorticoid receptor (GR). Together, these proteins make up the inactive GR, thus biochemically linking two families of proteins proposed to be involved in protein folding and assembly as well as two potent immunosuppressive modalities.

Calcineurin, a Ca2+, calmodulin-dependent protein phosphatase, was recently found to bind with high affinity to two different immunosuppressant binding proteins (immunophilins) with absolute dependence on the presence of the immunosuppressants FK506 or cyclosporin A (CsA) [Liu et al. (1991) Cell 66, 807-815]. The binding affinities of the immunophilin-drug complexes toward calcineurin and the stoichiometry of the resultant multimeric complexes have now been determined, and structural elements of FK506, CsA, and calcineurin that are critical for mediating their interactions have been identified. Analogues of FK506 (FK520, FK523, 15-O-demethyl-FK520) and CsA (MeBm2t1-CsA and MeAla6-CsA) whose affinities for their cognate immunophilins do not correlate with their immunosuppressive activities have been prepared and evaluated in biochemical and cellular assays. We demonstrate a strong correlation between the ability of these analogues, when bound to their immunophilins, to inhibit the phosphatase activity of calcineurin and their ability to inhibit transcriptional activation by NF-AT, a T cell specific transcription factor that regulates IL-2 gene synthesis in human T cells. In addition, FKBP-FK506 and CyP-CsA do not inhibit members of the PP1, PP2A, and PP2C classes of serine/threonine phosphatases. These data suggest that calcineurin is the relevant cellular target of these immunosuppressive agents and is involved in Ca(2+)-dependent signal transduction pathways in, among others, T cells and mast cells.

The 12-kDa FK506-binding protein (FKBP-12) is a cytosolic receptor for the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and subcellular localization of a 13-kDa FKBP (FKBP-13), which has a 21-amino acid signal peptide and appears to be membrane-associated. Although no internal hydrophobic region, and thus no transmembrane domain, is apparent within the 120 amino acids of mature FKBP-13, a potential endoplasmic reticulum retention sequence (Arg-Thr-Glu-Leu) is found at its C terminus. FKBP-13 has 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12; the N-terminal sequences are divergent, but the 92-amino acid C-terminal sequence of FKBP-13 has 46 identical and 20 related residues when compared with FKBP-12. The conserved residues that comprise the drug binding site and rotamase active site of FKBP-12 are completely conserved in FKBP-13. Therefore, the three-dimensional structures of FKBP-12 and the FKBP-12/FK506 complex are likely to be excellent models of the corresponding FKBP-13 structure.

Investigations of the actions and interactions of the immunophilin ligands FK506, cyclosporin A (CsA), rapamycin, and 506BD suggest that complexes of FK506 with an FK506-binding protein or of CsA with a cyclophilin (CsA-binding protein) inhibit the T-cell receptor-mediated signal transduction that results in the transcription of interleukin 2. Now we report an identical spectrum of activities of FK506, CsA, rapamycin, and 506BD on IgE receptor-mediated signal transduction that results in exocytosis of secretory granules from the rat basophilic leukemia cell line RBL-2H3, a mast cell model. Both FK506 and CsA inhibit receptor-mediated exocytosis (CsA IC50 = 200 nM; FK506 IC50 = 2 nM) without affecting early receptor-associated events (hydrolysis of phosphatidylinositol, synthesis and release of eicosanoids, uptake of Ca2+). In contrast, rapamycin and 506BD, which share common structural elements with FK506, by themselves have no effect on IgE receptor-mediated exocytosis. Both compounds, however, prevent inhibition by FK506 but not by CsA. Affinity chromatography with FK506, CsA, and rapamycin matrices indicates that the same set of immunophilins present in RBL-2H3 cells have been found in Jurkat T cells and calf thymus; however, the relative amounts of these proteins differ in the two cell types. These results suggest the existence of a common step in cytoplasmic signaling in T cells and mast cells that may be part of a general signaling mechanism.

The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. (1989) Nature (London) 337, 476-478)] involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.