From The Burnham Institute, La Jolla, California 92037
Received for publication, November 30, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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Bag1 is a regulator of heat shock protein 70 kDa
(Hsp70/Hsc70) family proteins that interacts with steroid
hormone receptors. Four isoforms of Bag1 have been recognized: Bag1,
Bag1S, Bag1M (RAP46/HAP46), and Bag1L. Although Bag1L, Bag1M,
and Bag1 can bind the androgen receptor (AR) in vitro, only
Bag1L enhanced AR transcriptional activity. Bag1L was determined to be
a nuclear protein by immunofluorescence microscopy, whereas Bag1,
Bag1S, and Bag1M were predominantly cytoplasmic. Forced nuclear
targeting of Bag1M, but not Bag1 or Bag1S, resulted in potent AR
coactivation, indicating that Bag1M possesses the necessary structural
features provided it is expressed within the nucleus. The ability of
Bag1L to enhance AR activity was reduced with the removal of an
NH2-terminal domain of Bag1L, which was found to be
required for efficient nuclear localization and/or retention. In
contrast, deletion of a conserved ubiquitin-like domain from
Bag1L did not interfere with its nuclear targeting or AR regulatory
activity. Thus, both the unique NH2-terminal domain and the
COOH-terminal Hsc70-binding domain of Bag1L are simultaneously required
for its function as an AR regulator, whereas the conserved
ubiquitin-like domain is expendable.
Steroid receptors play a crucial role in the development and
maintenance of many organs. The androgen receptor
(AR)1 is a ligand-activated
transcription factor that is a member of the nuclear receptor
superfamily (1). One tissue that exhibits profound dependence on the
ligand for this nuclear hormone receptor is the prostate gland. In
normal prostate, androgens induce production of growth factors in the
stromal cells that cause growth of the luminal secretory epithelial
cells (reviewed in Ref. 2). The AR controls gene expression programs in
these epithelial cells, resulting in the expression of various
proteins characteristic of the differentiated state including
prostate-specific antigen (3). Tumors develop from the prostatic
epithelial cells and ultimately become independent of the androgenic
hormones. Endocrine therapy, either by reducing the levels of androgen
or by blockading androgens at the level of the AR, usually results in a
favorable clinical response and a dramatic regression of prostate
cancer due to apoptotic cell death (4). However, after an
initial response to androgen ablation, the prostate cancers eventually become unresponsive if endocrine therapy is continued long term (5).
It remains an open question whether the AR continues to play a role in
hormone-independent prostate cancers. Many hormone-insensitive tumors
have been found to retain a wild-type AR gene. Moreover, the AR gene is
sometimes amplified, or its transcriptional activity may be increased
in advanced prostate cancer (6). Therefore, a need exists to understand
more about the factors that control the functions of the AR so that the
mechanisms responsible for resistance to endocrine therapy can be
revealed and eventually alleviated.
In the absence of the ligand, AR and most steroid hormone receptors are
maintained in an inactive state complexed with heat shock proteins.
Upon binding of the cognate ligand, the receptor dissociates from the
inactive complex and translocates to the nucleus in which it binds
specific response elements in the promoter and/or enhancer regions of
responsive genes (reviewed in Ref. 7). Once bound to the nuclear
response element, the nuclear receptor up-regulates or down-regulates
transcription by transmitting signals directly to the transcriptional
machinery via direct protein-protein interactions. In addition, another
class of proteins called coactivators is recruited and serves as
bridging molecules between the transcription initiation complex and the
nuclear receptor (reviewed in Ref. 8).
Recently, an isoform of the human Bag1 protein (known as Bag1L) has
been reported to bind the AR and enhance transcriptional activity in
the presence of the ligand (9). Bag1 contains a COOH-terminal
"BAG" domain that binds the ATPase domain of heat shock
protein 70 kDa (Hsp70/Hsc70) family proteins (10-14) and modulates the
activity of Hsc70/Hsp70 family chaperones in vitro and
in vivo. Through this interaction with Hsc70, Bag1 is able to interact with a variety of intracellular proteins and regulate diverse cellular processes relevant to cancer including cell division, cell survival, and cell migration (15-20). However, it is also possible that other non-Hsc70-binding domains in the
NH2-terminal portion of Bag1 mediate interactions with
target proteins, thus providing a mechanism for directing Hsc70 family
chaperones to specific proteins in the cells. For example, Bag1
contains a ubiquitin-like (UBL) domain, which has been proposed to
permit its direct binding to the 26 S proteosome (21). However, the
significance of this and other NH2-terminal regions in Bag1
for transactivation of AR or other steroid hormone receptors is unknown.
It has been shown that at least four isoforms of Bag1 protein can arise
from alternative initiation of translation within a common mRNA:
Bag1S, Bag1, Bag1M (RAP46/HAP46), and Bag1L (22, 23). These isoforms
all contain the Hsc70-binding BAG domain near the COOH terminus as well
as the upstream UBL domain, but they differ in the lengths of their
amino-terminal regions. Additional motifs have been recognized within
the NH2-terminal segment of the Bag1 proteins including
candidate nuclear localization sequences (NLS) and variable numbers of
TXSEEX repeat sequences (23, 24). Bag1L, the
longest isoform, contains both an SV40-LargeT-like and
nucleoplasmin-like candidate NLS preceded by a unique ~50 amino
acid-domain. This Bag1 isoform is predominantly nuclear (23). Bag1M
(RAP46/HAP46) contains only a portion of the candidate NLS and has been
shown to reside in the cytosol unless stimulated to traffic into the
nucleus by associating with other proteins, such as the glucocorticoid
receptor (24). Bag1 and the shorter and rarer isoform of Bag1S are
predominantly found in the cytosol (23).
Bag1 proteins have been reported to interact with and regulate the
activity of several members of the nuclear receptor superfamily. For
example, Bag1M and Bag1L have been found to repress the activity of the
glucocorticoid receptor (24, 25), and Bag1 represses the
transcriptional activity of retinoic acid receptors (27). Conversely,
Bag1L but not Bag1M or Bag1 can potentiate the transcriptional activity
of the AR (9). In this report, we have extended structure-function analysis of Bag1 protein with respect to their regulation of the AR.
Plasmids--
The plasmids pcDNA3-Bag1L, pcDNA3-Bag1L_C,
and pcDNA3-Bag1 have been described previously (9).
pcDNA3-Bag1M was generated from pcDNA3-Bag1/Bag1M (9) by
mutating the initiation codon that gave rise to Bag1 from an ATG to an
ATC so that this construct now can give rise only to Bag1M.
The cDNAs encoding various fragments of Bag1 were generated by
polymerase chain reaction from the plasmid pcDNA3-Bag1L (9) using
the following forward (F) and reverse (R) primers containing EcoRI and XhoI sites: Bag1S,
5'-GGGAATTCGCCACCATGGCGGCA-3' (F1) and
5'-CCCTCGAGTCACTCGGCCAGGGCAAAG-3' (R1); Bag1L
The polymerase chain reaction products were digested with
EcoRI and XhoI and then directly cloned into the
EcoRI and XhoI sites of the mammalian expression
vector pcDNA3 (Bag1S and Bag1L
The GST-Bag1 fusion proteins were generated from pGEX-4T-Bag1L,
pGEX-4T-Bag1L
The reporter pLCI plasmid contains the full-length mouse mammary tumor
virus long terminal repeat sequence linked with the chloramphenicol
acetyltransferase (CAT) gene (28, 29). The pSG5-AR plasmid contains the
cDNA for the wild-type AR (28).
Cell Culture--
The monkey kidney COS-7 cell line was obtained
from the American Type Culture Collection (Manassas, VA). Cells were
maintained in a humidified atmosphere with 5% CO2 in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 3 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin (Life Technologies, Inc.). One day prior to
experiments, cells were transferred into charcoal-treated fetal bovine
serum (CT-FBS) and Dulbecco's modified Eagle's medium minus Phenol
Red to reduce background levels of steroids. R1881 (PerkinElmer Life
Sciences) was dissolved in ethanol and added to the cultures at
a minimum dilution of 0.0001% (v/v). Control cells received an
equivalent amount of solvent only.
Transfections and Enzyme Assays--
COS-7 cells at 60%
confluency in 12-well plates (Nunc) were transfected by a lipofection
method. 1.1 µg of DNA was diluted into 176.5 µl of Opti-MEM (Life
Technologies, Inc.) and combined with 3 µl of LipofectAMINE (Life
Technologies, Inc.) in 185.5 µl of Opti-MEM. After incubation for 20 min, 0.375 ml of Opti-MEM was added, and the mixtures were overlaid
onto monolayers of cells. After culturing with 5% CO2 for
5 h at 37 °C, 0.75 ml of Opti-MEM containing 20% CT-FBS was
added to the cultures. At 32-36 h after transfection, cells
were stimulated with 0.1 nM R1881. Cell extracts were
prepared 48 h after transfection. For reporter gene assays, cell
lysates were made as described previously (30), and assays for
In Vitro Binding Assays--
The AR was in vitro
translated in reticulocyte lysates (TNT lysates, Promega)
containing [35S]methionine and then preincubated with 10 nM R1881 for 30 min. Glutathione S-transferase
(GST) fusion proteins were immobilized on glutathione-Sepharose and
blocked in NET-N buffer (20 mM Tris, pH 8.0, 20 mM NaCl, 1 mM EDTA) containing 0.1% Nonidet
P-40 and 15% milk for 30 min. The immobilized GST proteins (10 µg)
were incubated for 2 h with 10 µl of R1881 treated in
vitro translated AR in NET-N buffer containing 0.1% Nonidet P-40,
proteinase inhibitors, and 10 nM R1881. The beads were
washed three times in NET-N buffer containing 0.5% Nonidet P-40 and
then boiled in Laemmli SDS sample buffer. The use of equivalent amounts
of intact GST fusion proteins and successful in vitro
translated of the AR was confirmed by SDS-PAGE analysis using Coomassie
Blue staining or autoradiography, respectively.
Immunofluorescence--
Transfected COS-7 cells were fixed with
a Hormone Binding Assay--
COS-7 cells at 60% confluency in
10-cm2 plates were transiently transfected either with
empty vectors or a combination of expression vectors encoding for AR,
Bag1L, or Bag1 by a lipofection procedure. 10 µg of DNA was incubated
with 26 µl of LipofectAMINE (Life Technologies, Inc.) for 20 min, and
the mixtures were overlaid onto monolayers of cells. After culturing
with 5% CO2 at 37 °C for 5 h, 1 volume of Opti-MEM
containing 20% charcoal-stripped fetal bovine serum (CT-FBS) was added
to the cultures. Cells were transferred into 24-well plates 1 day later
and grown in CT-FBS medium for an additional 24 h. To determine
the hormone binding affinities of the transfected AR, cells were
incubated for 2 h with increasing concentrations (0.1-10
nM) of [3H]R1881 (86 BAG Domain of Bag1 Is Sufficient to Bind AR in Vitro--
The
human Bag1 protein exists as four isoforms (Fig.
1A), which all contain the
same COOH-terminal Hsc70-binding domain and an upstream ubiquitin-like
domain but differ in the lengths of their NH2-terminal
regions (23). The Bag1L and Bag1M (RAP46) isoforms of the human Bag1
protein have been previously shown to bind the AR in vitro
(9, 25). To determine whether other isoforms of the human Bag1 protein
could interact with the AR, we performed in vitro protein
interaction assays. The Bag1 isoforms were fused to GST and incubated
with in vitro translated radiolabeled AR. As shown in Fig.
1B, the AR specifically interacted with Bag1L and Bag1 but
not with the control proteins GST and GST-CD40. Mutants of Bag1L and
Bag1, which lack the COOH-terminal Hsc70-binding domain ( Nuclear Targeting of the Cytoplasmic Bag1 Is Insufficient for
Potentiating AR Activity--
Although all isoforms of Bag1 interact
with the AR in vitro, only the Bag1L protein significantly
enhances the transcriptional activity of the AR in vivo (9).
Because the ligand-bound AR is localized to the nucleus (7), only
isoforms of the Bag1 protein that are nuclear would be expected to
enhance the transcriptional activity of the AR. Therefore, we checked
the compartmentalization of the Bag1 proteins by immunofluorescence.
COS-7 cells were transfected with plasmids encoding the various Bag1
isoforms followed by immunostaining with an anti-Bag1 monoclonal
antibody and analysis by confocal laser-scanning microscopy. Bag1L is
exclusively a nuclear protein, whereas all other Bag1 isoforms are
predominantly cytosolic (Fig. 2,
left panels). These findings are consistent with the
presence of both nucleoplasmin-like and SV40-LargeT-like
nuclear-targeting sequence in the Bag1L protein (22, 23) but not Bag1M,
Bag1, or Bag1S. Although the Bag1M (RAP46/HAP46) isoform contains a region of basic residues suggestive of a nuclear targeting sequence (25), it is evidently transported inefficiently into the nucleus. Interestingly, the Bag1L protein may be associated with nuclear substructures given the speckled pattern of the immunofluorescence observed (Fig. 2).
The difference in cellular distribution of the Bag1 proteins represents
a possible explanation for the inability of the cytosolic Bag1 isoforms
to enhance the transcriptional activity of the AR. To address this
issue, we constructed plasmids that express Bag1L, Bag1M, Bag1, or
Bag1S fused to SV40-LargeT-like nuclear-targeting sequences. Nuclear
localization of these NLS-Bag1 proteins was verified by transfecting
COS-7 cells with plasmids expressing the nuclear-targeting Bag1
isoforms and then immunostaining with an antibody that recognizes Bag1
(Fig. 2, right panels). Confocal laser-scanning microscopy
analysis revealed that all the Bag1 isoforms are located exclusively in
the nucleus of transfected cells.
The effects of these nuclear-targeted Bag1 isoforms on AR
transcriptional activity were then tested by transfection into COS-7 cells together with an AR-expressing plasmid and an ARE-containing reporter gene. The cells were stimulated with R1881 to activate AR. As
shown in Fig. 3A, the fusion
of heterologous nuclear-targeting sequences to the 5' end of Bag1L did
not alter its ability to enhance the transcriptional activity of the
AR. Targeting of Bag1M to the nucleus but not Bag1 or Bag1S was
sufficient to enhance the transcriptional activity of the AR. Taken
together, these results suggest that the first 70 amino acids of Bag1L
are expendable for AR coactivation, whereas the
NH2-terminal region of Bag1M corresponding to amino acids
71-115 of Bag1L is required in conjunction with nuclear localization
for enhancing AR function.
Immunoblot analysis showed that the levels of all Bag1 isoforms
produced in cells (except Bag1S) were comparable to the
nuclear-targeted isoforms, excluding quantitative differences in the
levels of these proteins as a trivial explanation for the results (Fig. 3B). The difference in the level of expression of Bag1S and
NLS-Bag1S makes it difficult to conclude that NLS-Bag1S is without
effect on AR-mediated transactivation. The absence of Bag1S from the nucleus would argue against its role in transactivation.
The NH2-terminal Region of Bag1L Is Required for
Efficient Nuclear Localization--
The observation that Bag1L is the
only isoform that is nuclear suggests that the unique
NH2-terminal domain of Bag1L might perform a role in
nuclear targeting or retention. We therefore generated two
NH2-terminal truncation mutants of Bag1L lacking either the
first 16 amino acids of Bag1L (Bag1L
Both the Bag1L
To contrast these subcellular localization results with coactivation
function, COS-7 cells were cotransfected with the AR, an ARE-containing
reporter gene, and plasmids encoding Bag1L, Bag1L
To confirm this finding, we fused a nuclear-targeting sequence to
Bag1L, Bag1L The Ubiquitin-like Domain of Bag1L Is Not Required for Enhancing
Transcriptional Activity of AR--
The Bag1 protein contains a UBL
domain that is conserved within the Bag1 homologues of
Caenorhabditis elegans and Schizosaccharomyces pombe (32). To explore whether this region has functional
significance in the enhancement of AR-mediated transcription, we
constructed a deletion mutant of Bag1L lacking this region
(Bag1L Bag1 Proteins Do Not Alter the Affinity of the AR for Its
Ligand--
Heat shock proteins and other molecular chaperones are
required for placing steroid hormone receptors into a state that is competent to bind steroid ligands (33, 34). The ability of Bag1
proteins to bind and modulate the function of Hsp70/Hsc70 family
molecular chaperones (10), therefore, could conceivably alter the
ability of AR to bind androgenic hormones. Therefore, we determined the
hormone binding affinity of the AR in whole cells in the absence or
presence of overexpressed Bag1 or Bag1L. As depicted in Fig.
6A, the presence of elevated
levels of either Bag1 or Bag1L did not significantly influence the
amount of hormone bound to the receptors at any of the hormone
concentrations tested. In addition, as revealed by Scatchard analysis
(Fig. 6, B-D), neither Bag1 nor Bag1L significantly altered
the apparent equilibrium binding constant (Kd)
for R1881. Therefore, Bag1L exerts its influence on AR-mediated
transcription at a stage other than ligand binding.
The data presented here confirm that Bag1L is the only isoform of
Bag1 capable of enhancing the transcriptional activity of the AR (9).
Thus, despite evidence that Bag1M and Bag1 can interact with the AR
in vitro, only Bag1L interacts with AR in cells (9) and
alters its transactivation function. Because Bag1L is the only isoform
that is constitutively present in the nucleus, one possible explanation
is that the higher nuclear levels of Bag1L may be responsible for its
physical and functional interactions with AR complexes. Previous
studies have shown that Bag1L does not alter the
ligand-dependent nuclear translocation of the AR (9).
The appendage of an exogenous nuclear-targeting sequence to
Bag1M, Bag1, and Bag1S is sufficient to force these proteins into the
nucleus. This forced nuclear targeting bestowed upon Bag1M but not Bag1
or Bag1S the ability to coactivate the AR. Therefore, it appears that
additional structural differences in Bag1L and Bag1M compared with Bag1
and Bag1S play an important role in the differential effects of these
proteins on the AR. Recent papers have suggested that the
TXSEEX repeat found in 8, 8, and 2 copies, respectively, in Bag1L, Bag1M, and Bag1 is required for repression of
the transcriptional activity of the glucocorticoid receptor (24). Thus,
these TXSEEX motifs may be important for
functional collaboration of Bag1 with AR as both Bag1L and Bag1M are
effective at potentiating AR function when expressed within the
nucleus, whereas Bag1 is not.
By deleting the NH2-terminal region of Bag1L, we
demonstrated a function for this unique proline-rich domain for the
first time. Subcellular localization experiments indicated that
although Bag1L and Bag1L In contrast, deletion of the UBL domain from Bag1L did not impair
subcellular targeting or functional collaboration with the AR. A wide
variety of proteins have been shown to contain UBLs (reviewed in Ref.
35). These domains can mediate direct interactions with subunits of the
proteosome that recognize polyubiquitin chains on proteins that have
been targeted for destruction. Recently, Bag1 was reported to bind the
26 S proteosome in vitro. The UBL domain is found within all
four Bag1 isoforms and is conserved in the Bag1 homologues of other
species including the yeast S. pombe and the nematode
C. elegans (32), implying an evolutionarily conserved role
for this domain in some aspect of Bag1 function. However, deletion of
the UBL domain did not abrogate the stimulatory effect of Bag1L on AR
function. Thus, whatever the function of the conserved UBL domain of
Bag1 may be, it is expendable for coactivation of steroid hormone receptors.
Similar to the NH2-terminal unique domain, the
COOH-terminal region of Bag1 that is required for Hsc70 binding was
found to be essential for potentiation of AR activity. The data
reported here and elsewhere indicate that the COOH-terminal
Hsc70-binding domain of Bag1 and Bag1L is required for interactions
with AR in vitro and for coimmunoprecipitation of Bag1L with
AR from cell lysates (9). Moreover, we presented novel evidence here
that the BAG domain of Bag1 is sufficient for association with the AR
in vitro. It has therefore been postulated that the
interaction of Bag1L with the AR may involve Hsp70. Hsp70/Hsc70 along
with Hsp90 and Hsp56 are involved in maintaining nuclear receptors in
an inactive conformation in the cytoplasm. Thus, it is possible that
Hsc70/Hsp70 family molecular chaperones provide a bridge between the AR
and Bag1L. Alternatively, Hsp70 and AR may compete for binding to the
BAG domain as has been determined recently for Hsp70 and Raf-1
(36).
Molecular chaperones perform several important functions in the
regulation of steroid hormone receptors. For example, a variety of
chaperones including Hsc70/Hsp70 is required to achieve receptor conformations that are competent to bind steroid ligands. Thus, enhanced ligand binding represents one possible explanation for the
mechanism by which Bag1L potentiates AR function. If true, however, we
would have expected the other isoforms of Bag1 also to potentiate AR
activity, given that translation and folding of the AR occurs in the
cytosol where the unliganded AR resides in a complex with Hsp90 and
other chaperones. Thus, the data reported here demonstrating a
lack of effect of Bag1 on the ligand binding affinity of the AR are
consistent with the observed differences in the ability of nuclear and
cytosolic isoforms of Bag1 to enhance AR activity. In addition, because
heat shock proteins sequester AR in the cytosol in an inactive state
until bound by specific steroid ligands, it was also possible that Bag1
proteins might regulate cytosol to nuclear translocation of AR,
however, we previously demonstrated that this is not the case (9).
Given that Bag1L does not alter the affinity of AR for steroid ligands
and does not modulate nuclear translocation of AR, we speculate that it
potentiates AR function either by affecting coactivator binding to AR
or by altering AR affinity for DNA-binding sites. Interestingly, it has
been reported that Bag1M is able to bind to DNA and stimulate basal
transcription machinery directly in vitro (25). Thus, Bag1L
interactions with AR could conceivably increase the affinity of
AR-containing transcription complexes for DNA, thereby enhancing
transcriptional output of AR-responsive genes. However, given that we
failed to see any affect of Bag1L on the binding of the AR to an
ARE in gel retardation assays (data not shown), this observation
alone cannot account for the unique effects of Bag1L on AR.
Taken together, the data reported here imply that at least two domains
within Bag1L are important for its functional collaboration with the
AR, namely the TXSEEX repeats and the
COOH-terminal BAG domain, which binds Hsp70/Hsc70 family
molecular chaperones. Multiple explanations can be envisioned for how
these two domains might participate in the regulation of AR. For
example, both of these domains might directly or indirectly bind AR. In
this regard, it may be relevant that several coactivators of nuclear
receptors, such as GRIP-1 and SRC-1, make multiple interactions
with nuclear receptors through different domains (26, 37). However, it is equally probable that the BAG domain alone mediates interactions of
Bag1L with AR, whereas the TXSEEX repeats
associate with other proteins such as coactivators. Difficulties in
producing soluble stable TXSEEX repeats alone
have precluded us from distinguishing these two mechanisms to date.
Another aspect of Bag1L effects on AR is that they may be indirect,
involving Bag1L/Hsc70-induced conformational changes in AR that enhance
its interactions with coactivators or reduce interactions with corepressors.
Given that only the Bag1L isoform of Bag1 collaborates functionally
with the AR, one mechanism by which tissues could alter their
responsiveness to androgens is by adjusting the levels of the Bag1L
protein produced. The human bag1 mRNA can generate up to
four protein products through alternative mRNA translation from
various canonical AUG or noncanonical CUG codons (22, 23). The
mouse bag1 mRNA similarly can produce up to three
protein products but lack the AUG that is responsible for generating
Bag1M (RAP46) (23). In human tissues and tumor cell lines, it has also
been shown that Bag1 and Bag1L are by far the most abundant isoforms of
Bag1 produced with little or no Bag1M or Bag1S present (23).
Interestingly, whereas Bag1 is ubiquitously expressed throughout the
organs of mice and humans, Bag1L production is tissue-specific and
found predominantly in hormone-sensitive tissues, such as the testis,
ovary, breast, and prostate. Also, in human tumor cell lines, levels of
Bag1L tend to be highest in hormone-dependent cancers, such
as prostate (AR), breast (estrogen receptor (ER), progesterone receptor
(PR), and lymphoid (glucocorticoid receptor) malignancies in
which steroid hormones are known to play major roles in the regulation
of cell growth, differentiation, and death. Given that Bag1L enhances
the transactivation function of AR and that it has been shown to render
AR less inhibitable by anti-androgenic drugs (9), it will be
interesting to determine whether the levels of Bag1L change during the
progression of prostate cancers as they undergo conversion from
hormone-responsive to hormone-refractory disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-50, primer 5'-GCGGAATTCGCCACCATGACTGCCAGC-3' (F2) and R1; Bag1L
1-16,
5'-GGGAATTCGAGCGGATGGGTTCCCG-3' (F3) and R1; Bag1M
C83,
5'-GGGAATTCATGAAGAAGAAAACCCGGCGCC-3' (F4) and
5'-CCCCTCGAGTCAAAAACCCTGCTGGATTCCAG-3' (R2); and Bag1C83
5'-GGGAATTCCTGCCCAAGGATTTGCAAGCTG-3' and R1.
1-50) or pcDNA3-Myc
(Bag1L
1-16, Bag1M
C83, and Bag1C83).
C, pGEX-4T-Bag1, pGEX-4T-Bag1
C, pGEX-4T-Bag1M, pGEX-4T-Bag1M
C83, and pGEX-4T-1-Bag1C83. These plasmids were all
generated by subcloning the appropriate cDNAs from pcDNA3 clone
into the EcoRI and XhoI sites of pGEX-4T-1
(Amersham Pharmacia Biotech). To generate the nuclear-targeted Bag1
proteins, the appropriate cDNA was subcloned from pcDNA3 clone
into the EcoRI and XhoI sites of pcDNA3-NLS
(generated by the insertion of an oligonucleotide containing the
SV40-LargeT-like NLS into the
HindIII-EcoRI sites of pcDNA3.1).
-galactosidase and CAT activity were performed. All transfection experiments were carried out in duplicate, repeated at least three times, and normalized for
-galactosidase activity.
20 °C chilled mix of methanol and acetone (1:1) for 2 min at
20 °C. After fixation the cells were blocked with phosphate-buffered
saline containing 3% bovine serum albumin, 2% FBS, and 0.1% goat
serum and then incubated for 4 h at 20 °C with anti-Bag1
antibody (Dako Corp., Carpinteria, CA) diluted 1:50 in blocking
solution (31). After this incubation, cells were rinsed three times for
10 min with phosphate-buffered saline at 20 °C and then incubated
with fluorescein isothiocyanate-conjugated anti-mouse IgG (Dako Corp.),
diluted 1:50 in blocking solution for 2 h at 37 °C. Excess
secondary antibody was thoroughly washed off with phosphate-buffered
saline. The slides were then treated with Mowiol containing
1,4-diazabicylo[2.2.2]octane, and glass coverslips were
applied. The stained slides were observed using a laser-scanning
confocal microscope (Bio-Rad 1024MP).
Ci/nmol, PerkinElmer
Life Sciences) in the presence or absence of a 100-fold molar
excess of cold R1881. Cells were then washed three times with ice cold
phosphate-buffered saline, and radioactivity was determined by
scintillation counting. Specific binding was calculated by subtracting
the counts/min of cells transfected with a control plasmid from the
counts/min of samples transfected with the AR expression plasmid that
was treated with the same concentration of the hormone. The results
represent the means of three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C), were
unable to interact with the AR in these assays, indicating that the BAG
domain is required for interactions with the AR. Moreover, a GST-fusion
protein containing only the last COOH-terminal 83 amino acids,
GST-Bag1C83, was sufficient for binding to AR under these conditions
(Fig. 1C). Therefore, we conclude that the BAG domain of
Bag1 protein is necessary and sufficient for associating with the
AR.
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Fig. 1.
The BAG domain is necessary and sufficient
for interacting with the AR in vitro.
A, a schematic shows the isoforms of the Bag1
protein, Bag1L, Bag1, Bag1M, and the following deletion mutants:
Bag1 C and Bag1L
C, which lack the COOH-terminal last 47 amino
acids; Bag1M
C83, which lacks the COOH-terminal last 83 amino acids;
and Bag1C83, which contains only the last 83 amino acids. The regions
encoding the candidate nuclear localization sequences (black
box), TXSEEX repeat (downward diagonal
striped box), ubiquitin-like domain (gray box), and BAG
domain (upward diagonal striped box) have been indicated.
The sequence numbers all refer to the nucleotide
numbers of Bag1L and indicate the start codon position for each isoform
of Bag1 with respect to Bag1L. B, the interaction of the AR
with various isoforms of Bag1 or COOH-terminal deletion mutants of
these Bag1 proteins was tested by in vitro binding assays.
The Bag1 isoforms and COOH-terminal mutants were expressed as GST
fusion proteins and incubated with AR in vitro translated in
the presence of [35S]methionine. GST and GST fused to the
cytoplasmic domain of CD40 were included as negative controls.
GST-Bag1S failed to fold properly and could not be tested.
C, the interaction of the AR with deletion mutants of Bag1
was tested by in vitro binding assays. The Bag1M
C83 and
Bag1C83 were expressed as GST fusion proteins and incubated with AR
that was prepared by in vitro translation in the presence of
[35S]methionine.
View larger version (61K):
[in a new window]
Fig. 2.
Bag1L is the only isoform of
Bag1 that is nuclear. COS-7 cells were transfected with 0.5 µg
of pcDNA3-Bag1L, pcDNA3-Bag1M, pcDNA3-Bag1,
pcDNA3-Bag1S, pcDNA3-NLS-Bag1L, pcDNA3-NLS-Bag1M,
pcDNA3-NLS-Bag1, or pcDNA3-NLS-Bag1S. At 30 h
post-transfection, cells were fixed, stained with a monoclonal
antibody recognizing Bag1, and visualized using a laser confocal
microscope. The nucleus and nucleoli are immunopositive in all
transfectants involving NLS-targeted proteins (right
column).
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Fig. 3.
Targeting of Bag1M but not the shorter Bag1
isoforms to the nucleus confers the ability to enhance AR-mediated
transactivation. A, COS-7 cells were transfected with
0.06 µg of pSG5-AR, 0.5 µg of pLCI, 0.04 µg of
pCMV- -galactosidase, and various amounts of one of the following
Bag1 expression plasmids: pcDNA3-Bag1L, pcDNA3-Bag1M,
pcDNA3-Bag1, pcDNA3-Bag1S, pcDNA3-NLS-Bag1L,
pcDNA3-NLS-Bag1M, pcDNA3-NLS-Bag1, or pcDNA3-NLS-Bag1S. Total
DNA was maintained at 1.1 µg by the addition of pcDNA3 control
plasmid. At 30 h after transfections, cells were stimulated with 1 nM R1881. Cell extracts were prepared and assayed for CAT
and
-galactosidase activity at 40 h after transfection
(mean ± S.E., n = 2). B, COS-7 cells
were transfected with one of the following Bag1 expression plasmids:
pcDNA3-Bag1L, pcDNA3-Bag1M, pcDNA3-Bag1, pcDNA3-Bag1S,
pcDNA3-NLS-Bag1L, pcDNA3-NLS-Bag1M, pcDNA3-NLS-Bag1, or
pcDNA3-NLS-Bag1S. At 30 h after transfection, cells were lysed
in radioimmune precipitation buffer. Cell extracts (25 µg of total
protein) were subjected to SDS-PAGE/immunoblot assay and probed with an
antibody to Bag1. The Bag1 expression plasmid pcDNA3-Bag1M produces
both the Bag1M and Bag1 proteins due to translational initiation from
an internal AUG in Bag1M.
1-16) or retaining the
nuclear-targeting sequences of Bag1L but lacking the
NH2-terminal 50 amino acids that differentiate it from the
Bag1M protein (Bag1L
1-50).
1-16 and Bag1L
1-50 proteins retain the candidate
nuclear-targeting sequences of Bag1L and therefore should target to
nuclei similar to Bag1L. To explore this option, COS-7 cells
were transfected with plasmids encoding Bag1L
1-16, Bag1L
1-50, or Bag1L, and the localization of the resulting proteins was determined by immunofluorescence confocal microscopy. Bag1L and Bag1L
1-16 exhibited essentially the same compartmentalization pattern within the
cell (Fig. 4B, upper
panels), demonstrating a nuclear speckled pattern of
immunostaining. In contrast, the Bag1L
1-50 protein was more
promiscuous in its subcellular localization. Whereas Bag1L
1-50 was
found in the same nuclear substructures as Bag1L, the protein was also
found in a diffuse cytosolic staining pattern in transfected cells.
This finding suggests that amino acids 17-50 of Bag1L may contain
sequences necessary for optimal retention of Bag1L in the nucleus.
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Fig. 4.
The NH2 terminus of Bag1L
is required for its targeting/retention in the nucleus.
A, a schematic is presented showing deletion
mutants of Bag1L, which are lacking the NH2- terminal
16 (Bag1L 1-16) or 50 amino acids
(Bag1L
1-50). B, COS-7 cells were
translated with the following Bag1 expression plasmids: pcDNA3-Bag1L,
pcDNA3-Bag1L
1-16, pcDNA3-Bag1L
1-50, pcDNA3-NLS-Bag1L,
pcDNA3-NLS-Bag1L
1-16, or pcDNA3-NLS-Bag1L
1-50. At 30 h
post-transfection, cells were fixed, stained, and examined by laser
confocal microscopy. C, COS-7 cells were transfected as in
A, and after 30 h the cells were lysed in radioimmune
pre cipitation buffer. Cell extracts (25 µg of total protein)
were subjected to SDS- PAGE/immunoblot assay and probed with an
antibody to Bag1. D, COS-7 cells were transfected with
0.06 µg of pSG5-AR, 0.5 µg of pLCI, 0.04 µg of
pCMV-
-galactosidase, and various amounts of the Bag1 expression
plasmids used in B. Total DNA was maintained at 1.1 µg by
the addition of pcDNA3 control plasmid. At 30 h after
transfection, cells were stimulated with 1 nM R1881. Cell
extracts were prepared and assayed for CAT and
-galactosidase
activity at 40 h after transfection (mean ± S.E.,
n = 2).
1-16, or
Bag1L
1-50. The transfected cells were then stimulated with the
synthetic androgen R1881. Bag1L
1-50 but not Bag1L
1-16 exhibited
a decreased ability to enhance the transcriptional activity of the AR
(Fig. 4D). Taken together, these observations suggest that
the correct nuclear targeting/retention of Bag1L is required for
optimal functional interactions of Bag1L and the AR.
1-16, and Bag1L
1-50 and transiently transfected the
NLS-fusion constructs into COS-7 cells. Confocal immunofluorescence analysis revealed that NLS-Bag1L, NLS-Bag1L
1-16, and
NLS-Bag1L
1-50 exhibit essentially the same nuclear pattern within
the cell (Fig. 4B, lower panels). Targeting of
Bag1L and Bag1L
1-16 to the nucleus did not significantly alter
their effect on the AR, however, nuclear-targeting of Bag1L
1-50
markedly improved its ability to enhance AR transcriptional activity
(Fig. 4D). Immunoblot analysis revealed that all Bag1 isoforms were expressed at similar levels (Fig. 4C).
UBL) (Fig. 5A). COS-7
cells were cotransfected with plasmids expressing Bag1L
UBL, AR, and
an ARE-containing reporter gene plasmid and then stimulated with R1881.
As illustrated in Fig. 5B, deletion of the UBL domain from
Bag1L did not alter its ability to enhance AR-mediated transcription in
reporter gene assays, suggesting that this region of Bag1L is not
required for functional interactions of Bag1L and the AR. Immunoblot
analysis showed that the levels of Bag1L
UBL produced in cells was
comparable to Bag1L (Fig. 5C). COS-7 cells were transfected with plasmids encoding Bag1L
UBL or Bag1L, and the localization of
the resulting proteins was determined by immunofluorescence confocal
microscopy. Bag1L and Bag1L
UBL exhibited essentially the same
compartmentalization pattern within the cell (Fig. 5D), demonstrating a nuclear speckled pattern of immunostaining.
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Fig. 5.
Deletion of the ubiquitin-like domain
of Bag1L does not alter its ability to enhance AR-mediated
transactivation of an ARE-containing reporter. A, a
schematic is presented showing a deletion mutants of Bag1L
that lacks the ubiquitin-like domain. B, COS-7 cells were
transfected with 0.06 µg of pSG5-AR, 0.5 µg of pLCI, 0.04 µg of
pCMV- -galactosidase, and various amounts of pcDNA3-Bag1L or
pcDNA3-Bag1L
Ub. Total DNA was maintained at 1.1 µg by the
addition of pcDNA3 control plasmid. At 30 h after
transfection, cells were stimulated with 1 nM R1881. Cell
extracts were prepared and assayed for CAT and
-galactosidase
activity at 40 h after transfection (mean ± S.E.,
n = 2). C, COS-7 cells were transfected
as in B, and after 30 h the cells were lysed in
radioimmune precipitation buffer. Cell extracts (25 µg of total
protein) were subjected to SDS-PAGE/immunoblot assay and probed
with an antibody to Bag1. D, COS-7 cells were transfected as
in B, and at 30 h post-transfection, cells were fixed,
stained with a monoclonal antibody recognizing Bag1, and visualized
under a laser confocal microscope.
View larger version (28K):
[in a new window]
Fig. 6.
Effect of Bag1 and Bag1L on R1881 binding
affinity of the AR. COS-7 cells were transfected with either an
empty vector or with expression vectors encoding AR and Bag1 or Bag1L.
At 2 days after transfection, cells were incubated in the indicated
concentration of [3H]R1881 in the presence or absence of
a 100-fold molar excess of cold R1881. The bound radioactivity was
calculated by subtracting the amount of radioactivity incorporated in
the presence of competitor from the amount incorporated in the absence
of competitor. A, bound [3H]R1881 as a
function of hormone concentration in cells transfected with AR alone
(diamonds) or in combination with either Bag1
(crosses) or Bag1L (squares). B-D,
Scatchard analyses of the data represented in A, indicating
the calculated Kd for R1881 of AR in the presence or
absence of Bag1 and Bag1L. Data represent the mean ± S.E. of
three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-16 were localized to nuclear
substructures, Bag1L
1-50 protein was present diffusely throughout
the cells. Therefore, the region between amino acids 17 and 50 of
Bag1L was found to be required for its efficient import into nuclei or
retention within the nucleus. We speculate that this
NH2-terminal region of Bag1L participates in interactions
with the nuclear proteins that serve to anchor Bag1L firmly in the nucleus.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Chawnshang Chang for AR and ARE-CAT plasmids and Rachel Cornell for manuscript preparation.
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FOOTNOTES |
---|
* This work was supported in part by the United States Army Department of Defense Prostate Cancer Research Program Grant DAMD17-98-1-8584, the National Institutes of Health/National Cancer Institute Grant CA-67329, the Susan G. Komen Breast Cancer Foundation (to D. A. K.), The Schweizerische Stiftung fuer medizinisch-biologische Stipendien (to B. A. F.), and the State of California Cancer Research Program Grant CCRP99-00567V-10110).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to the work.
§ Current address: Dept. of Molecular Biology, University of Zurich, Winterthurerstrasse 190, 857 Zurich, Switzerland.
¶ To whom correspondence should be addressed: The Burnham Inst., 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax: 858-646-3194; E-mail: jreed@burnham-inst.org.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010841200
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ABBREVIATIONS |
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The abbreviations used are: AR, androgen receptor; Hsp, heat shock protein; UBL, ubiquitin-like; NLS, nuclear localization sequences; CAT, chloramphenicol acetyltransferase; CT-FBS, charcoal-treated fetal bovine serum; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; ARE, androgen response element.
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REFERENCES |
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