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Keywords:

  • androgen-insensitivity syndrome;
  • nuclear localization signal;
  • nuclear receptor;
  • prostate cancer;
  • transcription memory

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Sequence and structural anomalies in the gene for androgen receptor (AR) or its protein are associated with a range of clinical manifestations. Observations from living cells have shown that AR translocates to the nucleus upon ligand binding, where it forms typical ‘nuclear foci’ that are considered as sites of gene transcription. Recently, we reported the ligand-mediated association of AR with mitotic chromatin and suggested its role in ‘transcription memory’, proposing a ‘biopit model’. In the present study, we show that each of the AR domains is obligatory for its association with mitotic chromatin and also that full-length AR is necessary for efficient association. In addition, deletion or point mutations in bipartite nuclear localization signal (NLS) revealed impaired localization, ‘nuclear foci’ formation and abolished AR binding with mitotic chromatin. Interestingly, well-characterized AR-NLS mutants associated with the manifestation of pathological conditions (prostate cancer and androgen-insensitivity syndrome) exhibited differential behaviour on mitotic chromatin and also impaired receptor localization and ‘nuclear foci’ formation. Finally, we report that, in addition to its functions in nuclear import, DNA binding, acetylation, N/C-termini interactions and transactivation, the AR-NLS region also functions as ‘mitotic chromatin binding-determining region’ and has a novel role in the regulation of the AR association with mitotic chromatin.


Abbreviations
AIS

androgen-insensitivity syndrome

AR

androgen receptor

DBD

DNA-binding domain

DHT

dihydrotestosterone

GFP

green fluorescent protein

LBD

ligand-binding domain

NLS

nuclear localization signal

NTD

amino-terminal transactivation domain

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The human androgen receptor (AR), one of the 48 members of the nuclear receptor superfamily, plays a key role in the development of male secondary sexual characteristics and the growth of the prostate gland by executing the biological functions of androgens [1-6]. Anomalies in gene for AR or its protein are reflected by many pathological conditions, which primarily include prostate cancer (CaP) [4, 7-16], androgen-insensitivity syndrome (AIS) [2, 3, 11-13, 17-19], male breast cancer [20, 21] and spinal and bulbar muscular atrophy (Kennedy disease) [15, 22, 23]. Additionally, modest enhancements in AR protein levels are also known to play a critical role in the progression of hormone refractory or hormone-independent CaP [24, 25].

Similar to other members of the nuclear receptor superfamily, AR also contains four major functional domains: an amino-terminal transactivation domain (NTD), a central DNA-binding domain (DBD), the hinge region and a carboxy-terminal ligand-binding domain (LBD) [5, 26-30]. The NTD and C-terminal domain undergo ligand-mediated N/C termini interactions, which play an important role in the stabilization and transcription functions of AR [28, 31-33]. For nuclear import, AR contains a bipartite nuclear localization signal (NLS) that overlaps the DBD-hinge region [34-36]. The previously defined DBD-hinge NLS [34-36] displays features that are typical of a classical bipartite NLS (617RKCYEAGMTLGARKLKK633) found in proteins such as nucleoplasmin [37, 38]. Interestingly, the discovery of a second NLS in the LBD of another nuclear receptor (NR), glucocorticoid receptor, has also been reported [39, 40]. Consideration of the structural conservation of LBDs among different nuclear receptors suggested that AR might also contain a second NLS located within its LBD [38]. Poukka et al. [41] have also suggested a second NLS of AR in its LBD. Recently, Kaku et al. [42] have reported a domain-specific NLS and the import of AR.

Unliganded AR is a cytoplasmic protein and remains associated with heat shock protein 90 and several other chaperone proteins. Androgen binding leads to the dissociation of heat shock proteins, which is simultaneously accompanied by a conformational change of the receptor protein, resulting in translocation to the nucleus and the formation of subnuclear foci [32, 43-48]. In the nucleus, the receptor protein binds as a homodimer to the specific DNA sequences termed the ‘androgen response element’ of androgen-responsive genes and subsequently modulates their transcription by recruiting the coregulatory proteins [26, 28, 29, 32, 43-46, 48]. The nuclear import of AR is crucial for its functions and direct action on target genes. However, the specific importins and the mechanisms required for the nuclear import of AR have only recently been investigated in significant detail [42, 49]. AR import is mediated through the classical pathway that employs the binding of importin-α to the bipartite NLS of AR after androgen exposure, as recognized by importin-β, and translocation is facilitated by the GTPase Ran gradient [42, 45-51].

Many studies have revealed that mutations in the gene for AR, ranging from a single nucleotide mutation to complete gene failure, give rise to diseases that include AIS, CaP and spinobulbar muscular atrophy [11-13, 18, 23, 52-54]. A number of mutations have been reported within the putative NLS sequence of AR that are associated with AIS or CaP [7, 10-13, 15-19, 49, 52-54]. Several studies have revealed that mutant AR variants obtained from both AIS and CaP patients can be characterized by their abnormal intracellular localization and a lower capacity for ligand-dependent translocation compared to wild-type. AR mutants fail to achieve normal nuclear import and generally exhibit an atypical intracellular aggregation profile [18, 42, 49, 52, 53]. However, the nature of intracellular aggregation is not well characterized. Disease-imparting mutations within the NLS of AR have been shown to reduce the binding affinity to importin-α and, subsequently, retard nuclear import. Unexpectedly, however, the transcriptional activity of these mutants varies widely [49]. Moreover, several lysine residues within the AR NLS (629RKLKK633) are subjected to acetylation by the transcriptional coactivators p300 (PCAF) and Tip60 (HTATIP), which in turn alter the transactivation functions of AR [30, 55-62]. Considering that these residues serve a role in both acetylation and binding to importin-α, it is important to explore how mutations within this region and other parts of the hinge region of AR influence the subsequent association with mitotic chromatin. Determination of the mechanism for the mitotic chromatin association of AR and its mutants will not only provide us with the potential targets of androgen-mediated regulation, but also contribute to our understanding of the influence that androgens have on AR-related diseases [63].

In a previous study [64, 65], we reported the association of NR super-family members (AR, oestrogen receptor-α, and pregnane X receptor) with mitotic chromatin throughout mitosis in living and fixed cells using green fluorescent protein (GFP)-tagged receptors and indirect immunocytochemistry respectively. It was postulated that the docking of NRs to the condensed mitotic chromatin platform has important physiological ramifications. In this context, gene book-marking and BIOPIT (biomolecular imprints offered to progeny for inheritance of traits) hypotheses were proposed to explain the physiological significance of the mitotic chromatin association of transcription factors [63-68]. It was reported that, during mitosis, ‘interphase nuclear foci’ co-migrate with condensing chromatin as transcription units to the progeny cells, ensuring that the progenitor cells transmit a biomolecular blueprint of transcription status to subsequent generations to express and sustain their characteristic proteome [64]. The ‘biopit model’ attempted to explain how the disruption of ‘biopit markings’ by therapeutic anti-hormones or endocrine disruptors over prolonged periods may lead to an erosion of cellular transcription memory with deleterious cellular consequences [63, 64]. Although the binding of ligand-activated AR to mitotic chromatin during mitosis has been reported [64], there are several questions that warrant detailed investigation. For example, which domain(s) of AR are responsible for binding with mitotic chromatin? What factors regulate receptor–chromatin interactions? Are there any inter- or intra-molecular sequence determinants that assist the AR association with mitotic chromatin? Has AR any functional role in chromatin condensation or decondensation processes during the cell cycle? Abnormal receptor localization and intracellular dynamics appears to be affected under pathological conditions. This implies that the intracellular status of AR with respect to mitotic and interphase chromatin (i.e. when mutations in AR are the cause of pathogenesis) needs to be determined. Therefore, it is pertinent to unravel these mysteries by deriving our understanding from pre-existing knowledge of the receptor mutations implicated in receptor dependent pathogenesis. We hypothesize that studies in this direction may unravel some of the interesting inter- and/or intra-molecular determinants that are responsible for aberrant receptor dynamics and chromatin association, thereby highlighting the causative events of AR-related pathogenesis.

To undertake a systematic approach, we have extensively employed fluorescent protein tags and determined the role(s) of the AR functional domains and the NLS with respect to the distribution of the receptor in living cells during interphase and mitosis. Furthermore, we have shown that full-length AR is essential for the association with mitotic chromatin (chromosomes) and also that the bipartite NLS is a key determinant in executing this phenomenon because NLS deletion mutants of AR were devoid of any association with mitotic chromatin.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Every functional domain contributes to the AR association with mitotic chromatin

In recent studies, we have established the constitutive and agonist-mediated association of some NRs (pregnane X receptor, AR and oestrogen receptor-α) with mitotic chromatin throughout all stages of cell division. Observations made in a CaP line (LNCaP expressing endogenous AR), a liver cancer cell line (HepG2 stably expressing AR) and COS-1 cells transiently expressing GFP-tagged receptor showed that the phenomenon of receptor association with mitotic chromatin is neither cell-specific, nor a result of the overexpression of receptor protein [64, 65, 67].

To investigate whether any specific domain or motif of AR has a role in the association with mitotic chromatin, we performed a systematic study of AR domains using a multitude of deletion mutants tagged with fluorescent proteins and expressed in COS-1 cells. A schematic presentation of the protein expression profile of all of the ‘domain deletion mutants’, point mutants and mutants with a minor deletion in the functional bipartite NLS overlapping the DBD and hinge region, as well as leucine residues flanking the bipartite NLS, is provided in Fig. 1. The protein expression profile (western blot analysis) of GFP-tagged AR and its mutants indicates that the all of the NLS mutants of AR used express receptor protein efficiently and differ only moderately in their expression levels. We observed that GFP-NTD and GFP-ΔLBD constructs expressed truncated receptor protein at a higher level compared to full-length GFP-AR and GFP-ΔDBD (Fig. 1B). GFP-LBD and RFP-ΔNTD bands could not be detected because this part of the receptor protein lacks the epitopes for the AR antibody. However, GFP-LBD expression was confirmed using GFP antibodies (Fig. 1C). β-actin is used as a loading control (Fig. 1B,C). GFP-tagged wild-type AR, ΔNTD, ΔDBD and LBD were found predominantly in the cytoplasmic compartment during interphase in dihydrotestosterone (DHT)-untreated cells (Fig. 2A). Furthermore, these four receptor variants did not bind to mitotic chromatin in the absence of DHT (Fig. 2B). DHT-activated wild-type GFP-AR was imported into the nucleus within 60 min and organized in the nucleoplasm to form ‘nuclear foci’ (Fig. 2A). In mitotic cells, wild-type DHT-bound GFP-AR was found to be clearly associated with mitotic chromatin throughout the mitotic stages. For clarity, only the metaphase stages of mitosis are shown in Fig. 2B. In the presence of DHT, RFP-ΔNTD was also imported into the nucleus and formed ‘nuclear foci’, although not as prominently as wild-type AR (Fig. 2A). However, DHT-bound RFP-ΔNTD did not show any apparent association with mitotic chromatin (Fig. 2B). These observations indicate the involvement of AR-NTD for the association with mitotic chromatin. After 4 h of treatment with DHT, GFP-ΔDBD was partitioned into the nuclear and cytoplasmic compartment (N=C). The receptor was distributed in the nucleoplasm or cytoplasm as dotted structures, in the form of ‘cytoplasmic bodies’ and ‘nuclear bodies’ that were large in size, unlike the ‘nuclear foci’ with wild-type AR. The cytoplasmic bodies were large in size and more numerous than the nuclear bodies (Fig. 2A). Because GFP-ΔDBD does not contain the classical NLS, it is suggested that GFP-ΔDBD could be imported by another less characterized NLS in the LBD [41]. Similar to RFP-ΔNTD, DHT-treated GFP-ΔDBD also failed to associate with mitotic chromatin (Fig. 2B). After 4 h of DHT treatment, GFP-LBD was localized equally in the nucleus and cytoplasm (N=C) of cells and was distributed homogenously (Fig. 2A). DHT-treated GFP-LBD also did not associate with mitotic chromatin (Fig. 2B). Although LBD does not have a classical bipartite NLS, it undergoes nuclear import, presumably utilizing a second NLS present in the LBD [41, 42].

image

Figure 1. Schematic representation of AR constructs with disease-inflicting and introduced deletion mutations used in the present study and their protein expression profile by western blot analysis. (A) Human AR amino acid sequence corresponding to the second zinc finger (boxed) with the C-terminal extension to the hinge region expanded [28, 41]. Arginine (R) and lysine (K) residues are shown in bold, and the mutations in the bipartite nuclear localization signal [35, 41, 49] and the adjacent leucine (L) residues are depicted in blue. TAD, transcription activation domain; H, hinge region; RFP, red fluorescent protein; A, alanine; C, cysteine; D, aspartic acid; G, glycine; M, methionine; P, proline; Q, glutamine; S, serine; T, threonine; W, tryptophan; Y, tyrosine. The lower panel shows the schematic representation of the AR domain deletion mutants constructed as fluorescent protein-tagged AR chimeras. (B) Protein expression profile of all of the GFP/RFP-tagged AR plasmids (wild-type, mutants and truncated) in COS-1 cells by western blotting. All of the GFP-tagged AR protein bands with NTD were detectable, except GFP-LBD and RFP-ΔNTD lacking the NTD domain. However, both plasmids express GFP-LBD and RFP-ΔNTD proteins, as observed in living cells by fluorescence microscopy. (C) Western blot of GFP-AR (full-length) and GFP-LBD expressed in COS-1 cells using anti-GFP sera. β-actin was used as a loading control.

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image

Figure 2. Chromatin binding response of AR domain deletion mutants during interphase and mitosis. COS-1 cells were transiently transfected with 500 ng of fluorescent protein tagged wild-type AR and its domain deletion mutants (ΔNTD, ΔDBD, LBD, NTD and ΔLBD) plasmids and incubated in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide: ethanol (vehicle) for control or with 10−8 m of DHT. After 4 h of incubation with DHT, cells were observed, counted and recorded under a fluorescence microscope. Hoechst was used as a fluorescent dye to visualize corresponding nuclei/mitotic chromatin. (A) Interphase localization of untreated and DHT-treated wild-type AR and AR domain deletion mutants (B) Localization of untreated and DHT-treated AR and its domain deletion mutants during mitosis. (C) Localization of AR domain deletion mutants (NTD and ΔLBD) during interphase and metaphase. In the set of images, the left panel shows the GFP/RFP fluorescence emitted from the tagged receptors, the middle panel shows the Hoechst-stained DNA of the corresponding cells and the right panel shows the merged images of the green/red and blue fluorescence. Quantitative data and further details about the constructs are provided in Table 1. Scale bar = 5 μm.

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By contrast to other AR domain deletion mutants, NTD and ΔLBD do not contain the LBD for ligand binding. Thus, both mutants do not respond to DHT. GFP-NTD was observed to be distributed in both the cytoplasmic and nuclear compartment (N=C) (Fig. 2C). Interestingly, in the cytoplasmic compartment, GFP-NTD was distributed homogenously, whereas it formed large nuclear bodies in the nuclear compartment in almost 70% of observed cells. The remaining 30% of cells showed a homogenous distribution in the nucleus. The N=C localization pattern of GFP-NTD indicates that it may have a third NLS in AR, as suggested by Kaku et al. [42]. During mitosis, GFP-NTD also failed to associate with mitotic chromatin (Fig. 2C).

In subsequent experiments, GFP-ΔLBD was found to be localized predominantly in the nucleus of interphase cells. It was organized into ‘nuclear foci’ similar to the wild-type GFP-AR (Fig. 2C). GFP-ΔLBD showed varied results compared to other domain deletion mutants in mitotic cells. Interestingly, GFP-ΔLBD was observed to be associated with mitotic chromatin, albeit weakly in approximately 70% of cells. In the remaining 30% of mitotic cells, GFP-ΔLBD did not show any significant association with mitotic chromatin (Fig. 2C).

Intervention with the bipartite NLS sequence disrupts the AR interaction with mitotic chromatin

Because NLS occupies a critical position in the DBD-hinge region of the receptor it was important to explore whether the mutations within NLS and the neighbouring hinge region (which have been shown to alter nuclear import) can also alter the receptor binding to mitotic chromatin. Accordingly, GFP-AR NLS mutants (RK617,18GA, RK617,18SA, M624D, Δ629-633 and LLL634,7,9AAA plasmids) were transiently expressed in COS-1 cells and cultured in steroid-free medium for 24 h. All of the mutants of the NLS region used in the present study are shown in Fig. 1. After receptor expression, cells were treated either with dimethylsulfoxide : ethanol (vehicle) as a control or with 10−8 m of DHT. In the unliganded condition, all of the NLS mutants were localized in the cytoplasmic compartment of interphase cells and none of them were found to be associated with mitotic chromatin (Fig. 3). After 4 h of incubation with DHT, RK617,18GA and RK617,18SA were localized predominantly as the N=C distribution pattern in approximately 65% of cells (Fig. 3A). After DHT treatment, nuclear import of the mutants was conspicuously slow compared to wild-type AR, which reached completion within 60 min. All of the cells showed larger cytoplasmic and nuclear bodies for the expressed receptor, which are sometimes referred to as aggregations. DHT-bound M624D showed the N>C localization pattern in 80% of cells with nuclear bodies. DHT-treated Δ629-633 showed the C>N and N=C localization pattern in 60% and 30% of cells, respectively, with the formation of cytoplasmic and nuclear bodies. Nuclear transfer of LLL634,7,9AAA after DHT treatment was slow but almost complete. Mutant LLL634,7,9AAA was observed to be localized in the N and N>C pattern in 60% and 30% of cells, respectively, with the formation of ‘nuclear foci’ after 4 h of DHT treatment (Fig. 3A). Interestingly, during mitosis, all DHT-activated AR NLS mutants (RK617,18GA, RK617,18SA, M624D and Δ629-633) failed to bind to mitotic chromatin, except LLL634,7,9AAA (Fig. 3B and Table 1). Leucine residues flanking the bipartite NLS mutant LLL634,7,9AAA indicate a role for the NLS in mitotic chromatin association because NLS mutants failed to achieve the mitotic chromatin association. DHT-activated RK617,18GA formed dot-like structures in mitotic cells (Fig. 3B). Similar experiments were performed in the human HEK-293 cell line with GFP-tagged AR (wild-type) and its NLS mutants (RK617,18GA, RK617,18SA, M624D, Δ629-633, KKK630,2,3AAA and LLL634,7,9AAA plasmids) to confirm that the results observed in COS-1 cells are not artefacts of the overexpression of GFP-tagged AR (wild-type) or mutant proteins and that this phenomenon is not specific to COS-1 cells only. The results obtained in HEK-293 cells are in agreement and comparable with the results obtained from COS-1 cells (Fig. S1). Therefore, the results observed in COS-1 cells are not artefacts of the overexpression of GFP-tagged AR (wild-type) or its mutant proteins.

Table 1. Localization of wild-type GFP-AR and its mutants in COS-1 cells during interphase and mitosis. K630A and KKK630,2,3AAA are shown with lesser [56, 60] or hyper- [60] activity depending on promoter choice. C, cytoplasmic; N, nuclear. –, Unavailability of related information for the mutant
GFP/RFP-tagged proteinTranscriptional activityAcetylation statusDNA bindingInterphase localizationCytoplasmic bodiesNuclear foci/bodiesMitotic chromatin association
+DHT±DHT±DHT– DHT+DHT (4 h)+DHT (4 h)±DHT (4 h)– DHT+DHT
  1. a

    DNA-binding demonstrated by substituting a lysine residue with valine instead of alanine.

Wild-type ARActive [41,49]Yes [55–58,62]Yes (strong) [58–62]CNNoFociNoYes (strong)
ΔNTDLess activeNo [55]Yes (weak)CNNoFociNoNo
ΔDBDInactiveNoCC=NYesBodiesNoNo
LBDInactiveNo [55]NoCC=NNoNoneNoNo
NTDInactiveNo [55]NoC=NC=NNoBodiesNoNo
ΔLBDLess active [41]Yes [55]Yes [41]NNNoFoci∼70% Yes (weak)∼70% Yes (weak)
AR NLS mutants
Region of mutation: 616LRKCYEAGMTLGARKLKKLGNLKL639
RK617,18GALess active [41]CC=NYesBodiesNoNo
RK617,18SALess active [41]CC=NYesBodiesNoNo
M624DInactive [49]CN>CNoBodiesNoNo
Δ629-633Hyperactive [41,61]No [55,61,62]Yes (weak) [61]CC>NYesBodiesNoNo
K630AHyperactive [49,59]No [55,56,62]Yes (weak) [61] aCN>CYesFociNo∼50% (weak)
K632AHyperactive [49,59]No [55,56,62]Yes (weak) [61] aCN>CNoFociNo∼50% (weak)
K633AHyperactive [49,59]No [55,56,62]Yes (weak) [61] aCN>CNoFociNo∼50% (weak)
KKK630,2,3AAALess active [49,60]No [55,60]Yes (weak) [60,61]CC>NYesBodiesNo< 50% (weak)
LLL634,7,9AAAActive [41]Yes [41]CN>CYesFociNoYes (strong)
AR NLS mutants associated with pathological phenotypes
L616P (AIS)Inactive[49]CN>CYesBodiesNoNo
R617P (AIS)Inactive[49]Yes (weak) [52,53]CN=CYesBodiesNoNo
C619Y (CaP)Inactive[49]Yes (weak) [52,53]CN>CYesBodiesNoNo
R629W (AIS)Hyperactive [49,59]CN>CNoFociNo∼50% (weak)
R629Q (CaP)Hyperactive [49,59]Yes [59]CN>CYesFociNo∼50% (weak)
K630T (CaP)Hyperactive [49,58,59]Yes [58,62]Yes [58,59]CN>CNoFociNo∼50% (weak)
image

Figure 3. Mutations in the functional bipartite NLS in the hinge region of AR abort the receptor interaction with mitotic chromatin. COS-1 cells were transiently transfected with 500 ng of the indicated GFP-AR NLS mutant plasmids and incubated in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide: ethanol (vehicle) for control cells or with 10−8 m of DHT. After 4 h of incubation with DHT, cells were visualized and recorded under a fluorescence microscope. Hoechst was used as a fluorescent dye to stain corresponding nuclei/mitotic chromatin. (A) Localization of untreated and DHT-treated NLS mutants of AR in interphase cells. (B) Mitotic localization of untreated and DHT-treated NLS mutants of AR. The left panels show the GFP-tagged AR mutants, the middle panels show the Hoechst-stained DNA of the corresponding cells and the right panels show the merged images for fluorescence. Quantitative data and further details about the constructs are provided in Table 1. Scale bar = 5 μm.

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Lysine[RIGHTWARDS ARROW]alanine mutations in AR NLS impede or prevent AR binding to mitotic chromatin

Mutations within AR NLS (629RKLKK633) and other parts of the hinge region affect nuclear import and acetylation-dependent functions [30, 49, 55-62]. Therefore, it was important to explore whether these mutations can also influence the receptor binding with mitotic chromatin. To investigate the effect of lysine residues on the binding of AR with mitotic chromatin, COS-1 cells were transfected with lysine[RIGHTWARDS ARROW]alanine mutants of GFP-AR (i.e. K630A, K632A, K633A and triple null KKK630,2,3AAA) and cultured in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide : ethanol (vehicle) for control cells or with 10−8 m of DHT. Under unliganded conditions, all of the lysine[RIGHTWARDS ARROW]alanine mutants of AR NLS were localized in the cytoplasm of interphase cells and did not associate with mitotic chromatin (Fig. 4). After 4 h of DHT treatment, K630A, K632A and K633A showed predominantly nuclear (N>C) localization, with the formation of ‘nuclear foci’ in interphase cells. By contrast, the NLS triple null KKK630,2,3AAA mutant showed C>N localization, with the formation of cytoplasmic bodies in interphase cells. After 24 h of DHT treatment, KKK630,2,3AAA localization was still C>N, whereas mutants K630A, K632A and K633A were imported completely to the nuclear compartment. In DHT-treated cells, all of the lysine[RIGHTWARDS ARROW]alanine mutants of AR NLS showed weak binding with mitotic chromatin in approximately 50% mitotic cells. In the remaining 50% of cells, none of the lysine[RIGHTWARDS ARROW]alanine mutants bound to the mitotic chromatin (Fig. 4).

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Figure 4. Lysine[RIGHTWARDS ARROW]alanine (mutation) in AR NLS reduces or prevents the AR association with mitotic chromatin. COS-1 cells were transiently transfected with 500 ng of GFP-AR Lys mutants (K630A, K632A, K633A and KKK630,2,3AAA plasmids) and incubated in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide: ethanol (vehicle) for control cells or with 10−8 m of DHT. After 4 h of incubation with DHT, cells were observed and recorded under a fluorescence microscope. Hoechst was used as a fluorescent dye to visualize corresponding nuclei/mitotic chromatin. (A) Subcellular localization of untreated and DHT-treated Lys[RIGHTWARDS ARROW]Ala mutants of AR-NLS in interphase. (B) Mitotic localization of untreated and DHT-treated Lys[RIGHTWARDS ARROW]Ala mutants of AR-NLS. The left panels show the GFP-tagged AR mutants, the middle panels show the Hoechst-stained DNA of the corresponding cells and the right panel shows the merged images for fluorescence. Quantitative data and further details about the constructs are provided in Table 1. Scale bar = 5 μm.

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AR mutants associated with the pathological phenotype (AIS and CaP) reveal a differential binding response on mitotic chromatin

The point mutations within the AR-NLS region (616–634) that are associated with the clinical manifestations of AIS and CaP include L616P (AIS), R617P (AIS), R629W (AIS), C619Y (CaP), R629Q (CaP) and K630T (CaP). These GFP-tagged AR point mutants were transiently expressed in COS-1 cells and examined for their localization and ligand responses. All of the GFP-AR mutants associated with AIS and CaP localized in the cytoplasm of interphase cells when in an unliganded state and did not associate with mitotic chromatin (Figs 5 and 6). After 4 h of DHT treatment, the L616P mutant receptor was observed to be predominantly nuclear (N>C), with cytoplasmic and nucleoplasmic bodies that were larger in size compared to the ‘nuclear foci’ of wild-type AR. Mutant L616P receptor was slowly but completely imported into the nucleus after 24 h of incubation with DHT. During mitosis, DHT-bound L616P receptor failed to bind to the mitotic chromatin, even after 24 h of incubation (Fig. 5). The R617P mutant was localized in the N=C distribution pattern after 4 h of DHT treatment, exhibiting atypical large cytoplasmic bodies and larger nuclear bodies in the nucleoplasm during interphase. Mutant R617P was completely imported into the nucleus after 24 h of DHT incubation. During mitosis, DHT-activated R617P mutant did not associate with mitotic chromatin but formed dot-like structures in the cytosol of mitotic cells (Fig. 5). DHT-activated R629W mutant showed a predominantly nuclear (N > C) distribution pattern in interphase cells, whereas it showed a weak association with mitotic chromatin in approximately 50% of mitotic cells. The remaining cells did not show any association of the receptor with mitotic chromatin (Fig. 5).

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Figure 5. Response of AIS-associated AR point mutants on mitotic chromatin. COS-1 cells were transiently transfected with 500 ng of GFP-AR AIS point mutant constructs (L616P, R617P and R629W) and incubated in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide: ethanol (vehicle) for control cells or with 10−8 m of DHT. After 4 h of incubation with DHT, cells were recorded under a fluorescence microscope. Hoechst was used as a fluorescent dye to visualize corresponding nuclei/mitotic chromatin. (A) Interphase localization of untreated and DHT-treated AR AIS mutants. (B) Mitotic localization of untreated and DHT-treated AR AIS mutants. In the ‘INTERPHASE and ‘METAPHASE’ images, the left panels show the GFP-tagged AIS-associated AR mutants, the middle panels show the Hoechst-stained DNA of the corresponding cells and the right panel shows the merged images for fluorescence. Quantitative data and additional details about the constructs are provided in Table 1. Scale bar = 5 μm.

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image

Figure 6. Response of CaP-associated AR point mutants on mitotic chromatin. COS-1 cells were transiently transfected with 500 ng of GFP-AR CaP point mutants (C619Y, R629Q and K630T plasmid constructs) and allowed to express the receptor in steroid-free medium for 24 h. After receptor expression, cells were treated with dimethylsulfoxide: ethanol (vehicle) for control cells or with 10−8 m of DHT. After 4 h of incubation with DHT, cells were recorded under a fluorescence microscope. Hoechst was used as a fluorescent dye to visualize corresponding nuclei/mitotic chromatin. (A) Subcellular localization of untreated and DHT-treated AR CaP mutants in interphase cells. (B) Localization of untreated and DHT-treated AR CaP mutants during mitosis. The left panels show the GFP-tagged AR mutants, the middle panels show the Hoechst-stained DNA of the corresponding cells and the right panel shows the merged images for fluorescence. Quantitative data and further details about the constructs are provided in Table 1. Scale bar = 5 μm.

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DHT-treated C619Y, R629Q and K630T AR mutants previously implicated with CaP [49, 53, 59, 61, 69, 70] exhibited a predominantly nuclear (N>C) distribution in interphase cells. The C619Y and R629Q mutants formed larger cytoplasmic bodies and ‘nuclear foci’. DHT-activated C619Y mutant did not associate with mitotic chromatin, whereas DHT-activated R629Q and K630T mutants bound weakly with mitotic chromatin in approximately 50% of mitotic cells. The remaining cells did not show any association of these mutants with mitotic chromatin (Fig. 6).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study employing fluorescent protein-tagged AR, its domain deletion mutants and various NLS mutants, we have shown that functional domains and the bipartite NLS of AR govern the receptor intracellular distribution and ligand response in living cells. Furthermore, we have determined that full-length AR is necessary for the mitotic chromatin association and that its bipartite NLS overlapping the hinge region is apparently the key determinant in executing this phenomenon because NLS deletion mutants abolished any association. No domain by itself was capable of associating with mitotic chromatin. It is therefore reasonable to presume that each of the AR domains may contribute to the basic conformity of the AR protein with respect to its successful association with mitotic chromatin.

Using GFP-tagged AR domain deletion mutants, we have demonstrated that, in the presence of DHT, none of these mutants associated with mitotic chromatin, except GFP-ΔLBD, which was associated in approximately 70% of mitotic cells, albeit weakly. These results indicate that each of the major domains (NTD, DBD and LBD) plays a significant role in the interactions between DHT-induced AR and mitotic chromatin. It is well known that NR-DBD binds with chromatin on DNA response elements in interphase cells [28, 31, 71]. Therefore, it is apparent that AR-DBD may also be required for direct binding with mitotic chromatin, as in the case of interphase chromatin. This also explains why DHT-induced GFP-ΔDBD (i.e. that lacks DBD) could not associate with mitotic chromatin. Similarly, the failure of the DHT-activated RFP-ΔNTD association with mitotic chromatin may be explained on the basis of a loss of the N/C terminal interaction and protein conformational changes because it lacks NTD [31, 33, 71]. GFP-LBD and GFP-NTD domain mutants lack NTD.DBD and DBD.LBD domains, respectively. Therefore, the observed receptor abortion from mitotic chromatin may be attributed primarily to the absence of DBD and other domains. Unlike the other domain mutants, the GFP-ΔLBD domain mutant has been shown to associate sparingly with mitotic chromatin in approximately 70% of mitotic cells. This behaviour of GFP-ΔLBD with mitotic chromatin may be the result of a lack of stable binding with mitotic chromatin. This is substantiated by studies where LBD has been reported to stabilize AR binding with chromatin [31, 71]. Moreover, LBD is also involved in the N/C terminal interaction of AR, which plays a key role in interphase chromatin binding and receptor function. Taken together, the results from experiments utilizing AR domain deletion mutants suggest that each functional domain (NTD, DBD and LBD) has some critical role providing competency to AR for an association with mitotic chromatin. Hence, for efficient mitotic chromatin binding, all of the domains of AR are essential, implying that the overall protein conformation and other structural features may be a prerequisite for intramolecular interactions. An overall outline of the localization pattern of different AR domain deletion mutants and their behaviour with mitotic chromatin is provided in Table 1.

Nuclear import is a necessary event in the regulation of AR transcriptional functions. The NLS overlapping the DBD and hinge region plays a critical role in the regulation of nuclear import of receptor and in stabilizing its binding to DNA [49, 55, 57, 62, 72]. A major question that arises is whether these functional deviations are reflected in ‘nuclear foci’ formation and mitotic chromatin binding responses. Accordingly, we used several mutants of AR NLS and the hinge region (i.e. that have been shown to alter nuclear import) to investigate whether NLS mutants also alter receptor binding to mitotic chromatin. Interestingly, in our more defined studies using GFP-AR NLS mutants (RK617,18GA, RK617,18SA, M624D and Δ629-633), we showed that mutations in the bipartite NLS region disrupt the DHT-induced AR binding with mitotic chromatin. By contrast, DHT-bound LLL634,7,9AAA mutant has been shown to retain a mitotic chromatin associating property. Because LLL634,7,9AAA mutant flanking the bipartite NLS binds with mitotic chromatin, it indicates the involvement of the NLS in mitotic chromatin association because all other NLS mutants failed to achieve this association. These results confirm the role of NLS in imparting the property to the receptor for an association with mitotic chromatin. Furthermore, the success and failure of binding to mitotic chromatin may also be attributed to (a) changes in the receptor conformation; (b) a reduction in intramolecular interactions; (c) the stability of AR on mitotic chromatin; and (e) intermolecular interactions with some other adapter molecules that are required for AR targeting. First, it may be reasonable to speculate that the mutations in the NLS region may alter the protein conformation in such a way that it cannot establish a ‘good-fit’ relationship with the binding sites on mitotic chromatin. Second, the NLS region overlaps with both the DBD and the hinge region and it is evident that this region provides stability and flexibility to the receptor–chromatin interactions [28, 34, 59, 61, 62]. Therefore, the mutations in this region can diminish the AR and mitotic chromatin interactions. Third, the existence of some adapter molecules (e.g. importin-α/β) that may be interacting with the NLS region for the nuclear import of receptors is plausible. At least in one instance, the involvement of importin-α/-β in protein targeting to mitotic chromatin has been reported for human chromokinesin-like DNA binding protein [73].

DHT-activated GFP-AR mutants with lysine[RIGHTWARDS ARROW]alanine mutations in the NLS region demonstrate reduced binding with mitotic chromatin. These results further substantiate the involvement of the NLS region in the regulation of the association of AR with mitotic chromatin. A failure in association may be explained by the reduced interaction between mitotic chromatin and the receptor or participating adaptor molecules. It is reasonable to presume that post-translational modifications such as acetylation at lysine residues may represent other factor contributing to the AR association with mitotic chromatin.

Furthermore, AR NLS mutants associated with pathological manifestations for AIS and CaP have shown a differential response on mitotic chromatin. All of the DHT-bound AR mutants of AIS and CaP exhibit delayed nuclear import and an atypical organization as cytoplasmic and nuclear bodies. The results from receptor import, utilizing disease-inflicting mutants of AR, are in agreement with previous studies [49, 53, 54, 59, 61, 70]. We observed that DHT-activated L616P (AIS), R617P (AIS) and C619Y (CaP) mutants do not exhibit binding with mitotic chromatin. Nonetheless, DHT-activated R629W (AIS), R629Q (CaP) and K630T (CaP) have shown reduced binding with mitotic chromatin in only approximately 50% of mitotic cells, with the remaining cells exhibiting no such association. Interestingly, the AIS and CaP mutants that have mutations located in DBD do not show any binding with mitotic chromatin, whereas the other AIS and CaP mutants with mutations located in the hinge region exhibit reduced binding in approximately 50% of mitotic cells. In conclusion, these results indicate the involvement of DBD in mitotic chromatin binding and a role for the NLS (hinge region) in providing stability to the receptor with mitotic chromatin.

Moreover, NLS mutants have shown a slower nuclear import in response to DHT, exhibiting atypical accumulation as cytoplasmic bodies (aggregations) and nuclear bodies (Table 1). Whether the appearance of these features and localization can be attributed to alterations in receptor conformation, solubility, stability or intermolecular interactions needs to be investigated. Nonetheless, from these observations, it is conceivable that this aberrant subcellular localization of AR mutants could be one plausible reason for the pathogenesis of several AR-related diseases [18, 49, 54, 59, 61, 69, 70, 74]. Interestingly, by observing the subcellular localization of receptor and the presence or absence of typical ‘nuclear foci’, it was possible to predict the transcription status of AR (Table 1). Similarly, from the transcription status, it was possible to predict the success or failure of the AR association with mitotic chromatin.

N/C-termini interactions have been shown to stabilize AR binding to interphase chromatin [31, 32, 61, 62, 72]. We speculate that such interactions may also be required for the AR association with mitotic chromatin. However, the phenomenon of receptor association with mitotic chromatin needs further investigation by the introduction of effective mutations influencing N/C-terminal interactions.

In conclusion, we have shown that none of the AR domains by itself was capable of associating with mitotic chromatin. Similarly, it was evident that the absence of any major receptor domain either abolished or weakened the chromatin association. It was apparent that, for efficient mitotic chromatin binding, all of the domains of AR are essential and the overall protein conformation or structural features may be a perquisite for these intramolecular interactions. A subsequent detailed analysis using several NLS mutants indicated that the bipartite NLS region of AR functions as the ‘mitotic chromatin binding-determining region’ that has a distinct and novel role in the regulation of the AR association with mitotic chromatin. The possibility of the involvement of post-translational modifications such as acetylation, phosphorylation or methylation in the mitotic chromatin binding-determining region, as well as some potential adaptor molecules such as importins or coregulatory proteins in mediating the AR association with mitotic chromatin, cannot be ruled out. These are some of the potential avenues that require rigorous investigation. It is also possible that the impairment of ligand-dependent nuclear translocation, mislocalization, ‘nuclear foci’ formation and a failure of AR association with mitotic chromatin may be central to AR-related pathogenesis such as AIS and CaP. Also, it appears that complete ‘nuclear foci’ formation is essential for the AR association with mitotic chromatin. Future investigations into the mechanisms of these events are expected to provide further insight into the pathogenesis of AR-mediated diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Biochemicals

DHT, Escort III, Hoechst, horseradish peroxidase-conjugated antirabbit secondary serum raised in goat and mammalian cell culture reagents were procured from Sigma Chemicals Co. (St Louis, MO, USA). Plastic wares were purchased from Corning Costar Corp. (Lowell, MA, USA). Steroid-stripped serum and fetal bovine serum was purchased from Hyclone (Logan, UT, USA). Restriction enzymes were obtained from New England Biolabs (Beverly, MA, USA). Antibodies against full-length human AR and β-actin were developed in our laboratory and have been described previously [64]. GFP polyclonal antibodies are a product of Abcam (Cambridge, MA, USA) and were a kind gift from S. K. Dhar (JNU, New Delhi, India). All other general chemicals used were of analytical grade and procured from different commercial sources.

Plasmids

GFP-tagged human AR (GFP-AR) and its mutants GFP-ΔLBD, GFP-NTD, GFP-ΔDBD, GFP-LBD, GFP-AR RK617,8GA, GFP-AR-Δ629-633 and GFP-AR LLL634,7,9AAA were gifts from O. A. Janne (University of Helsinki, Finland) and have been reported previously [41, 64]. AR NLS mutants GFP-AR M624D, GFP-AR K630A, GFP-AR K632A, GFP-AR K633A, GFP-AR KKK630,2,3AAA and RK617,8SA, as well as AR NLS mutants associated with pathological phenotypes GFP-AR L616P (AIS), GFP-AR R617P (AIS), GFP-AR R629W (AIS), GFP-AR C619Y (CaP), GFP-AR R629Q (CaP) and GFP-AR K630T (CaP), were gifts from D. E. Neal (Cancer Research UK Cambridge Research Institute, Cambridge, UK) and have been described previously [49]. Human AR-DBD.LBD was isolated from GFP-AR and subcloned in frame between HindIII and BamHI restriction sites of pDsRed-Express-C1 (BD Biosciences, Franklin Lakes, NJ, USA) and is termed RFP-ΔNTD.

Mammalian cell cultures and transient plasmid transfection

ATCC cell lines COS-1 and human HEK-293 were obtained from the National Cell Repository (NCCS, Pune, India). COS-1 and HEK-293 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 μg·mL−1 penicillin and 100 μg·mL−1 streptomycin (complete medium). The cultures were maintained in a humidified incubator maintained at 5% CO2 and a 95% air atmosphere at 37 °C. Transient DNA transfections in COS-1 or HEK-293 cells were performed using Escort III reagent in accordance with the manufacturer's instructions.

Fluorescence microscopy of living cells

COS-1 or HEK-293 cells were seeded into 35-mm plates and allowed to grow to 70–80% confluency. The next day, cells were transfected with 500 ng of plasmids using Escort III reagent. After 12–15 h of transfection incubation, cells were supplemented with 5% steroid stripped serum DMEM followed by treatment with 10−8 m DHT prepared in dimethylsulfoxide : ethanol (1 : 1). This concentration of DHT has been extensively used previously and is acceptable in experiments relating to mammalian cell culture as performed in laboratories working in the area of intracellular dynamics and the transcriptional response of AR [18, 32, 43]. After the treatment period, events for nuclear translocation, sub-nuclear compartmentalization and mitosis were evaluated under a fluorescence microscope. To facilitate visualization of the nucleus, Hoechst (final concentration 1 μg·mL−1) was added to living cells at least 1 h before imaging. The fluorescent cells were viewed and imaged using an upright fluorescence microscope (model 80i; Nikon, Tokyo, Japan) equipped with water immersion objectives and connected to cooled charge-coupled device digital camera (model Evolution VF; Media Cybernatics, Inc., Bethesda, MD, USA). Cell images were captured and analyzed with image proplus, version 5.0 (Media Cybernatics, Inc.). Images were processed using standard image-processing techniques.

Quantification of fluorescent cells in interphase and mitosis

For statistical analysis of the subcellular localization and association of GFP-tagged wild-type AR and its mutants with mitotic chromatin, the receptors expressing interphase and mitotic cells were counted in the absence and in presence of 10−8 m DHT. At any time point, 100 interphase and 50–100 mitotic cells expressing GFP-AR and mutants were scored for subcellular localization and mitotic chromatin association, respectively. Hoechst was used as a fluorescent dye to visualize and identify the interphase nuclei or mitotic DNA. Fluorescence was considered nuclear (N) when it was exclusively in the nucleus or cytoplasmic (C) when present exclusively in the cytoplasm. When the protein was present primarily in the nucleus or primarily in the cytoplasm, it was considered N>C and C>N, respectively. When the fluorescence was uniformly divided between the nucleus and the cytoplasm, it was classified as N=C [67].

Western blotting

COS-1 cells were seeded into six-well culture plates and allowed to grow to 70–80% confluency. The next day, cells were transfected with 500 ng of each of the GFP/RFP-tagged AR plasmids used in the present study using Lipofetamine 2000 reagent (Invitrogen Corporation, Carlsbad, CA, USA). After expression for 24 h, cells were washed twice with cold NaCl/Pi, scraped and collected by centrifugation at 500 g for 5 min at 4 °C. The pellets were resuspended in lysis buffer (20 mm Tris–HCl, pH 8, 1 mm EDTA, 300 mm NaCl, 0.1% NP-40, 0.5 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride and 1× protease inhibitor cocktail) and cell lysates were centrifuged at 15 000 g for 10 min at 4 °C after 20 min of incubation on ice. Equal amount of proteins were finally dissolved in SDS/PAGE sample buffer, denatured by heating at 95 °C for 5 min and resolved by 10% SDS/PAGE. Proteins were electroblotted onto a poly(vinylidene difluoride) membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA). After protein transfer, the blot was blocked with 5% nonfat dry milk in NaCl/Tris for 2 h at room temperature and then incubated overnight with AR antiserum at dilution of 1 : 8000 or anti-β-actin serum at a dilution of 1 : 2000 at 4 °C. GFP antibodies was used at a dilution of 1 : 5000 for 1 h at room temperature. The membrane was then washed three times with NaCl/Tris with 0.1% Tween-20 and incubated for 1 h with a 1 : 10 000 dilution of horseradish peroxidase-conjugated antirabbit serum. The bound antibody complexes were detected using enhanced chemiluminescence.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The research work reported was financially supported by a research grants to R.K.T. from the Council of Scientific and Industrial Research [CSIR grant no. 37(1405)/10/EMR-II) India]. Sanjay Kumar acknowledges CSIR for the award of Senior Research Fellowship. Infrastructural and instrumentation facilities at AIRF-JNU, DST-purse, UGC capacity build-up and UGC-SAP (DRS-I) support to our centre are all gratefully acknowledged. The authors declare that there are no conflicts of interest.

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  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12046-sup-0001-FigS1.zipZip archive705KFig. S1. Mutations or deletion in the functional bipartite NLS in the hinge region of AR abort receptor binding with mitotic chromatin.

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