The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo
2003; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.1093/emboj/cdg108
ISSN1460-2075
AutoresDaniel L. Riggs, Patricia J. Roberts, Samantha C. Chirillo, Joyce Cheung‐Flynn, Viravan Prapapanich, Thomas Ratajczak, Richard F. Gaber, Didier Picard, David F. Smith,
Tópico(s)Transgenic Plants and Applications
ResumoArticle3 March 2003free access The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo Daniel L. Riggs Daniel L. Riggs Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Patricia J. Roberts Patricia J. Roberts Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Samantha C. Chirillo Samantha C. Chirillo Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Joyce Cheung-Flynn Joyce Cheung-Flynn Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Viravan Prapapanich Viravan Prapapanich Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Thomas Ratajczak Thomas Ratajczak Western Australian Institute for Medical Research, University of Western Australia, The Queen Elizabeth II Medical Centre, Nedlands, WA, 6009 Australia Search for more papers by this author Richard Gaber Richard Gaber Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL, 60208 USA Search for more papers by this author Didier Picard Didier Picard Department of Cell Biology, University of Geneva, 1211 Geneva 4, Switzerland Search for more papers by this author David F. Smith Corresponding Author David F. Smith Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Daniel L. Riggs Daniel L. Riggs Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Patricia J. Roberts Patricia J. Roberts Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Samantha C. Chirillo Samantha C. Chirillo Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Joyce Cheung-Flynn Joyce Cheung-Flynn Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Viravan Prapapanich Viravan Prapapanich Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Thomas Ratajczak Thomas Ratajczak Western Australian Institute for Medical Research, University of Western Australia, The Queen Elizabeth II Medical Centre, Nedlands, WA, 6009 Australia Search for more papers by this author Richard Gaber Richard Gaber Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL, 60208 USA Search for more papers by this author Didier Picard Didier Picard Department of Cell Biology, University of Geneva, 1211 Geneva 4, Switzerland Search for more papers by this author David F. Smith Corresponding Author David F. Smith Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA Search for more papers by this author Author Information Daniel L. Riggs1, Patricia J. Roberts1, Samantha C. Chirillo1, Joyce Cheung-Flynn1, Viravan Prapapanich1, Thomas Ratajczak2, Richard Gaber3, Didier Picard4 and David F. Smith 1 1Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, 85259 USA 2Western Australian Institute for Medical Research, University of Western Australia, The Queen Elizabeth II Medical Centre, Nedlands, WA, 6009 Australia 3Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL, 60208 USA 4Department of Cell Biology, University of Geneva, 1211 Geneva 4, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1158-1167https://doi.org/10.1093/emboj/cdg108 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. Introduction Steroid receptors require continuous interactions with molecular chaperones to establish and maintain competence as ligand-dependent transcription factors (Pratt and Toft, 1997; Cheung and Smith, 2000). Studies of the assembly of receptor–chaperone complexes in cell-free systems have revealed an ordered dynamic pathway involving the obligate participation of major chaperone components (Hsp40, Hsp70 and Hsp90) and various co-chaperones. Native Hsp90–receptor complexes typically contain one of the Hsp90-bound immunophilins: the FK506-binding proteins FKBP52 and FKBP51, the cyclosporin A-binding protein cyclophilin 40 (CyP40) or the protein phosphatase PP5. A common feature of these co-chaperones is a tetratricopeptide repeat (TPR) domain, which forms the Hsp90-binding site, linked to another functional domain, a peptidylprolyl isomerase/drug-binding domain in the FKBPs and CyP40 or a protein phosphatase domain in PP5. In cell-free assembly reactions, the immunophilins are not required for receptors to gain hormone-binding ability (Dittmar and Pratt, 1997; Kosano et al., 1998), but immunophilins do influence steroid response pathways. Receptor-associated FKBP52 (Czar et al., 1995; Galigniana et al., 2001 and references therein) and PP5 (Dean et al., 2001) may play a role in nuclear targeting of glucocorticoid receptor (GR). Overexpression and structural alterations of FKBP51 may largely account for the cortisol insensitivity observed in many New World primates (Reynolds et al., 1999; Scammell, 2000). Additionally, human FKBP51 can lower GR hormone binding and transactivation (Denny et al., 2000) and is transcriptionally upregulated by glucocorticoids (Baughman et al., 1995). Still, detailed analysis of the function of individual immunophilins in vertebrate cells is confounded by the co-expression of several immuno philins at relatively high levels, and so we sought an alternative model system. Genetic approaches to analyzing the roles of chaperones in steroid hormone signaling have favored the genetically tractable yeast Saccharomyces cerevisiae. Although yeasts do not express steroid receptors, co-transformation of S.cerevisiae with cDNAs for a steroid receptor and a suitable hormone-responsive reporter is sufficient to establish a biological response (Schena and Yamamoto, 1988). Selected gene disruptions in S.cerevisiae have provided some of the best evidence for the physiological roles of Hsc82/Hsp90 (Picard et al., 1990a; Bohen and Yamamoto, 1993; Nathan and Lindquist, 1995), Ydj1/Hsp40 (Caplan et al., 1995; Kimura et al., 1995) and the co-chaperones Sti1/Hop (Chang et al., 1997) and Sba1/p23 (Freeman et al., 2000) in steroid hormone signaling. In another case, disruption of the gene for Cpr7, one of two CyP40 homologs in yeast, reduces glucocorticoid responses (Duina et al., 1996). Saccharomyces cerevisiae also contains an ortholog for mammalian PP5 (PPT1/YGR123C), but there have been no reported characterizations of the role of this gene in steroid signaling. Finally, whereas S.cerevisiae harbors several small FKBP proteins, it does not contain any of the large TPR-containing FKBPs that bind to Hsp90. Thus S.cerevisiae provides a null background for testing in vivo the function of FKBP52 and FKBP51 in steroid signaling. Results Saccharomyces cerevisiae reporter model To assess the in vivo function of receptor-associated immunophilins, we constructed S.cerevisiae reporter strains that harbor expression plasmids for a vertebrate steroid receptor, one of the receptor-associated human immunophilins and a lacZ reporter plasmid (Figure 1A). Hormone signaling in S.cerevisiae has previously been measured by assaying the accumulation of the reporter gene product β-galactosidase at a fixed time interval after hormone addition (ranging from 1 to 12 h). While the amount of β-galactosidase is strongly influenced by the rate of synthesis, other factors, such as cell density, growth rate and stability, also influence its levels. Taking advantage of a commercially available β-galactosidase assay that is rapid and sensitive, we modified the typical assay to measure responses soon after hormone addition while accounting for cell growth. Figure 1.Measurement of hormone-induced reporter activity. (A) Yeast strains were transformed with a hormone receptor expression plasmid, an immunophilin expression plasmid and a plasmid carrying a corresponding reporter gene. The receptor and immunophilin genes are transcribed from the strong constitutive glycerol phosphate dehydrogenase promoter. The reporter plasmid contains the lacZ gene transcribed from a truncated yeast cytochrome C1 promoter (PCYC1) downstream of receptor-specific hormone response elements (HRE). Receptor complexes, which contain yeast Hsp90 and other chaperones plus an immunophilin, are activated by hormone binding. The active receptor binds to HRE and recruits RNA polymerase complex (RNAP) to drive transcription from the reporter plasmid. (B) β-galactosidase was induced in the GR reporter strain by addition of deoxycorticosterone (H) at the concentrations indicated. β-galactosidase activities, measured in chemiluminescent relative light units (RLU), are plotted as a function of OD600. (C) To generate a DOC dose–response curve for the reporter gene expression rate, the slope (ΔRLU/ΔOD600) was calculated for the linear portion of each induction curve in (B) and plotted relative to hormone concentration. Download figure Download PowerPoint Attention was focused on the GR reporter system since this has been the most thoroughly investigated steroid receptor system in yeast models. First, we established a dose–response curve for hormone-induced β-galactosidase activity (Figure 1B and C). Replicate cultures were treated with a range of concentrations of deoxycorticosterone (DOC), a favored GR agonist in yeast models (Garabedian and Yamamoto, 1992; Kralli et al., 1995). The optical density at 600 nm (OD600) of the culture, a measure of yeast number and thus total protein, was monitored and aliquots were assayed for β-galactosidase activity. By plotting the β-galactosidase activity of each sample against its OD600, plots of β-galactosidase synthesis relative to total protein synthesis are generated (Figure 1B). After an initial 20–30 min lag, the rate of β-galactosidase expression, as reflected by the linear slope of each plot, remains constant for several hours. To generate a dose–response curve (Figure 1C), the calculated slope for each data set was plotted relative to DOC concentration. Since immunophilin influences on hormone signaling are likely to be most physiologically relevant at a limiting concentration of hormone and 25 nM DOC is as near as practical to the physiological range of GR agonists while still being sufficient for reliable data generation, 25 nM was selected as the hormone concentration used in most single-dose assays, but similar results were obtained in experiments using 50 or 100 nM concentrations of hormone. Immunophilin influence on hormone responses To test whether the receptor-associated immunophilins influence basal hormone signaling, we compared GR reporter strains transformed with an empty vector or with vectors individually expressing the human immunophilins FKBP51, FKBP52, PP5 or CyP40, or overexpressing CPR7, a yeast ortholog of CyP40 (Figure 2A). Reporter activity increased ∼2-fold over basal levels with any of the immunophilins, but only FKBP52 conferred a markedly higher response to hormone. Since the limited enhancement of DOC response seen with FKBP51, which is most closely related to FKBP52, is typical of the other immunophilins, FKBP51 served as a control for FKBP52-specific effects. To determine whether the immunophilins differentially influence the steady state level of GR protein, extracts from cells lacking GR or GR-plus cells expressing FKBP52, FKBP51 or no added immunophilin were compared by western immunostain (Figure 2B). GR protein level was increased 2- to 3-fold in the presence of FKBP52, but an equal increase was observed with FKBP51. Perhaps elevation of GR protein could account for the modest increase in hormonal responses observed with each immunophilin (Figure 2A), but this does not account for the greater potentiation observed with FKBP52. Figure 2.Potentiation of GR signaling by the immunophilins. (A) Reporter expression rates of the GR reporter cells transformed with an empty vector or with plasmids expressing FKBP52, FKBP51, CPR7, CyP40 or PP5. Each bar is the average expression rate (±SD) from quadruplicate cultures of a representative isolate. (B) Steady state levels of GR in the parental strain W303a and GR reporter strains in FKBP51, FKBP52 or control vector backgrounds. Extracts from each strain were analyzed by western blotting for GR (upper panel) and for the ribosomal protein L3 as a loading control (lower panel). (C) Similar to (A) except that GR reporter cells were transformed with a single vector or two vectors (52+52, 51+52 or 52+PP5) for co-expression of immunophilins. (D) For each strain shown in (C), the total cell extract was immunostained for the proteins indicated. Download figure Download PowerPoint Since FKBP51 has been shown to inhibit GR activity in mammalian systems, we anticipated that an attenuation of GR signaling might be observed in the yeast model, but this was not the case (Figure 2A). This unexpected finding suggested that the effect of FKBP51 observed in mammalian systems might be an indirect consequence of blocking FKBP52 potentiation. To test whether FKBP51 can block this potentiation, immunophilins were co-expressed in the GR reporter strain (Figure 2C). Co-expression of two FKBP52 plasmids did not significantly increase potentiation observed with a single plasmid. However, co-expression of FKBP51 with FKBP52 efficiently blocked potentiation. Since FKBP51 competes with FKBP52 for Hsp90 binding and incorporation into GR complexes, simple displacement of FKBP52 from GR complexes might account for the loss of potentiation. This possibility is discounted by the observation that PP5, which also competes for Hsp90 binding and assembly with receptor complexes, fails to block FKBP52-mediated potentiation. Western immunoassays were performed to show that the FKBP52 protein level in dual transformations is similar to the level obtained in yeast expressing FKBP52 alone (Figure 2D). Therefore it appears that FKBP51 functions in a specific and efficient manner to counteract the effects of FKBP52 on GR signaling. FKBP52 and GR hormone binding To gain a better understanding of how FKBP52 enhances GR signaling, reporter gene expression was measured over a broad range of DOC concentrations to generate full hormone response curves for the FKBP strains and the vector control strain (Figure 3A). In the presence of FKBP52, there is a distinct shift in the hormone response curve with a DOC EC50 of ∼70 nM, 3-fold less than the FKBP51 strain. In this respect, FKBP52 appears to mimic the effect of a GR mutation GR-F620S, a serine substitution for phenylalanine at position 620, which was identified in yeast genetic screens for elevated hormone responsiveness (Garabedian and Yamamoto, 1992). To determine whether FKBP52 also potentiates GR-F620S, similar hormone response curves were generated for cells expressing this receptor (Figure 3B). As with wild-type GR, the GR-F620S response was further shifted in the presence of FKBP52 (EC50 ≈ 15 nM), suggesting that the mechanism for enhanced basal hormone binding in GR-F620S is distinct from the mechanism for FKBP52-dependent potentiation. Figure 3.Hormone dose–response curves. (A) GR reporter cells expressing FKBP51 (GR/51) or FKBP52 (GR/52), or transformed with empty vector (GR/vector), were treated over a range of DOC concentrations. Reporter expression rates are normalized to the rate at the highest hormone concentration. Each data point is the average expression rate (n = 4 ± SD) that is representative of two independent isolates. (B) Reporter cells expressing wild-type GR or the F620S GR mutant were transformed with an expression plasmid for FKBP51 or FKBP52. Typical hormone dose–response curves are shown. Download figure Download PowerPoint The left shift in the DOC dose–response curve stimulated by FKBP52 is consistent with an increase in GR hormone-binding affinity. To test this more directly, radiolabeled hormone-binding assays were performed on intact cells. To minimize the technical challenges others have encountered with GR-binding measurements in yeast, mutant GR-F620S was used since it has a higher affinity for hormone and also responds to FKBP52 in a similar manner to wild-type GR. Generating full-saturation binding curves was still not practical, and so we compared the levels of bound hormone at 25 nM, 250 nM and 2.5 μM [3H]corticosterone. At the lowest concentration, corticosterone binding was 5-fold greater in the presence of FKBP52 (21 ± 2 fmol/OD unit cells) compared with FKBP51 (4 ± 1 fmol/OD unit). At the highest concentration, the difference was reduced to <1.5-fold (549 ± 110 versus 388 ± 43 fmol/OD unit), indicating that binding in both strains approached a similar saturation level. This hormone-binding pattern is fully consistent with an FKBP52-dependent increase in GR hormone-binding affinity, as opposed to an increase in receptor number. As seen with wild-type GR (Figure 2B), western analysis confirmed that the steady state levels of GR-F620S protein were identical in the two FKBP strains (data not shown). We conclude that FKBP52 stimulates an increase in GR hormone-binding affinity to account, at least in part, for the potentiation of reporter activity. Selectivity of FKBP52 for GR Each of the receptor-associated immunophilins can assemble with any of the five steroid receptors, although there are clear preferences for particular immunophilins in different receptor complexes (Barent et al., 1998). To determine whether FKBP52 enhances all steroid receptors, FKBP52 was tested in yeast reporter strains expressing GR, estrogen receptor (ER) or chimeric receptors (Figure 4). In contrast with GR reporter cells, no potentiation of ER activity was observed in yeast expressing FKBP52. Similarly, FKBP52 did not enhance reporter gene activity stimulated by androgen or progesterone receptor (data not shown). Based on the different results with GR and ER, we took advantage of chimeric receptors in which ligand-binding domains (LBDs) were exchanged between these receptors (Bunone et al., 1996) to examine whether FKBP52 potentiation maps to the GR LBD or to the GR DNA-binding domain (DBD) and upstream sequences (Figure 4). Clearly, potentiation is specific for the GR LBD, as is consistent with our observation that FKBP52 elevates GR hormone-binding affinity. Figure 4.Selectivity of FKBP52-dependent potentiation. (A) Yeast reporter strains were prepared that express human ER (oval features), rat GR (rectangular features) or the ER.GR or GR.ER chimera. The approximate positions for the DBD (solid fill) and the LBD (hatched fill) in each receptor construct are illustrated. (B) Reporter strains expressing FKBP51 or FKBP52 or transformed with empty vector were compared for hormone-dependent β-galactosidase activity. The background level of reporter activity is shown for uninduced vector-only cells. Other bars represent reporter activity in vector, FKBP51 and FKBP52 cells induced with the following hormone concentrations: GR strains, 25 nM DOC; ER strains, 10 pM 17-β-estradiol; GR.ER, 450 pM 17-β-estradiol; ER.GR, 400 nM DOC. These data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. Download figure Download PowerPoint In addition, we tested for FKBP effects on hormone-independent reporter activity by co-expressing FKBP52 or FKBP51 with GR-N525, a GR truncation mutant lacking an LBD that constitutively transactivates the GRE-regulated reporter (Picard et al., 1990a). Neither FKBP affected the constitutive level of β-galactosidase activity mediated by GR-N525 (results not shown). These observations highlight the role of the LBD in FKBP52-mediated potentiation and minimize the likelihood that FKBP52 acts additionally on the DBD or at the level of general transcriptional enhancement. Mechanism of FKBP52 action To assist in the design of FKBP52 mutants for functional mapping, the crystal structure of FKBP51 (Sinars et al., 2002) was used as a guide (Figure 5A). FKBP51 contains three major structural domains: two FKBP12-like domains, termed FK1 and FK2, plus a typical Hsp90-binding TPR domain. Peptidylprolyl isomerase (PPIase) activity and FK506 binding are restricted to FK1 (Sinars et al., 2002). The domain organization of FKBP52 is likely to resemble FKBP51 since they share ∼75% sequence similarity. Figure 5.The role of Hsp90 binding in FKBP52-mediated potentiation. (A) Major structural domains of human FKBP51. FKBP51 contains two FKBP12-like domains, FK1 and FK2, that have predominant β-sheet structure. FK1 forms the FK506-binding site and has PPIase activity; the 80S and 40S loop regions characteristic of FKBP12 are indicated. Six adjacent amino acids in the FK1 domain (broken lines) that correspond to the 40S loop of FKBP12 are unresolved. The N- and C-terminal ends of the molecule, as indicated, are also unresolved. FK2, which is structurally similar to FK1, lacks PPIase and FK506-binding activities. The C-terminal TPR domain is predominantly α-helical and forms the Hsp90-binding site; mutation of Lys-352 within the TPR domain will abrogate Hsp90 binding. FKBP52 is likely to have an overall structure very similar to FKBP51. (B) The FKBP52 point mutant K354A (corresponding to Lys-352 in FKBP51) was compared with wild-type FKBP52 for binding to Hsp90. Radiolabeled FKBP52 and FKBP52-K354A were prepared in vitro (first two lanes); aliquots of each synthesis mixture were tested for co-immunoprecipitation with Hsp90 complexes (final two lanes). Samples were separated by SDS–PAGE and visualized by Coomassie Blue staining (upper panel) or autoradio graphy (lower panel). (C) Hormone-induced β-galactosidase activity in GR reporter cells containing vector or plasmids expressing FKBP52 or FKBP52-K354A. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. Download figure Download PowerPoint Does the increase in GR hormone-binding affinity require FKBP52 binding to Hsp90? As discovered by Chinkers and colleagues (Russell et al., 1999), a K354A point mutation in the TPR domain of FKBP52 (see Figure 5A for the equivalent position in FKBP51) abrogates binding to Hsp90 (Figure 5B). When FKBP52-K354A is substituted for wild-type FKBP52 in GR reporter cells, potentiation is virtually abolished (Figure 5C). This observation is consistent with previous evidence that the receptor-associated immunophilins primarily enter receptor complexes indirectly through interactions with Hsp90 (Renoir et al., 1990; Pratt and Toft, 1997). Is the TPR domain sufficient for potentiation of GR activity? To address this question and extend the functional mapping of FKBP52, a series of FKBP52 domain truncation mutants (Figure 6A) were tested. All the domain truncations completely lacked the potentiation activity observed with full-length FKBP52 (Figure 6B). This indicates that the TPR alone is not sufficient and suggests an additional requirement for other domains. To test this possibility further, we took advantage of the structural similarity between FKBP52 and FKBP51, which lacks potentiation activity, to generate chimeric constructs in which the FK1 domain was exchanged (Figure 6A). Substituting FKBP51-FK1 for FKBP52-FK1 largely abrogated potentiation (Figure 6C, chimera 2), whereas the converse substitution on FKBP51 (chimera 1) caused a gain in potentiation that approximates half the activity seen with FKBP52. Western immunostains for each FKBP were performed to verify that wild-type and chimeric proteins are expressed at similar levels. The steady-state level of chimera 1, which is more active in potentiation of GR signaling than FKBP51 or chimera 2, is ∼30% lower than FKBP51; if anything, this suggests that the ability of chimera 1 to enhance GR responses may be underestimated. Chimera 2 levels are similar to FKBP52 and so loss of potentiation does not correspond to reduced protein. In another experiment, the N-terminal 30 amino acids preceding FK1 were exchanged with no effect on potentiation (results not shown). These observations demonstrate that the FK1 domain of FKBP52 is required for potentiation of GR signaling. Figure 6.Mapping of FKBP52 domains required for potentiation. (A) Domain structures are illustrated for full-length FKBPs, FKBP52 truncation mutants and FKBP chimeric proteins. (B) Reporter expression rates were measured in separate GR reporter strains containing empty vector or expressing the indicated immunophilin product. Expression data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. Expression levels for FKBP52 and truncation mutants were compared by western immunostaining. Owing to the loss of individual epitopes in truncation mutants, two anti-FKBP52 antibodies (Hi52b and Hi52d) were used along with an L3 probe as loading control. (C) Reporter expression rates were similarly determined in GR reporter strains expressing wild-type proteins or full-length chimeras containing the FK1 domain from FKBP52 (chimera 1) or FKBP51 (chimera 2). Expression of each form was verified by western immuno staining with anti-FKBP51 antibodies FF1 or Hi51b and anti-FKBP52 Hi52c. Note that FF1, which has an epitope in FK1 of FKBP51, detects wild-type FKBP51 and chimera 2, Hi51b, whose epitope is in the C-terminal region of FKBP51, detects FKBP51 and chimera 1, and Hi52c, with an epitope in FK1 of FKBP52 detects wild-type FKBP52 and chimera 2. Immunostaining of L3 (asterisk) provided an internal loading reference for each sample. Download figure Download PowerPoint Pratt and colleagues have reported that the FK1 region of FKBP52 binds to dynein and have proposed that, through the mutual interaction of FKBP52 with dynein and receptor-bound Hsp90, FKBP52 assists in transporting GR from the cytoplasm to the nucleus (Pratt et al., 1999). Saccharomyces cerevisiae contains a single gene for dynein (DYN1), and cells containing a dyn1 null allele are viable (Eshel et al., 1993; Saunders et al., 1995). To test whether DYN1 contributes to FKBP52-dependent potentiation, GR reporter strains were constructed in the dyn1 null background and compared with reporter strains in the wild-type background (Figure 7A). FKBP52-dependent potentiation was unaffected by the absence of dynein. Figure 7.Roles of dynein binding and PPIase activity in potentiation. (A) Reporter expression in cells containing empty vector, FKBP51 or FKBP52 in a wild-type DYN1 or dyn1-null background in response to 50 nM DOC. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. (B) Cultures of GR reporter cells expressing the indicated immunophilin were treated with FK506 (10 μg/ml) for 30 min before addition of DOC (50 nM) and measurement of reporter activity (A and B normalized to the level of expression in vector-only cells). (C) Expression rates in GR reporter cells containing empty vector, FKBP51, FKBP52 or the PPIase-deficient mutant FKBP52-FD67DV. All data are the average (n = 4 ± SD) of duplicate assays from two independent isolates. The inset shows a western immunostain for FKBP52 or L3 which compares FKBP52 expression levels in the wild-type and mutant strains. Download figure Download PowerPoint Since PPIase activity resides in FK1, its potential role was examined both pharmacologically and genetically. The immunosuppressive drug FK506 binds FK1 and efficiently blocks the PPIase activity of FKBP52 (Peattie et al., 1992). Addition of FK506 to cells prior to hormone addition completely blocked FKBP52-mediated enhancement of reporter expression (Figure 7B). However, apart from inhibition of PPIase activity, FK506 could, conceivably, sterically interfere with FK1 interactions or alter the conformation of FK1 and disrupt interactions that occur away from the PPIase active site. As an alternative approach, we tested the FKBP52 mutant FD67DV, which has substitutions in two residues that are highly conserved in the FKBP family and are critical for
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