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Endocrinology, doi:10.1210/en.2008-0168
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Endocrinology Vol. 149, No. 11 5724-5734
Copyright © 2008 by The Endocrine Society

A Novel FK506-Like Binding Protein Interacts with the Glucocorticoid Receptor and Regulates Steroid Receptor Signaling

Hayley D. McKeen1, Kerry McAlpine1, Andrea Valentine, Derek J. Quinn, Keeva McClelland, Christopher Byrne, Martin O'Rourke, Sheila Young, Christopher J. Scott, Helen O. McCarthy, David G. Hirst and Tracy Robson

School of Pharmacy, Queen’s University, Belfast BT9 7BL, Northern Ireland

Address all correspondence and requests for reprints to: Dr. Tracy Robson, School of Pharmacy, McClay Research Centre, Queen’s University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland. E-mail: t.robson{at}qub.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TWO LARGEST families of immunophilins, namely the cyclophilins and FK506-binding proteins (FKBPs), are characterized by a conserved domain that is the binding site for immunosuppressive drugs and has peptidylprolyl isomerase (PPIase) activity (reviewed in Ref. 1). Three high molecular weight immunophilins, FKBP51, FKBP52, and cyclophilin 40 (CyP-40), have been well characterized in steroid receptor-heat shock protein (hsp)-90 complexes interacting via tetratricopeptide repeat (TPR) domains (2). In the last few years another FKBP-like (FKBPL) protein has been isolated (3). However, it is a divergent member of this group with shared homology mostly in the C-terminal TPR domain, important for the interactions with hsp90, although there is still some weak homology spanning the PPIase domain (3, 4). FKBPL was found to have homology with yeast and human proteins including serine and threonine protein phosphatases, Sti/p60/Hop and the immunophilins FKBP52 and CyP-40 (4). FKBPL exhibits significant homology to the immunophilins FKBP52 and CyP-40 at the C terminus, with 26 identical and 19 similar amino acids in this TPR domain (4).

The FKBPL gene was initially identified in a screen to establish genes whose expression levels were affected by low dose ionizing radiation. FKBPL mRNA was down-regulated at low doses (0.05–1.0 Gy; maximally at 0.2 Gy); however, doses of 2 and 4 Gy did not affect its expression (3). Mimicking the effects of radiation using FKBPL antisense-targeted oligonucleotides, led to protection from radiation-induced damage and increased cell survival (4, 5). More recently, its role as a stress response-related protein was further established when Jascur et al. (6) demonstrated that FKBPL (WISp39) interacted with hsp90 via its TPR domain and was key to the stabilization of newly synthesized p21. FKBPL did not affect the rate of mRNA synthesis, stability, or translation of p21. However, the data suggest that FKBPL prevented proteasomal degradation of p21 and the stabilization of p21 was dependent on the recruitment of hsp90 to the complex. Down-regulation of p21 results in a decrease in cell cycle-arrested cells and an increase in cell survival (7). Although not discussed by Jascur et al. (6), the down-regulation of FKBPL by ionizing radiation or antisense oligonucleotides is consistent with a subsequent down-regulation of p21 and the observed increase in cell survival as described by Chu et al. (7). In a more recent study, overexpression of FKBPL was shown to decrease proliferation by inducing a G0/G1 block (8).

The interaction of FKBPL with hsp90 and its homology with the immunophilins suggests that it may also be a component of the steroid receptor-hsp90 complexes such as glucocorticoid receptor (GR)/hsp90 complexes, although its role in the process has not yet been established. Unliganded GR resides mainly in the cytoplasm in close proximity to chaperone proteins including hsp90. Upon hormone binding, GR translocates to the nucleus where it binds to high-affinity target sites, glucocorticoid response elements (GREs) on chromatin, resulting in altered transcriptional activity of target gene promoters (9, 10, 11, 12). Translocation of the GR on hormone binding from the cytoplasm to the nucleus is dependent on the hsp90 chaperone machinery.

The assembly of steroid receptor complexes involves multiple chaperone and cochaperone proteins such as hsp90, hsp40, hsp70, hsp90/70 organizing protein Hop, and p23 (reviewed in Ref. 13). Mature hsp90-steroid receptor complexes also contain at least one immunophilin: FKBP52, FKBP51, CyP-40, or protein phosphatase 5. Hsp90 has the ability to bind to only one of these TPR immunophilins at a time, suggesting four distinct heterocomplexes (14). Complexes with each of these immunophilins have been immunoadsorbed from the cytosol of cells with the GR (reviewed in Ref. 15). These heterocomplexes have also been shown to contain the motor protein dynein (16). Galigniana et al. (17) suggested that the hsp90 binding immunophilins also bind dynamitin, a 50-kDa subunit of the dynein-associated dynactin complex, directly through their PPIase domain, in turn linking the hsp90 heterocomplex to dynein.

Chen et al. (18) provided the first evidence for the need of TPR immunophilins in steroid receptor signaling. They observed that overexpressing the TPR domain fragment of the PP5 protein inhibited GR transcriptional activity. Composition and assembly of the large steroid receptor complex appeared to remain unaffected by the binding of immunosuppressant drugs within FKBP52 and CyP-40; this provided evidence that the PPIase activity of these immunophilins was not required to enable correct folding for binding of hormone to the ligand binding domain (19).

Although FKBPL’s role in radiation stress signaling and p21 regulation in association with hsp90 has been well established (4, 5, 6), its homology to other members of the immunophilin family suggest a further role in steroid receptor signaling. Here we explore this hypothesis and demonstrate, for the first time, that FKBPL plays an important role in steroid receptor complexes and signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The materials used were four-well chamber slides (Lab-Tek, East Sussex, UK), anti-FKBPL rabbit polyclonal IgG (PTG Labs, Chicago, IL), anti-GR mouse monoclonal IgG and antidynactin p50 mouse monoclonal IgG (BD Biosciences, Oxford, UK), anti-hsp90{alpha} rabbit polyclonal IgG (Biomeda, Foster City, CA), mouse monoclonal anti-{alpha}-tubulin (Sigma, Dorset, UK), Alexa Fluor goat antirabbit (GAR) 488, Alexa Fluor donkey antimouse (DAM) 488, Alexa Fluor DAM 594, and Alexa Fluor DAM 594 (all Molecular Probes, Paisley, UK), Vectashield mounting medium with propidium iodide (Vector Labs, Peterborough, UK), dexamethasone (Sigma), Complete, EDTA-free; protease inhibitor cocktail tablets (Roche, West Sussex, UK), Dharmafect (Dharmacon, Lafayette, CO), oligofectamine and Lipofectamine Plus (Invitrogen, Paisley, UK).

Cell culture
All cell lines were obtained from the American Tissue Culture Collection (Manassas, VA). DU145 cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (FCS). For dexamethasone experiments, DU145 cells were incubated with phenol red-free RPMI 1640 supplemented with 10% charcoal stripped FCS for 24 h before ligand addition. V79 and L132 cells were maintained in MEM (Invitrogen) supplemented with 10% FCS. Cells were grown as monolayers incubated at 37 C under 5% CO2 and subcultured every 3–4 d to maintain exponential growth.

FKBPL overexpression and knockdown transfections
For overexpression analysis, cells were transfected with pcDNA3.1/FKBPL, pcDNA3.1 empty vector control, or pACT/GR using lipofectamine plus reagent according to the manufacturer’s instructions. For knockdown analysis, small interfering (si) control nontargeting siRNA no. 1, 5'-UAGCGACUAAACACAUCAA-3' (Dharmacon), and FKBPL siRNA, 5'-CGCUUGAGCUGGAAGUAAGtt-3' (Ambion, Warrington, UK) were transfected using Dharmafect (DU145) or oligofectamine (L132) reagents according to manufacturer’s instructions. Cells were assayed 72 h after transfection.

Colocalization assay
DU145 cells were plated on chamber slides, fixed in 4% paraformaldehyde for 20 min at room temperature, washed with ice-cold PBS, and permeabilized with 2% BSA containing 0.1% Triton X-100 for 20 min at room temperature. FKBPL was visualized with an anti-FKBPL rabbit polyclonal IgG (dilution 1:50) and an Alexa Fluor GAR 488 secondary antibody. GR was detected with an anti-GR mouse monoclonal IgG (dilution 1:30); dynamitin was detected with an antidynactin p50 mouse monoclonal IgG (dilution 1:50) with an Alexa Fluor DAM 488 as the secondary antibody. For dual staining, Alexa Fluor GAR 488 was used as secondary against FKBPL; tubulin was visualized with an anti-{alpha}-tubulin mouse monoclonal (1:500) and Alexa Fluor DAM 594 was used as a secondary against GR, dynamitin, and tubulin (dilution 1:250). Negative controls were also analyzed to ensure that the secondary antibodies did not bind nonspecifically. Slides were visualized using a Leica confocal system TCS Sp2, (Leica, Buckinghamshire, Germany). A x40 magnification oil immersion Plan Apochromatic objective was used.

Bimolecular fluorescence complementation (BiFC) assay
BiFC was used as previously described by Hu et al. (20) to assess potential protein interactions between FKBPL or the FKBPL PPIase fragment (amino acids 1–155) with hsp90 and dynamitin. FKBPL or the PPIase fragment were cloned in frame into the pC158A/B [C terminus of green fluorescent protein (GFP), amino acids 158–242] plasmid at the SFI1 restriction sites (A/B) downstream of the GFP fragment. Hsp90 and dynamitin were cloned in frame into the pN157A/B plasmid (N terminus of GFP incorporating amino acids 1–157) also at the SFI1 restriction sites. A positive interaction brings together the N and C termini of the GFP protein, resulting in green fluorescence in situ. V79 cells were plated on to glass coverslips for each transfection condition and incubated overnight at 37 C with MEM containing 10% FCS. Transfections were carried out using lipofectamine plus reagent in accordance with manufacturer’s instructions. After transfection (48 h), cells were fixed in 4% paraformaldehyde as described before and mounted using Vectashield mounting medium with or without propidium iodide. Fluorescence was visualized on a Leica confocal system TCS Sp2 microscope.

Coimmunoprecipitation and Western blotting
For endogenous coimmunoprecipitation, a T75 tissue culture flask of either L132 or DU145 cells was lysed in 500 µl lysis buffer [20 mM Tris HCl (pH 7.4), 1% Igepal, 12 mM sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM sodium molybdate, 1 complete EDTA-free tablet protease inhibitor]. For exogenous interactions, 2 x 105 L132 cells were transfected with pcDNA3.1/FKBPL and pACT/GR according to the manufacturer’s instructions and lysed 24 h later in 250 µl lysis buffer. Precleared lysates were incubated with antibody bound protein G-Sepharose beads (Cancer Research, London, UK) at 4 C overnight. The beads were washed five times in lysis buffer, suspended in 50 µl 2x Laemmli buffer, and subjected to SDS-PAGE. For FKBPL overexpression studies, DU145 and L132 cells were transfected and cells lysed in 200 µl 2x Laemmli buffer. Western blotting analyses were carried out with anti-FKBPL rabbit polyclonal (dilution 1:2,000) together with antirabbit IgG horseradish peroxidase (HRP)-linked whole antibody secondary (dilution 1:10,000) or anti-GR mouse monoclonal (dilution 1:2,000) together with antimouse IgG HRP-linked whole antibody secondary (dilution 1:15,000), or antidynactin p50 mouse monoclonal primary antibody (dilution 1:500) together with an antimouse IgG HRP-linked whole antibody or anti-hsp90{alpha} rabbit polyclonal antibody (1:1,000) together with an antirabbit IgG HRP-linked whole antibody (dilution 1:7,500), as described previously. As a loading control for overexpression assays, anti-β-actin mouse monoclonal antibody (dilution 1:2500) was used with antimouse IgG HRP-linked whole antibody as described previously. Immunoreactive bands were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Dublin, Ireland) according to the manufacturer’s instructions. For overexpression assays, statistical analysis was carried out by one-way and two-way ANOVA.

Translocation studies
DU145 (2 x 105) cells were plated on chamber slides or 35-mm dishes and incubated for 24 h. After incubation, the medium was replaced with medium containing either 1 µM dexamethasone or methanol (vol/vol) as a vehicle control and cells incubated at 37 C for 0, 10, and 30 min. Cells were fixed, permeabilized, and labeled for FKBPL and GR and visualized by confocal microscopy as described before. Each time point was scored for FKBPL and GR translocation using a scoring system that had been previously described by Harrell et al. (21); a score of 4 indicated nuclear localization much greater than cytoplasmic; 3, nuclear fluorescence greater than cytoplasmic fluorescence; 2, nuclear fluorescence equal to cytoplasmic fluorescence; 1, cytoplasmic fluorescence greater than nuclear fluorescence; and 0, cytoplasmic fluorescence much greater than nuclear fluorescence. Statistical significance was analyzed by the nonparametric Mann Whitney U test. To determine GR translocation by Western blot, cytoplasmic and nuclear fractions were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) after vehicle control or 1 µM dexamethasone treatment and immunoblotted as described above.

Reporter gene luciferase assay
Transcriptional activities of FKBPL were measured by a luciferase reporter gene assay. DU145 and L132 cells (2 x 104) were grown in maintenance media for 24 h. Cells were transfected with the luciferase reporter construct, JH4-GRE-oct6-Luc (a kind gift from Dr. Albert O. Brinkmann, University Medical Center, Rotterdam, The Netherlands), pcDNA3.1/FKBPL, or pcDNA3.1 empty vector control and pBIND Renilla plasmid (Promega, Southampton, UK) as a transfection efficiency control. For dexamethasone experiments, doses of 10–9, 10–8, 10–7, or 10–6 M were added immediately after transfection. GRE luciferase reporter activity was measured 24 h after transfection using the Dual-Glo luciferase assay system (Promega) according to the manufacturer’s instructions. For each sample, GR transactivity was corrected for Renilla transfection efficiency. Statistical analysis was performed using one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FKBPL interacts with the components of steroid hormone receptor complexes
FKBPs modulate steroid receptor signaling by facilitating their movement along microtubules in response to ligand binding. This is achieved via the PPIase domain of FKBP proteins binding to dynamitin, whereas the TPR domain of FKBPs is bound to the molecular chaperone hsp90, tethered to the hormone receptor. To assess whether FKBPL displayed similar properties to the FKBPs in this role, we first assessed its localization relative to these proteins within the cell using immunocytochemistry. DU145 cells were used for this investigation because they possess relatively high levels of functional GR. These cells were also ideal due to their larger size enabling a clear distinction between nuclear and cytoplasmic compartments microscopically for assessing translocations from cytoplasm to nucleus. FKBPL was found to localize to both the cytoplasm and the nucleus. Some cells appeared to have mainly cytoplasmic staining with some granular nuclear staining (Fig. 1AGo). Other cells revealed FKBPL localization that was mainly nuclear with some diffuse cytoplasmic staining (data not shown). These results suggest that FKBPL may shuttle between the cytoplasm and the nucleus. GR staining in cells maintained in RPMI 1640 with 10% FCS indicated nuclear and cytoplasmic localization, with stronger nuclear staining being apparent (Fig. 1BGo). The motor protein dynamitin was found to localize diffusely throughout the cytoplasm (Fig. 1CGo). Neither of the secondary antibodies bound nonspecifically to cellular proteins or primary antibody.


Figure 1
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  		FIG. 1. Detection and colocalization of FKBPL, GR, dynamitin, and tubulin in DU145 prostate tumor cells maintained in medium with 10% FCS. DU145 cells were fixed, permeabilized, and then stained with anti-FKBPL rabbit polyclonal primary antibody and Alexa Fluor GAR 488 secondary antibody for FKBPL (green) (A) anti-GR monoclonal primary antibody and Alexa Fluor DAM 488 secondary antibody for GR (green) (B) and antidynamitin p50 monoclonal primary antibody and Alexa Fluor DAM 488 secondary antibody for dynamitin (green) (C). The cells were mounted using Vectashield mounting medium with propidium iodide (PI) as a nuclear stain (red). For colocalization studies, cells were fixed, permeabilized, and then stained with anti-FKBPL polyclonal primary antibody and Alexa Fluor GAR 488 secondary antibody for FKBPL (green) and anti-GR monoclonal primary antibody and Alexa Fluor DAM 594 secondary antibody for GR (red) (D) or antidynamitin p50 monoclonal primary and Alexa Fluor DAM 594 secondary antibodies for dynamitin (red) (E) or anti-{alpha}-tubulin monoclonal primary antibody and DAM Alexa Fluor DAM 594 secondary antibody for tubulin (red) (F) and mounted using Vectashield mounting medium. Colocalization of FKBPL and tubulin in L132 cells were also investigated using antibodies above (G). The cells were visualized using a Leica TCS Sp2 confocal microscope. A x40 oil immersion objective was used in conjunction with a x2 electronic zoom.

 
To detect any colocalization of the proteins, double staining was also carried out with Alexa Fluor secondary antibodies of differing emission and excitation properties, i.e. Alexa Fluor 488 and Alexa Fluor 594. In the dual-stained samples FKBPL and GR appeared to colocalize with mainly diffuse staining in the cytoplasm of the cells and stronger localization appearing in the nucleus of some cells (Fig. 1DGo). The cytoplasmic fraction of FKBPL appeared to colocalize with dynamitin in the cytoplasm of the DU145 cells (Fig. 1EGo). The cytoplasmic fraction of FKBPL was also found to partially colocalize with tubulin in some cells, with more concentrated FKBPL appearing around the boundaries of the nucleus (Fig. 1FGo). Colocalization of FKBPL and tubulin could also be detected in the L132 cell line in which microtubule filaments could be more easily identified (Fig. 1GGo). These results provided the first indication that FKBPL may be involved in the transport of steroid receptors to the nucleus via the motor protein dynamitin along the microtubule network.

We were able to confirm these interactions using immuno-precipitations. Endogenous GR (Fig. 2AGo), dynamitin, (Fig. 2BGo), and hsp90 (Fig. 2CGo) could be immunoabsorbed from complexes with FKBPL in DU145 cells. In L132 cells, dynamitin (Fig. 2DGo) and hsp90 (Fig. 2EGo) could be immunoabsorbed in complexes with FKBPL; however, the interaction with GR was detected only in cells overexpressing GR, when GR was immunoprecipitated and the Western blot probed for FKBPL (Fig. 2FGo). This was probably because the levels of GR in this cell line were much lower than in DU145 cells. It is of note that coimmunoprecipitation of FKBPL with dynamitin and GR cannot be confirmed in both directions (Fig. 2Go, B and F). This may be due to interference or masking of epitopes making it difficult for the second antibody to bind or it may suggest that that the stoichiometry of dynamitin to FKBPL precludes significant yield of the latter.


Figure 2
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  		FIG. 2. Coimmunoprecipitation of FKBPL with GR, dynamitin, and hsp90{alpha} in DU145 and L132 cells. Aliquots of DU145 (A–C) or L132 (D and E) cell lysate were immunoadsorbed to protein G-Sepharose beads with antibody against endogenous FKBPL, GR, dynamitin, or hsp90{alpha} and antirabbit IgG as a negative control (NC). The immune pellets were washed, and pellet-bound proteins were resolved by SDS-PAGE and Western blot analysis with antibody against FKBPL, GR, hsp90{alpha}, or dynamitin. F, Aliquots of L132 cell lysates transfected with pcDNA3.1/FKBPL and pACT/GR were immunoadsorbed to protein G-Sepharose beads with antibody against exogenously expressed FKBPL, GR, or antirabbit IgG as a negative control. Pellet bound proteins were resolved by SDS-PAGE and Western blot analysis with antibodies against FKBPL and GR.

 
Finally, the BiFC assay was used to further assess these interactions in a cell-based assay, in real time (as described in Materials and Methods; also see Ref. 20). A positive interaction between candidate proteins results in reconstitution of GFP and subsequent fluorescence that can be detected by fluorescent microscopy. Furthermore, this assay also allowed visualization of the cellular localization of these interactions. Cotransfection of pC158/FKBPL with either pN157/hsp90 (Fig. 3AGo) or pN157/dynamitin (Fig. 3BGo) resulted in reconstitution of GFP indicated by the presence of green fluorescence, providing further evidence of an interaction between FKBPL, hsp90, and dynamitin. Whereas the interaction between FKBPL and hsp90 occurred mainly in the cytoplasm of V79 cells (Fig. 3AGo), the interaction between FKBPL and dynamitin occurred at various locations within the cell population (Fig. 3BGo). In some cells an interaction could be detected in small discrete foci or vesicles throughout the cytoplasm (Fig. 3BGo, upper panel), whereas in other cells the interaction occurred in what appeared to be the endoplasmic reticulum with a ruffled appearance around the outside of the nucleus (Fig. 3BGo, lower panel). The PPIase domain of immunophilins is thought to be important for the interaction of these proteins with dynamitin. We therefore assessed whether the PPIase domain of FKBPL was responsible for the interaction between dynamitin and FKBPL as determined in Fig. 2Go. The reconstitution of whole GFP (Fig. 3CGo) after cotransfection of pC158/PPIase and pN157/dynamitin indicated that the FKBPL PPIase region interacted with dynamitin. The localization of the interaction appeared to be similar to that obtained for full-length FKBPL and dynamitin with some vesicular staining in the cytoplasm and concentrated staining around the nucleus, indicative of the endoplasmic reticulum. No GFP fluorescence was detected after cotransfection of the empty vector negative control plasmids, C158/FKBPL with pN157A/B, confirming that there was no nonspecific binding of the C and N terminus of GFP (Fig. 3DGo).


Figure 3
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  		FIG. 3. Localization of the interaction between FKBPL, hsp90, and dynamitin in V79 cells by bimolecular fluorescence complementation. V79 cells were cotransfected with pC158/FKBPL and pN157/hsp90 fusion constructs (A), pC158/FKBPL and pN157/dynamitin (B), pC158/PPIase and pN157/dynamitin (C), or empty vector controls pN157A/B and pC158A/B (D). The cells were fixed and mounted with Vectashield mounting media containing propidium iodide. Localization of the interaction was determined by visualization of the site of reconstitution of whole GFP by confocal microscopy. The cells were visualized using a Leica TCS Sp2 confocal microscope. A x40 oil immersion objective was used in conjunction with a x2 electronic zoom. SDS-PAGE and Western blot analysis were carried out on V79 cell lysates cotransfected with pC158/FKBPL and pN157/hsp90 fusion constructs (A) or pC158/FKBPL and pN157/dynamitin (B) to check for expression of the GFP fusion proteins using antibodies against FKBPL (60 kDa), dynamitin (70 kDa), or hsp90 (110 kDa).

 
FKBPL translocates to the nucleus in response to GR ligand
The GR translocates rapidly along the microtubules to the nucleus upon binding ligand. This rapid retrograde translocation occurs in less than 10 min and is aided by the hsp90-immunophilin steroid receptor complexes and requires intact microtubules (22, 23). We therefore assessed whether FKBPL translocated to the nucleus upon addition of the GR ligand dexamethasone at the same rate as the GR to help determine whether FKBPL was acting as a cochaperone in the hsp90-steroid receptor complexes. This was carried out by incubating DU145 cells with dexamethasone (1 µM) and assessing the localization of both FKBPL and GR at various time points using immunocytochemistry and confocal microscopy or Western blot.

At 0 min both FKBPL and GR were localized mainly in the cytoplasmic compartment of the cells (Fig. 4AGo). Within 10 min after dexamethasone treatment, a substantial amount of FKBPL and GR had translocated from the cytoplasm to the nucleus, and by 30 min both FKBPL and GR exhibited strong nuclear staining. The nuclear staining of both FKBPL and GR at the 30-min time point appeared very dense and in some cells appeared localized to specific areas within the nucleus. A scoring system described by Harrell et al. (21) was used to quantitate the translocation of FKBPL/GR after exposure to dexamethasone, compared with methanol vehicle control. Statistical analysis was carried out on the translocation score of FKBPL compared with vehicle only control using the nonparametric Mann Whitney U test. U values less than 965 were taken to be statistically significant at a 95% confidence interval. All U values were less than 965, indicating that the nuclear translocation of FKBPL was statistically significant at each time point compared with methanol vehicle control. The data shown are an average of two experiments, and 30 or more cells/data point per experiment were scored (Fig. 4BGo). The translocation score obtained for FKBPL was very similar to that for GR after dexamethasone treatment. This study suggests that FKBPL translocates to the nucleus at the same rate as GR upon dexamethasone treatment. In addition, we have been able to demonstrate that targeted siRNA knockdown of FKBPL in DU145 cells slows down the cytoplasmic to nuclear translocation upon ligand stimulation. We observe higher levels of cytoplasmic GR at 10 and 30 min after dexamethasone treatment in FKBPL knockdown cells compared with a nontargeted siRNA (Fig. 4CGo). This is further supported by a delay in GR movement into the nucleus after ligand stimulation; levels of nuclear GR do not reach a maximum until 30 min after ligand stimulation, whereas maximum levels of GR have already reached the nucleus within 10 min in the nontargeted siRNA-treated cells. These results suggest that inhibition of FKBPL perturbs GR translocation.


Figure 4
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  		FIG. 4. Cytoplasmic to nuclear translocation of GR and FKBPL after treatment with dexamethasone (Dex) in DU145 cells. DU145 cells were grown on chamber slides or 35-mm dishes and maintained in phenol-free RPM1 1640 medium with 10% charcoal-stripped FCS for 24 h and then treated with either 100% methanol vehicle control or 1 µM dexamethasone. For microscopy, cells were fixed in 4% paraformaldehyde solution, permeabilized, and stained for FKBPL with anti-FKBPL polyclonal primary antibody and GAR Alexa Fluor 488 secondary antibody (green); and GR with anti-GR monoclonal primary antibody and donkey antimouse Alexa Fluor 594 secondary antibody (red). The cells were mounted with Vectashield mounting medium onto microscope slides and visualized using a Leica TCS Sp2 confocal microscope. A x40 oil immersion objective was used in conjunction with a x2 electronic zoom. The images shown in A are representative images for each time point. The localization of GR and FKBPL was scored using a scoring system used by Harrell et al. (21 ). The data shown in B were based on two independent experiments consisting of 30 cells/time point per experiment. The Mann Whitney U test was used to determine significance of FKBPL translocation compared with vehicle control at each time point. **, Statistically significant at 95% confidence interval) (n = 2). For FKBPL knockdown studies, DU145 cells were transfected with nontargeting or FKBPL siRNAs and treated with vehicle control or 1 µM dexamethasone (Dex) and nuclear and cytoplasmic proteins isolated and immunoblotted for FKBPL and GR (C).

 
FKBPL overexpression modulates glucocorticoid receptor protein levels
Riggs et al. (24) observed that overexpression of the immunophilins FKBP51 and FKBP52, in yeast cells caused an increase in GR protein levels. Therefore, assessing GR protein levels after overexpression of FKBPL may provide further insight into the functional significance of the FKBPL in GR signaling. A notable decrease in GR protein levels was observed in L132 cells transfected with pcDNA3.1/FKBPL compared with empty vector controls at 0, 4, 24, and 48 h (Fig. 5AGo). Statistical analysis of densitometry values by two-way ANOVA indicated a highly significant average 0.3-fold decrease (P = 0.002) in GR protein levels in pcDNA3.1/FKBPL transfected cells compared with vector only controls over the entire time course of the experiment (Fig. 5BGo). Interestingly, in DU145 cells, overexpression of pcDNA3.1/FKBPL caused an increase in GR protein levels (Fig. 5CGo). Statistical analysis via two-way ANOVA indicated that across the time course of the experiment an average 1.65-fold increase in GR protein levels could be detected (P = 0.001; Fig. 5DGo). Furthermore, transfection of DU145 cells with a targeted FKBPL siRNA lead to a decrease in GR protein levels (Fig. 5CGo), again suggesting that FKBPL is important for regulating GR levels.


Figure 5
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  		FIG. 5. GR levels after FKBPL overexpression or knockdown studies. L132 (A and B) or DU145 (C and D) cells were transiently transfected with pcDNA3.1 or pcDNA3.1/FKBPL and samples taken at 0, 4, 24, and 48 h after a 6-h transfection. For FKBPL knockdown, DU145 cells were transfected with nontargeting or FKBPL siRNA and incubated for 72 h. The samples were collected in 2x Laemmli buffer and the lysate analyzed by SDS-PAGE and Western blot analysis. The amount of GR protein (94 kDa) was assessed by immunoblotting with anti-GR antibody, FKBPL (48 kDa) was immunoblotted by anti-FKBPL antibody to confirm overexpression or knockdown, and β-actin (42 kDa) was blotted for with anti-β-actin antibody as a protein loading control. The results shown are representative blots from three independent experiments. The bands produced by Western blot analysis were analyzed by densitometry. All values were calculated by correcting for protein loading using β-actin protein densitometry values. Results are expressed relative to the level of GR in vector only controls (vector = 1) at each time point. All data shown are the mean of three separate experiments ± SE (n = 3). *, P ≤ 0.05; ***, P ≤ 0.001.

 
FKBPL modulates the transcriptional activity of the GR
The observed translocation of FKBPL/GR to the nucleus in response to ligand and the FKBPL-mediated alteration in GR levels suggested that FKBPL may play a critical role in GR signaling. The effects of FKBPL overexpression on GR transcriptional activity was therefore assessed in L132 and DU145 cells after cotransfection of empty vector (pcDNA3.1) or pcDNA3.1/FKBPL with a glucocorticoid-responsive GRE-luciferase reporter construct (JH4-GRE-oct6-LUC) and the pBIND-Renilla expressing construct. Transcriptional activity was measured, after correction for transfection efficiency, as GRE luciferase reporter activity. FKBPL overexpression in L132 cells led to a 28% decrease in GR transcriptional activity, compared with empty vector controls (Fig. 6AGo), correlating with the fall in GR protein levels also observed. Furthermore, FKBPL siRNA knockdown (Fig. 6BGo) led to a 2-fold increase in GR transactivity in L132 cells (Fig. 6CGo). In DU145 cells, overexpression of FKBPL produced an increase in the level of GR transcriptional activity (Fig. 6DGo). The effect was further evaluated using various doses of dexamethasone because the modulation of GR transactivity by the immunophilin FKBP52, is dependent on dose (14). DU145 cells were maintained in steroid-free medium and the effects of FKBPL overexpression determined in the presence of 10–9, 10–8, 10–7, or 10–6 M dexamethasone for 24 h and the transcriptional activity of GR measured. In vector-only controls, a dexamethasone concentration of 10–7 M produced the highest transcriptional activity compared with vehicle controls. At this concentration FKBPL overexpression produced a highly significant 1.7-fold increase in GR transcriptional activity compared with empty vector controls (P < 0.01) (Fig. 6EGo). The data shown are the mean of two independent experiments, and each condition was performed in triplicate in each experiment ± SE. No significant difference was observed at any other dose of dexamethasone. The data suggest that modulation of FKBPL can perturb the transcriptional activity of the GR.


Figure 6
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  		FIG. 6. Overexpression of FKBPL modulates the transactivity of the GR in L132 and DU145 cells. L132 or DU145 cells were cotransfected with JH4-GRE-oct6-LUC, pBIND, and either pcDNA3 1 and pcDNA3 1/FKBPL or nontargeting and FKBPL siRNA. The measurement of GRE-driven luciferase luminescence served as an indicator of the level of GR transactivity and was corrected for Renilla transfection efficiency. All samples were calculated by subtracting background luminescence of nontransfected controls. A decrease in GR transactivity was observed in L132 cells overexpressing FKBPL compared with empty vector controls (n = 3) (A), whereas FKBPL knockdown (B) resulted in increased GR transactivity compared with nontargeting siRNA controls (C). An increase in GR transactivity was observed in DU145 cells overexpressing FKBPL compared with empty vector controls (D). GR transactivity was also assessed in DU145 cells after exposure to 10–9, 10–8, 10–7, or 10–6 M dexamethasone. All conditions were performed in triplicate for this experiment. The data are an average of two separate experiments ± SE (n = 2); a highly significant difference (***, P < 0.01) between vector-only and FKBPL transfected cells was observed at a concentration of 10–7 M dexamethasone (E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We tested the hypothesis that FKBPL may be involved in hsp90 steroid receptor complexes due to its homology with the immunophilins, FKBP52 and Cyp40, and because it interacted with the molecular chaperone hsp90 (6). We have demonstrated that FKBPL colocalizes and interacts with the glucocorticoid receptor and the dynein-associated protein, dynamitin. In addition to this, we have determined that FKBPL may be involved in regulation of cellular steroid receptor protein levels and transcriptional activity.

FKBPL was found to colocalize with GR, dynamitin, and the cytoskeletal element, tubulin. Like immunophilins, the colocalization of FKBPL with proteins involved in the dynamic retrograde transport of steroid receptor complexes to the nucleus suggests that FKBPL may also be involved in this process. The bimolecular fluorescence complementation provided further evidence that FKBPL binds hsp90 and dynamitin and suggests that the PPIase domain of FKBPL is important for the interaction with dynamitin. FKBPL and hsp90 interacted in the cytoplasm of V79 cells as expected because hsp90 is mainly a cytoplasmic protein. The localization of FKBPL and dynamitin appeared to be in the endoplasmic reticulum (ER) of V79 cells. This observation is in contrast to the localization of both endogenous dynamitin and FKBPL in Fig. 1Go. It has been reported that newly synthesized proteins are transported from the ER to Golgi along the microtubules using the minus-end directed dynein/dynamitin network (25). However, overexpression of dynamitin causes loss of cytoplasmic dynein heavy chain from the membrane of Golgi peripheral elements, therefore disrupting ER to Golgi transport of newly synthesized proteins (26). In our study, the overexpression of dynamitin resulted in the ER localization of FKBPL/dynamitin, possibly because of disruption of this transport system for the newly synthesized FKBPL/dynamitin. Thus, whether FKBPL and dynamitin would interact together under normal cell circumstances (i.e. normal protein processing) still needed to be addressed. Some of the cells cotransfected with FKBPL and dynamitin also exhibited much more vesicular GFP localization throughout the cytoplasm of the V79 cells. Because dynamitin overexpression also inhibits the movement of endosomes and Golgi vesicles (27), causing inhibition of protein transport to normal cellular locations, this may account for the vesicular fluorescence observed in some of the transfected cells. FKBPL and dynamitin may be interacting in the inhibited vesicles rather than being transported to their normal destination. However, these results are preliminary, and further experiments would be required as part of a follow-up study in which the cell biology and structure can be fully explored.

Dexamethasone is a widely known GR ligand causing GR translocation to the nucleus (17, 28) in preparation for activation or repression of GR target genes. This translocation occurs in a rapid manner (half time ~4.5 min) in cells with intact microtubules, and the GR is transported in a complex with chaperones including hsp90 and to date, one of four immunophilins, FKBP51, FKBP52, cyclophilin 40, or PP5 (22, 23). FKBPL may be involved in the transportation of GR to the nucleus upon hormone binding because immunocytochemistry and confocal analysis revealed that FKBPL translocated to the nucleus in the same manner as GR upon treatment with dexamethasone. Furthermore, FKBPL knockdown inhibited this movement to the nucleus, although some GR was still able to translocate to the nucleus, most likely via other immunophilins or because FKBPL levels were not completely knocked down. Studies have demonstrated that immunophilins link the hsp90-steroid receptor complexes to dynein, by binding dynamitin using their N terminus PPIase domains (16, 29, 30). However, the chaperone activity of both CyP-40 and FKBP52 has been shown to be independent of PPIase activity (31, 32). FKBPL has been shown to have weak homology to the PPIase domains of immunophilins; however, the ability of this region to bind dynamitin was suggested in the BiFC assay. A strong interaction between this domain of FKBPL and dynamitin indicated that the full-length FKBPL protein is likely to interact with dynamitin via its N terminus PPIase-like domain, although further investigations would be required to confirm this interaction. However, this result provides further evidence that FKBPL acts in a similar manner to immunophilins and is a probable cochaperone of hsp90-steroid receptor complexes, aiding the transportation of the mature complexes from the cytoplasm to the nucleus. The interaction between FKBPL and proteins involved in the steroid receptor complexes was also confirmed by immunoprecipitation, indicating that FKBPL exists in a native complex with GR.

Riggs et al. (24) determined that overexpression of the immunophilins FKBP52 and FKBP51 caused a 2- to 3-fold increase in GR protein levels in yeast, suggesting that immunophilins may regulate steroid receptor levels. We therefore determined whether FKBPL overexpression affected GR protein levels in DU145 and L132 cells. Overexpression of FKBPL in L132 cells resulted in a significant decrease in GR protein levels. Conversely, GR levels increased in DU145 cells after FKBPL overexpression; this was reversed after transfection of targeted FKBPL siRNA. Another TPR protein, ARA9 (XAP2), has been shown to perturb the protein levels of a specific receptor. In 2000 Meyer et al. (33) elucidated that cotransfection of aryl hydrocarbon receptor (AhR) and XAP2 into COS-1 cells caused an increase in AhR protein levels compared with cells transfected with AhR alone. XAP2, like FKBPL, is a homolog of the immunophilins and like FKBPL does not possess PPIase activity. Like XAP2 and the immunophilins, FKBPL may play a role in the regulation of GR protein levels in the cell. GR modulation by overexpression of FKBPL appeared to be cell line dependent, and it is possible that FKBPL may play a dual role in both stabilizing GR protein and preventing its degradation. FKBPL contains both estrogen response elements and GREs within its promoter (by bioinformatic analysis; our unpublished data), suggesting that it may be regulated by both estrogen and glucocorticoids, further supporting a role for FKBPL in steroid levels/signaling. Translocation of FKBPL to the nucleus in response to dexamethasone suggests that it may facilitate transport of these complexes via interaction with the motor protein dynamitin and thus may assist in GR transcriptional regulation. The transcriptional activity of the GR was therefore assessed after FKBPL overexpression. A statistically significant decrease in GR transactivity was observed in L132 cells transfected with FKBPL compared with vector-only controls, and this could be reversed with FKBPL siRNA knockdown. Overexpression of FKBPL in DU145 cells increased GR transactivity. Our data suggests this increase was dependent on the dose of steroid, also reported by Davies et al. (14), who demonstrated an increase in GR transactivity at 10–7 M dexamethasone but not at 10–8 or 10–9 M. Furthermore, they demonstrated that overexpression of FKBP52 in SK-N-MC, HEK, and HeLa cells produced no effect, supporting the lack of activity we saw in L132 cells. Nevertheless, the FKBPL-mediated modulation of GR transactivity corresponded with FKBPL-mediated modulation of GR levels in both cell types; in L132 cells a decrease in GR protein levels correlated with the decrease in GR transactivity, and in DU145 cells, an increase in GR protein levels was observed together with a dose-dependent increase in GR transactivity. These data suggest that by controlling GR levels, FKBPL can affect GR-mediated transactivity.

In summary, our findings demonstrate that FKBPL can be classed as a new member of the FKBP protein family with an important role in steroid receptor complexes and signaling. The growing diversity of the various TPR domain or immunophilin proteins that associate with hsp90-steroid receptor complexes may provide an integrated system for targeted movement of proteins to diverse sites of action within the cell or in different cell types. Because GR regulates genes controlling a wide variety of processes including development, metabolism, and immune responses the ability of FKBPL to modulate this process suggests its importance.


   Footnotes
 
This work was supported by the Biotechnology and Biological Research Council and the Health and Personal Social Services Research and Development Office, Northern Ireland.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 31, 2008

1 H.D.M. and K.M. are considered to have contributed equally to this manuscript. Back

Abbreviations: AhR, Aryl hydrocarbon receptor; BiFC, bimolecular fluorescence complementation; CyP-40, cyclophilin 40; DAM, donkey antimouse; ER, endoplasmic reticulum; FCS, fetal calf serum; FKBP, FK506-binding protein; FKBPL, FKBP-like; GAR, goat antirabbit; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HRP, horseradish peroxidase; hsp, heat shock protein; PPIase, peptidylprolyl isomerase; si, small interfering; TPR, tetratricopeptide.

Received February 6, 2008.

Accepted for publication July 21, 2008.


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 Discussion
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