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Prostate Research Group School of Surgical and Reproductive Sciences Medical School, University of Newcastle upon Tyne Newcastle upon Tyne, England NE2 4HH
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-dihydrotestosterone is a
phenomenon not fully understood. The two-hybrid interaction trap assay
has been used to isolate proteins that interact with the hAR in an
attempt to identify molecules involved in hAR transactivation and
movement. We have identified the actin-binding protein filamin, a
280-kDa component of the cytoskeleton, as an hAR interacting protein.
This interaction is ligand independent but is enhanced in its presence.
The functional significance of this interaction was analyzed using a
cell line deficient in filamin via transient expression of a green
fluorescent protein-hAR chimera. In filamin-deficient cells this
revealed that hAR remained cytoplasmic even after prolonged exposure to
synthetic ligand. Nuclear shuttling was restored when this cell line
regained wild-type expression of filamin. These data suggest a novel
role for filamin, implicating it as an important molecule in AR
movement from the cytoplasm to the nucleus. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The receptor becomes activated on binding of its cognate ligand,
5-dihydrotestosterone (DHT), generated from testosterone by membrane
bound 5-reductase. The hAR is a cis-acting transcription
factor essential for the growth and differentiation of cells within the
prostate and male external genitalia. It is also a pivotal molecule in
the development and progression of prostatic carcinoma.
Little is known about the mechanism of cytoplasmic translocation or associated molecules that coordinate movement of the activated hAR to the nucleus. Work with the hAR fused to a green fluorescent protein (GFP) reporter has allowed the study of the movement of the hAR in vivo. In Cos-7 cells, unliganded hAR is cytoplasmic and is fully translocated to the nucleus within 30 min of steroid addition (8). Although this study highlighted specific hAR kinetics, it did not reveal a molecular mechanism of receptor movement. Specific hAR kinetics has been linked to a bipartite nuclear targeting signal between the DNA binding domain and the hinge region of the hAR. A region of the hAR consisting of two clusters of basic amino acids, separated by 10 residues, is necessary for full receptor nuclear import, and is modulated by elements within the NH2 and carboxyl-terminal regions (9).
The role of the cytoskeleton in the trafficking of steroid hormone receptors has presented conflicting data. Studies involving a progesterone receptor mutant with an inactive karyophilic signal revealed that chemical disruption of the microtubule and actin network neither prevented nor delayed the hormone-dependent transfer of the PR mutant to the nucleus (10). Conversely, receptor trafficking studies involving the GR fused to a GFP highlighted the cytoskeletal network as an important structure in GR movement (11). Chemical disruption of the cytoskeleton by colcemid blocks the okadaic acid-dependent inhibition of hormone-dependent GR recycling to the nucleus. This implies that elements within the cytoskeleton are required for GR movement and that when the cytoskeleton is disrupted the normal shuttling of GR via cytoskeletal tracts utilizing cytoskeletal associating motor proteins is abrogated. A key molecule in this process is the cellular chaperone heat shock protein 90 (Hsp90) that forms a stable heterocomplex with the GR and has been shown to translocate to the nucleus with the receptor (12). The role of Hsp90 in this interaction may be to facilitate translocation by tethering the GR heterocomplex with the cytoplasmic movement machinery. Addition of okadaic acid may prevent the heterocomplex binding to these elements, allowing it to move to the nucleus via diffusion.
GFP-visualized movement of SHRs has also been performed previously for other members of the SHR superfamily (13, 14, 15, 16, 17). These dynamic studies have detailed the subcellular distribution of SHRs both before and after activation by ligand.
We have performed a yeast two-hybrid interaction trap assay (18) to isolate proteins that interact specifically with the hAR. The aim of this study was to identify novel hAR interacting proteins involved in either movement or signaling from the cytoplasm to the nucleus. A cDNA clone encoding a central portion of the cytoskeletal actin-binding protein filamin (ABP 280) was isolated as an hAR interacting protein. Using a GFP-hAR chimera, we have shown that hAR nuclear translocation is abolished in a cell line deficient in the cytoskeletal protein filamin, and hence that filamin appears to have a significant role to play in nuclear translocation of the hAR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). A separate
two-hybrid assay (data not shown) also identified 
-filamin (19) as
an hAR-interacting protein. Both interacting clones span an area of
high homology between the two isoforms overlapping highly conserved
IgG-like repetitive regions. Androgen receptor constructs comprising
either the DNA-binding domain or the ligand-binding domain produced
only basal levels of ß-galactosidase activity. This suggests that the
region of the AR involved with filamin interaction possibly requires
the three-dimensional (3-D) structure created via the juxtaposition of
the two adjacent domains.
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represents in vitro
transcribed/translated protein products before immunoprecipitation. A
minimal domain of interaction was sought using hAR truncations
corresponding to functional domains of the hAR. Figure 2B demonstrates
that the filamin clone
Fil1788-2121 interacts
with the full-length hAR (amino acids 1–918), the DNA-binding domain
and the steroid- binding domain (amino acids 559–918), and the
steroid-binding domain alone (amino acids 624–918). No interaction was
observed between
Fil1788-2121 and the
transactivation domain and the DNA-binding domain (amino acids 1–624).
To further minimize this domain of interaction, hAR truncations
encompassing the steroid-binding domain including the hinge region of
the hAR were assessed for their ability to coprecipitate. Figure 2C
shows that all the constructs containing the hinge region of the hAR
coprecipitate with
Fil1788-2121 including the
hinge region of the hAR alone (amino acids 616–674).
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Filamin and hAR Interact in Vivo
Immunoprecipitation and subsequent Western analysis using hAR and
filamin antibodies (Fig. 3) confirmed
this interaction. A mouse monoclonal antibody was used to
immunoprecipitate filamin protein from prostate LNCaP cells before
detection of hAR by Western analysis. A 98-kDa band, corresponding to
the hAR, was observed (lane 1), which was absent in control lanes
omitting extract or antibody (lanes 2 and 3, respectively). The normal
distribution of filamin has been well documented using
immunohistochemistry, occurring predominantly along stress fibers and
in the cell periphery (20). It is therefore likely that filamin-AR
interaction occurs primarily within the cytoplasm.
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). Deletion constructs in
pACT2 were tested for their ability to interact with hAR in yeast. The
ß-galactosidase activity of these clones was assessed in comparison
to the original filamin clone in pACT2. The relative ß-galactosidase
activities of these clones were quantified in a liquid LacZ assay (Fig. 4B). The minimal domain of interaction was shown to lie within a
150-amino acid fragment encompassing repeats 18 and 19 of the filamin
IgG-like repeats. This area of interaction is similar to that
identified for the glycoprotein Ib, which was found to interact
between repeats 17 and 20. The majority of the other proteins
identified as interactors of filamin appear to be focused around this
C-terminal portion of the protein, indicating that elements within
these IgG-like repeats are responsible for the specificity of
interaction with these varied cellular molecules.
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). COS-7 cells were transfected with
plasmids encoding wild-type hAR (50 ng) and
Fil1788-2121 (0–800 ng),
together with a luciferase reporter plasmid containing an enhancer
element comprising three androgen responsive elements (AREs), p
(ARE)3-Luc. As shown in Fig. 5, hAR is activated
by the synthetic androgen ligand, mibolerone. Increasing amounts of
Fil1788-2121, in the presence or absence of androgen,
cause a dramatic reduction in the level of hAR transactivation.
Parallel experiments, using wild-type filamin in place of
Fil1788-2121, only caused a small reduction in the
mibolerone-induced AR activation, observed at the highest doses of
filamin (data not shown). A negative control promoter (1,659 bp)
derived from the matrix metalloproteinase 2 (MMP2) promoter, which
lacks defined SHR-responsive DNA elements and is known to be
unresponsive to androgens (our unpublished data) was used to
demonstrate that
Fil1788-2121 had no effect
on the inhibition of transactivation via a nonspecific promoter element
(Fig. 5B). These results suggest that the truncated form of filamin,
Fil1788-2121, is acting as
a dominant negative protein by binding hAR and preventing activation of
target genes.
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activation of nuclear factor-
B and TRAF2
activation of the JNK/SAPK (c-jun N-terminal
kinase/stress-activated protein kinase) pathway is inhibited by
the addition of increasing amounts of filamin added in a similar
reporter gene assay. Interestingly, the filamin clone used encompassed
amino acids 1644–2118 of the full cDNA sequence, a fragment
overlapping the one identified in our study as an hAR interactor. It
would be tempting to speculate that the mechanism of filamin-induced
inhibition of target gene expression is similar in both systems.
A GFP-hAR Chimera Remains Cytoplasmic in a Filamin-Negative Cell
Line
To assess the functional significance of the filamin-hAR
interaction, the movement of the hAR in response to ligand was analyzed
in a cell line deficient in filamin. Previous reports regarding the GR
highlighted the importance of the cytoskeleton in the recycling of the
GR to the nucleus. A GFP-hAR fusion was used to visualize hAR
localization. An hAR construct was generated which lacked the
N-terminal transactivation domain
(GFP-hAR559-918),
previously shown to be constituitively nuclear in the absence of ligand
(22), to act as a positive control. A recent investigation with an
AR-GFP chimera reported that unliganded AR was cytoplasmic but rapidly
translocated to the nucleus upon addition of ligand (8). In the
filamin-deficient parental cell line (M2FIL-) we
observed the hAR to be cytoplasmic in location and remained cytoplasmic
even after prolonged exposure to synthetic ligand (Fig. 6, A and B). However, in a stably
transfected derivative of this cell line expressing filamin
(A7FIL+), the normal reported movement of the hAR
in response to ligand was observed. Unliganded cytoplasmic hAR quickly
translocated to a nuclear site upon addition of ligand (C and D).
Interestingly, the
GFP-hAR559-918 protein
remained in the cytoplasm (E) in the presence or absence of ligand in
M2FIL- cells. In contrast unliganded
GFP-hAR559-918 was
constituitively nuclear in A7FIL+ cells (F), as
previously reported (22). We also observed a shift in morphology in the
A7FIL+ cells, where the cells moved along their
axes to become more spherical in response to ligand. This effect has
been reported previously for the GR (23). In accordance with this
study, there appears to be an underlying nuclear organization of
GFP-hAR accumulation. A known nuclear localizing protein, Tip60 (tat
interacting protein 60) (24) was fused to a GFP to analyze the movement
of a non-steroid hormone receptor construct in the M2 and A7 cell
lines. No aberrant localization of this construct was observed in the
absence of filamin in the absence or presence of mibolerone (G and H).
Taken together, these results suggest that the cytoskeletal protein
filamin is important in the nuclear translocation of the activated
hAR.
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). Transient transfection of GFP-hAR and
p(ARE)3-Luc into M2FIL-
cells failed to induce a response to the synthetic ligand, Mibolerone.
In contrast, a similar transfection into A7FIL+
cells resulted in a 4.5-fold increase in luciferase activity in
response to ligand. These results demonstrate that GFP-hAR is
functional and that the inability of GFP-hAR to translocate to the
nucleus in filamin-deficient cells results in lower transcriptional
activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Filamin consists of three main isoforms, assigned , ß, and
. All three share a high degree of sequence homology due to their
role as F-actin cross-linking proteins within the cell. ABP 280 or
-filamin is the original isoform and is located telomeric to the
color vision locus (R/GCP) and centromeric to G6PD in Xq28 (27).
Filamin has previously been identified as interacting with the ß1-integrins, transmembrane molecules involved in cell-cell contact and transmembrane signaling (28). The integrins are also critical in the formation of focal adhesions, local areas on the cell membrane implicated in signal transduction events from the extracellular matrix to the cell nucleus. The structure of focal adhesions also links the integrins with F-actin and actin-associating proteins such as talin, vinculin, and filamin.
Another interacting partner of filamin is the stress-activated protein kinase SEK-1, responsible for signal transduction to downstream molecules JNK and c-Jun affecting cellular responses such as growth and differentiation (29). It is unclear as to the role of filamin in this signal transduction pathway, whether filamin itself is phosphorylated or is acting as an anchoring protein similar in function to JIP-1 (JNK inhibiting protein), which also allows cross-talk between the mitogen-activated protein kinase (MAPK) and SAPK pathways (30), remains to be determined.
More recent studies have identified caveolin-1, a cholesterol-binding integral membrane protein, as an interacting partner for filamin (20). The association between androgen-independent prostate cancer and high expression of caveolin-1 has been documented (31, 32). The identification of filamin as an hAR interactor highlights a potential link between caveolin-1, filamin, and the hAR in androgen-independent prostate cancer.
The melanoma cell line deficient in filamin expression has been well characterized elsewhere (33, 34). The majority of work on this cell line analyzed the cell plasma membrane activity relating to filamin expression. Cells deficient in filamin expression display an increased occurrence of membrane blebbing due to a decrease in the local actin polymerization rate. The retraction of these cell surface protrusions is dependent upon the establishment of a stable actin network, and the prolonged blebbing displayed by the filamin-deficient cell line is due to the local rate of actin polymerization being outpaced by the fluid-driven expansion of the cell membrane. This prolonged blebbing is abolished when filamin is reexpressed to wild-type levels and increases the rate of actin polymerization leading to increased protrusive activity, thus increasing the motility of the cell. The importance of filamin-dependent motility is highlighted in an X-linked male lethal condition known as periventricular heterotopia (PH). This condition arises due to a mutation in the filamin gene resulting in aberrant neural cell migration within the neural network (35; reviewed in Ref. 36).
This study implicates filamin in the cytoplasmic trafficking of the hAR. There has been evidence to suggest that SHRs interact with components of the cytoskeletal architecture, in particular the studies carried out on the GR (11) that demonstrated an intact cytoskeletal network is important in the shuttling function of the receptor. Disruption of the cyto-architecture rendered the GR unable to shuttle between the cytoplasm and the nucleus. This implies that there is a regulatory component within the cytoskeleton essential for the trafficking of molecules to the nucleus. Earlier work (37) presented the first evidence that SHRs associate with the actin cytoskeleton. This study demonstrated that the inactive 8S GR contacts the actin filaments via its binding to Hsp90. Hsp90, similarly to filamin, also has the ability to cross-link actin filaments (38). Upon hormone binding, the Hsp90-receptor complex dissociates and the receptor transforms into the activated complex able to bind glucocorticoid-responsive elements on target genes within the nucleus. The conclusions from this study were that the GR (and possibly other SHRs) bind the actin fibers via their Hsp90 moieties, and this binding anchors the inactive receptor and prevents translocation to the nucleus. Later work, again studying GR translocation, implicated the microtubule network as important in the mechanism of action of GR hormones (39).
The function of filamin, in the context of hAR cytoplasmic
trafficking, may involve the disruption of the high-affinity
association between Hsp90 and the receptor. In the absence of filamin,
the receptor-Hsp90 complex may remain anchored in an inactive state to
the actin filaments even in the presence of steroid and an available
nuclear localization sequence on the receptor. It is tempting to
speculate that filamin may be acting as a mediator between the receptor
and the molecular chaperone Hsp90 controlling the release of activated
receptor after ligand binding. Interaction between heat shock proteins
and filamin has been detailed (40). The small stress protein cvHsp has
been reported to interact with the C-terminal tail of -filamin in a
yeast two-hybrid assay. It is plausible that filamin may interact with
Hsp90 in a similar fashion in complex with the hAR.
This study has shown that the filamin-hAR interaction is not steroid dependent, suggesting receptor anchoring in the absence of ligand. In the presence of ligand the affinity of the interaction is increased and, after Hsp90 dissociation, filamin may be acting as a molecular chaperone, maintaining the active hAR in a stable conformation and fulfilling its other role as a signaling molecule interacting with components of the SAPK and MAPK pathways.
This study also has clinical implications. A prostate cancer susceptibility locus, termed HPCX, has recently been identified in the region of Xq27–28, where the filamin locus maps (41). Linkage analysis in 153 prostate cancer families over a 30 centimorgan (cM) region of HPCX has provided additional support for the existence of a prostate cancer susceptibility locus at Xq28 in a separate study (42). Our investigations highlight an altered response of the hAR to steroid in a cell line lacking expression of filamin, which is restored in its presence. This work provides an insight into hAR movement. Further investigation is required to ascertain whether this interaction extends to other members of the SHR superfamily and the significance of this interaction in clinical specimens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The 1.002-kb filamin clone (hereafter designated Fil1788-2121) in pACT2 was transformed back into the yeast strain PJ69–4A as previously described (44) with truncated forms of the hAR in pAS2–1. Transformants were grown on media lacking leucine and tryptophan, and resultant colonies were grown in selective media overnight at 30 C in the presence of 1 µM dihydrotestosterone. Samples were diluted to A600 0.2 and grown to an A600 of 0.6–0.8 and split into three 1-ml samples. Samples were subjected to liquid LacZ assays as described previously (45). Individual experiments were performed in triplicate.
In Vitro Interaction of hAR and Filamin
Fil1788-2121cDNA
was excised with EcoRI and HindIII digestion from
pAS2–1 and directionally cloned into pT7–7 or pRSETC
(Invitrogen, San Diego, CA) via EcoRI and
HindIII sites. hAR fragments used in the yeast
two-hybrid analysis were excised from pAS2–1 with EcoRI and
cloned into the EcoRI site of pRSETC. Constructs were
sequenced to confirm the maintenance of the open reading frame.
Template DNA was subjected to Geneclean (Anachem, Luton, UK) to remove any contaminant RNase, and the coupled Transcription/Translation Kit (Promega Corp., Madison, WI) was used for in vitro interaction analysis. Templates were labeled with 35S-methionine and the reaction was performed according to the manufacturer’s guidelines. After the 90-min incubation, samples were split and mixed equally, and 1 ml of reaction lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM Na3VO4, 0.5% Nonidet P-40, 1 mM phenylmethlsulfonyl fluoride, 1 mM dithiothreitol, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 25 µg/ml pepstatin) was added. Interacting proteins were precipitated and visualized as previously described (43).
Coimmunoprecipitation
Approximately 107 cells from an LNCaP,
hAR-positive cell line were washed in cold PBS, harvested, and lysed in
reaction lysis buffer on ice for 30 min. Lysates were centrifuged at
14,000 x g at 4 C for 10 min. Supernatants were
precleared with 25 µl protein G sepharose (PGS) after three
washes in reaction lysis buffer and rotated at 4 C for 4 h. PGS
and nonspecific bound protein were removed by centrifugation at
14,000 x g for 5 min. Immunoprecipitations were
performed with 4 µg of mouse monoclonal antifilamin antibody
(Chemicon Intl. Inc., Temecula, CA) or 4 µg of rabbit
polyclonal C-terminal AR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and incubated overnight at 4 C with
rotation. After incubation, a further 25 µl of PGS were added to
immunoprecipitate samples and returned to 4 C for 1 h with
rotation. PGS with bound protein complexes was recovered by
centrifugation at 14,000 x g for 5 min, and samples
were washed once in wash buffer A (PBS, 0.2% Triton-X-100, 350
mM NaCl) and twice in wash buffer B (PBS, 0.2%
Triton-X-100). Sample buffer containing 10% ß-mercaptoethanol was
added to recovered fractions. Samples were resolved on 10% denaturing
polyacrylamide gels for 30 min and transferred to nitrocellulose
filters. The membrane was probed with polyclonal AR (1:500) or
monoclonal filamin (1:1000) antibodies. Immunoreaction was visualized
using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) and developed on
radiographic film.
Deletion Constructs of Filamin Clone
Three deletion constructs of the
Fil1788-2121 in pACT2 were
constructed via PCR. Primers used to generate deletion constructs were:
Fil1788-2081, pACT2FSP
(CLONTECH Laboratories, Inc., Palo Alto, CA), 2R-5'-TGA
GCC CAC CAT AGC CTG C-3';
Fil1930-2081, 1F-5'-GGG
ACT ACA GCA TTC TAG TC-3', 2R-5'-TGA GCC CAC CAT AGC CTG C-3';
Fil1788-1930, pACT2FSP
(CLONTECH Laboratories, Inc.), 1R-5'-GAC TAG AAT GCT GTA
GTC CC-3'. Amplified inserts were cloned into pCR2.1
(Invitrogen) and subcloned into pACT2 via the
EcoRI site. Samples were sequenced to verify the open
reading frame. Deletion constructs were cotransformed with
AR559-918 construct in
pAS2–1 into yeast strain PJ69–4A, and resultant transformants were
assayed for ß-galactosidase activity as described previously
(45).
Transient Transfection
COS-7, M2, and A7 cells were grown in steroid-depleted
RPMI 1640 medium containing 10% FCS 48 h before transfection.
Cells were transfected with either pCDNA3-AR, pEGFP-Tip60, pEGFP-hAR,
pCMV-Fil1788-2121, or
pMMP2-Luc (donated by Dr. Y. Sun, Parke-Davis, Ann Arbor,
MI) and p(ARE)3-Luc (provided by Dr. D.
Gioeli, University of Virginia, Charlottesville, VA), as
indicated, using Superfect reagent (QIAGEN, Chatsworth,
CA). A total of 1.5 µg DNA was used per 35- mm well. Transfected
cells were cultured in steroid-depleted medium with or without
synthetic androgen, Mibolerone, where indicated. After 48 h
incubation, cells were harvested, and luciferase activities were
determined using a luciferase reporter system (Promega Corp.). pCMV-ß-gal was used as an internal control for
normalization of transfection efficiency.
Microscopic Analysis of GFP-hAR Chimera in M2 and A7 Cell
Lines
Full-length hAR (corresponding to residues 1–918) was cloned
into the XbaI site of the GFP plasmid pEGFP-C1
(CLONTECH Laboratories, Inc.) to generate
GFP-hAR1-918. Residues
559–918 of the hAR comprising DNA-binding and ligand-binding domains
of the hAR were cloned into the EcoRI site of pEGFP-C2
(CLONTECH Laboratories, Inc.) to produce
GFP-hAR559-918. Full-
length Tip60 was isolated as described previously (43) and cloned into
the BamHI site of pEGFP-C2 (CLONTECH Laboratories, Inc.). Human epithelial melanoma cell lines, M2 (filamin -ve)
and A7 (filamin +ve) (a kind gift from Dr. T. Stossel,
Harvard University, Cambridge, MA), were used in the GFP-hAR
translocation assays. Cell lines were grown overnight on 22 x 22
mm microscope cover slips in six-well cell culture plates. Cells were
transfected with 2 µg of
GFP-hAR1-918 or
GFP-hAR559-918 construct
using Superfect and cultured in RPMI 1640 medium for 24 h. Cells
were then placed in steroid-depleted media for a further 24 h and
exposed to 10 nM Mibolerone for 0, 5, and 20 min.
Coverslips were washed with sterile PBS and fixed in 100% methanol for
30 min at -20 C, dried, inverted on microscope slides, and mounted in
antifading medium (Shandon Southern Instruments, Inc., Sewickley,
PA). Slides were analyzed on an MRC 600 scanning laser confocal
microscope (Bio-Rad Laboratories, Inc., Richmond, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 31, 2000. Revision received June 12, 2000. Accepted for publication July 3, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. Blood 92:1268–1276 activation of SAPK in melanoma cells.
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