From the Max Planck Institute of Psychiatry,
Kraepelinstraße 10, D-80804 Munich and § Max Planck
Institute of Biochemistry, Am Klopferspitz 18a,
D-82152 Martinsried, Germany
Received for publication, November 25, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The co-chaperone BAG-1 is involved in the
regulation of steroid hormone receptors, including the glucocorticoid
receptor (GR). More recently, BAG-1 was found in the nucleus where it
decreases GR transactivation. Moreover, nonspecific DNA binding of
BAG-1 has been reported. We discovered that of the N-terminal part of BAG-1M, the first 8 amino acids are sufficient for DNA binding, containing a stretch of three lysines and a stretch of three arginines. Changing the spacing between these stretches had no effect on DNA
binding. Surprisingly, this small, nonsequence-specific DNA binding
domain was nonetheless necessary for the inhibitory function of BAG-1
for GR-dependent transcription, whereas the following serine- and threonine-rich
E2X4 repeat domain was not.
Mutational analysis of these two domains revealed that only mutants
retaining DNA binding capability were able to down-regulate GR-mediated transactivation. Intriguingly, lack of DNA binding could not be functionally rescued by BAG-1M harboring a point mutation abolishing interaction with hsp70. Thus, DNA binding and hsp70 interaction are
required in cis. We propose that the nonsequence-specific DNA-binding protein BAG-1 acts at specific chromosomal loci by interacting with other proteins.
BAG-1 is involved in the regulation of a diversity of
physiological processes, e.g. apoptosis, tumorigenesis, and
neuronal differentiation (1-4), by virtue of its ability to interact
with numerous regulatory proteins, including nuclear receptors. BAG-1 was originally identified as an associating factor of the
anti-apoptotic factor bcl2 (5) and, independently, as a protein called
RAP46 that associates with the glucocorticoid receptor
(GR)1 and other steroid
hormone receptors (6). It also is termed "hap46," hsp70- and
hsc70-associating protein (7). BAG-1 enhances the transcriptional
activity of the androgen receptor (8, 9), and in the case of the
vitamin D receptor evidence has been provided for both enhancement (10)
and inhibition (11) of transcriptional activity. In addition, BAG-1
interaction with the retinoic acid receptor (RAR) results in inhibition
of binding of RAR to its response elements on DNA and of
RAR-dependent transcription (12). Likewise, BAG-1 has been
described as a negative regulator of GR, because it binds to the hinge
region of GR and inhibits DNA binding and transactivation of the
receptor (13).
Before hormone activation, GR resides in the cytoplasm, where it
interacts with various molecular chaperones in a stepwise fashion to
attain the state competent for hormone binding (14-16). Central to
this folding process are heat shock protein (hsp) 90, hsp70, and
hsp70/hsp90 organizing protein (hop) (17-19), which bridges hsp70 and
hsp90 via its tetratricopeptide repeat domains (20). hsp90, hsp70, and
hop, possibly with the involvement of hsp40 (21), form an intermediate
complex with GR (15, 22), from which hsp70 and hop presumably
dissociate to allow entry of p23 and one of the large immunophilins to
the final complex, where GR gains hormone binding competence.
The chaperone activity of hsp70 can be modulated by BAG-1 and also by
hsp40, C terminus of hsp70-interacting protein, and hsp70-interacting
protein (hip). hsp40 enhances the ATPase activity of hsp70 in
vitro (23) and the hsp70-dependent refolding in mammalian cells (24, 25). hip also has been identified as a positive
regulator of hsp70 chaperone activity (26, 27). In contrast, the C
terminus of hsp70-interacting protein has been found to inhibit the
ATPase activity of hsp70 and to interfere with stable hsp70-substrate
complexes (28). All isoforms of BAG-1, i.e. the 50-kDa
BAG-1L, the 46-kDa BAG-1M, and the 33/29-kDa BAG-1S, which derive from
different translation initiation sites localized on the same gene
(29-31), have been described in several studies (27, 32-35) to
inhibit hsp70-dependent refolding activity in
vitro and in vivo. Moreover, BAG-1 was found to compete
with the stimulatory action of hip (27). hip, in turn, opposes the negative effect of BAG-1 on steroid binding of GR (36). The negative
effect of BAG-1 on steroid binding (37) was not observed by others
(38). This seeming discrepancy could be explained by different protein
concentrations of BAG-1 present in these assays, as the protein level
of BAG-1 has been shown to be crucial for its inhibitory action
(37).
Although all these reports strongly suggest a cytosolic effect of BAG-1
on GR folding and activity, there are also data supporting a nuclear
function of BAG-1. For example, BAG-1 has been reported to bind to DNA
in a nonsequence-specific manner and to stimulate DNA transcription
(39). Deletion of, or mutations within, the N-terminal 10 amino acids
of BAG-1 abolish its DNA binding (39). In addition, it has been shown
that BAG-1 is transported into the nucleus upon steroid binding and
nuclear translocation of GR (38). This nuclear translocation is
dependent on the C-terminal hsp70 binding domain of BAG-1. Moreover,
the inhibiting effect of BAG-1 on DNA binding of GR (13) can be
overcome in a cell-free system by supplementing with increasing amounts
of hsp70 (40).
Both C- and N-terminal deletions deprive BAG-1 of its ability to
inhibit GR function (38). Whereas an hsp70 interaction domain has been
identified and characterized by crystallography and NMR analysis in the
C-terminal region (41, 42), the function of the N-terminal part seems
less clear. It has been speculated that a serine- and threonine-rich
E2X4 domain may be necessary for the
inhibitory function of BAG-1 (38). However, it also is conceivable that
the described DNA binding domain is important for inhibition of GR function.
Here we present a detailed mutational analysis of the N-terminal part
of BAG-1 with the aim to 1) describe the unusual DNA binding domain in
more detail, and 2) dissect the contributions of the
E2X4 motif domain and the DNA
binding domain to the inhibition of GR-dependent
transcription. We demonstrate here that, surprisingly, the first eight
amino acids of BAG-1 are required for the inhibitory effect of BAG-1 on
GR, whereas the E2X4 domain is
dispensable. Moreover, the binding of BAG-1 to DNA is because of the
positive electric charge at the N terminus. Mutations that inhibit
binding to DNA cannot be functionally rescued by overexpressing BAG-1 with a point mutation abolishing its interaction with hsp70, indicating that the DNA binding and the hsp70 interaction domain must be present
in cis. Here we discuss a model in which the nonspecific DNA-binding motif of BAG-1 is essential for inhibiting GR function and
becomes specific due to interaction with other factors like GR.
Cell Culture and Transfection--
Human neuroblastoma SK-N-MC
cells (ATCC HTB-10) and COS-7 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum (FCS),
36 mg/liter sodium pyruvate, 100 units/ml penicillin, 100 µg/ml
streptomycin sulfate, 0.25 µg/ml amphotericin (all from Invitrogen),
and 4.5 g/liter glucose at 37 °C and 10% CO2.
Two days before transfection, cells were seeded into medium containing
10% charcoal-stripped, steroid-free FCS. Dextran T-70 (Amersham
Biosciences) was used for charcoal-stripping of FCS (43).
Cells were harvested at about 70-90% confluency, and about 0.5 to
1 × 107 cells were resuspended in 400 µl of
electroporation buffer (50 mM
K2HPO4, 20 mM KAc, pH 7.35, 25 mM MgSO4). 3.5 µg of steroid-responsive firefly luciferase reporter plasmid MTV-Luc (44), 4 µg of pRK7GR that
expresses human GR (45) from the cytomegalovirus promoter of the vector
pRK7 (46) or an HA-tagged version of it, 6 µg of either one of the
BAG-1 expression plasmids (Tables I and II) or the corresponding empty
expression vector (to keep the total amount of transfected DNA
constant), and as internal control plasmids either 3.5 µg of simian
virus 40 (SV40) promoter-driven BAG-1 Expression Plasmids--
A series of BAG-1 mutants was
generated by PCR and cloned into the mammalian expression plasmid
pRK5mcs. The plasmid pRK5mcs was derived from pRK5SV40PUR (46) by
cutting with the restriction endonucleases EcoRI and
HindIII and inserting an annealed linker oligonucleotide
with the sequences
5'-AATTCTCGAGATATCGGGCCCGGATCCGCGGCCGCTCGCGA-3' and
5'-AGCTTCGCGAGCGGCCGCGGATCCGGGCCCGATATCTCGAG-3' for the opposite strand. For cloning, the wild type and mutant sequences of BAG-1 were
amplified by PCR from the plasmid pMal-c2-Bag-1 (34). We chose the
vector pProExHTa (Invitrogen) to express BAG-1 proteins in bacteria.
Sequences of the oligonucleotides used are provided as Supplemental Material.
Luciferase and
When using the Renilla luciferase expression plasmid in
combination with the firefly reporter plasmid, cell extracts were scraped in 200 µl of a lysis buffer provided and prepared according to the manufacturer's recommendation (Promega Inc.), and firefly and
Renilla luciferase activities were measured on a 96-well
plate in 50 µl of cell extract in the automatic luminometer on a
96-well plate.
Protein Expression and Purification--
Histidine-tagged BAG-1
encoding plasmids were grown in pBL3Lys bacteria (Invitrogen). Lysis
and purification was performed using Ni-NTA-agarose according to the
manufacturer's recommendations (Qiagen). The histidine tag was removed
by cleavage with tobacco edge virus protease for 6 h at 30 °C
(Invitrogen). The cleaved proteins were rebound to Ni-NTA-agarose
columns again to remove traces of uncleaved proteins and purified
afterward with Bio-Spin columns (Bio-Rad) to remove
dithiothreitol from cleavage buffer and traces of liquid
columns. Protein concentrations were determined using the BCA Protein
Assay kit (Pierce).
DNA Binding and Gel Shift Assays--
DNA binding of BAG-1 was
essentially performed as described (39). Briefly, 1 or 2 µg of
purified and tobacco edge virus-cleaved BAG proteins (isoforms or
mutant) were incubated with 0.2 ng of 32P-end-labeled
125-bp Immunodetection of GR and BAG-1 Proteins--
25 µg of
total protein from whole cell lysates used for luciferase assays
was separated by SDS-PAGE under denaturing conditions (Lämmli
buffer containing 10% SDS). The proteins were transferred to
polyvinylidene difluoride membrane (Schleicher & Schüll). Nonspecific binding to the membrane was blocked by 5% nonfat milk in
Tris-buffered saline/Tween buffer, and then specific antibodies were
added. BAG protein was detected by addition of a monoclonal anti-BAG
antibody (anti-BAG-C16, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
followed by horseradish peroxidase-conjugated goat anti-rabbit antibody
(Sigma). The HA tag on GR was detected by a monoclonal anti-HA antibody
(Boehringer Ingelheim) followed by addition of horseradish
peroxidase-conjugated goat anti-rat antibody (Sigma). Signals were
visualized by enhanced chemiluminescence system solution (Amersham Biosciences).
Deletion of the N-terminal DNA Binding Domain of BAG-1 Abolishes
Its Inhibitory Function on GR, Whereas Deletion of the
E2X4 Motif Domain Does Not--
BAG-1S, the
short isoform of BAG-1, is unable to inhibit the transcriptional
activity of GR (38). The N-terminal amino acid stretch missing in
BAG-1S as compared with BAG-1M contains a serine- and threonine-rich
E2X4 repeat domain and a recently
described DNA binding domain (Table I)
(39). To clarify which features in the N terminus are required for
inhibition of GR, we created two deletion mutants, either missing the
E2X4 domain (BAG-1M
BAG-1M clearly inhibited GR-dependent reporter gene
transcription, as expected (Fig. 1A). To our surprise,
however, deletion of the E2X4 motif
domain had no influence on the ability of BAG-1 to inhibit GR-mediated
transcription. On the other hand, deletion of the N-terminal 10 amino
acids completely abolished the effect of BAG-1 on GR. The same pattern
of activity of the BAG-1 mutants was observed in COS-7 cells and HeLa
cells (data not shown). Because co-expression of various plasmids can
produce misleading results in case of expression interference (49), we
checked the relative amounts of GR and of BAG-1. The protein levels of
GR were similar in all the experiments (Fig. 1B). Likewise,
the levels of the expressed BAG-1 proteins were comparable
throughout our experiments (Fig. 1C). We did not detect
significant levels of endogenous BAG-1 in SK-N-MC or COS-7 cells.
However, endogenous BAG-1 was detected in HeLa cells (data not shown).
Thus, our results do not depend on the presence or absence of
endogenous BAG-1. We conclude that the domain recently put forward as
DNA binding domain of BAG-1 (39) is necessary for the inhibitory effect
of BAG-1 on GR.
Spacing of the Lysine and Arginine Stretch Is Not Important for DNA
Binding of BAG-1--
To characterize the DNA binding domain of BAG-1
in more detail, we created a series of additional mutants (Table
II); all proteins derived from BAG-1
mutants and isoforms in Tables I and II were expressed in bacteria and
purified (Fig. 2, A and B). As a reference, we mutated lysines 2, 3, and 4 to
alanines (BAG-1M KA), which has been described to abolish DNA binding
of BAG-1. To reduce the DNA binding domain essentially to the
positively charged amino acids lysine and arginine, we deleted amino
acids 9-67 (
The purified and cleaved proteins were used to examine their ability to
bind to DNA. The 125-bp fragment of an HindIII cleavage of
DNA from bacteriophage Mutants That Inhibit DNA Binding of BAG-1 also Abolish Its
Inhibitory Function on GR--
The results shown in Fig. 1 suggest
that DNA binding of BAG-1 is necessary for its inhibitory effect on GR
function. We set out to either strengthen this correlation or to prove
that, although this domain per se is necessary, it is not
DNA binding but some other property of this domain that causes the
inhibitory effect. Therefore, we constructed expression clones of all
mutants and tested them in our transient reporter gene assay as
described in Fig. 2. We find that BAG-1M
Therefore, the data in Figs. 1-3 demonstrate that all mutants of
BAG-1M that are able to bind to DNA inhibit the transcriptional activity of GR, whereas those that are unable to bind to DNA have no
influence on GR activity.
The DNA Binding and the hsp70 Interaction Domain of BAG-1 Need to
be Present in Cis to Inhibit GR Function--
Besides the N-terminal
domain, deletion of the C-terminal 70 amino acids also abolishes the
effect of BAG-1 on GR (40). Because these amino acids contain the
interaction domain with hsp70, it has been proposed that interaction
with hsp70 is necessary for the function of BAG-1. This raises the
question whether the domains responsible for interaction with hsp70 and
for binding to DNA are required to be present in cis.
Therefore, we first used a point mutation of BAG-1 that abolishes
interaction with hsp70 (R237A BAG-1M (42)). In our transient reporter
gene assay, this mutant abolishes the inhibition of GR activity by
BAG-1 (Fig. 4A, lane
4). This proves that the previously reported inability of a
C-terminally truncated BAG-1 protein to inhibit GR is actually due to
the inability to interact with hsp70 rather than to some other
concomitant consequence of the deletion. To test whether a BAG-1
protein that is unable to interact with hsp70, but has an intact DNA
binding domain, can rescue the inhibitory function of a DNA-binding
mutant bearing an intact hsp70 interaction domain, we concomitantly
expressed a DNA-binding mutant (KA) with an hsp70 interaction mutant in
our reporter assay. Although these proteins are expressed to a
comparable level as wild type BAG-1 (Fig. 4B), no inhibition
of GR-dependent reporter gene transcription was detected
(Fig. 4A, lanes 5 and 6). We also show
that the hsp70 interaction mutant does not prevent inhibition of GR by
BAG-1M (Fig. 4A, lanes 7 and 8). The
levels of GR were the same throughout our experimental conditions (Fig.
4C). We conclude that the N-terminal domain and the hsp70
interaction domain of BAG-1 need to be present at the same time and on
the same molecule.
BAG-1 displays a remarkable functional versatility by
participating in regulation of apoptosis via interaction with
Bcl-2, by functioning as a modulator of chaperone activity via
interaction with hsp70, by enhancing transcription in general (50), by
regulating neuronal differentiation (3), by mediating stress signaling via regulating Raf-1/ERK (51), and by modulating nuclear
receptor-dependent transcription (shown here and see Refs.
9, 12, 13, and 40). This functional diversity suggests that BAG-1 uses
different domains for different functions. For example, it has been
reported recently (52) that distinct BAG-1 isoforms have different
anti-apoptotic functions. In the present study, we focus on the
requirements for BAG-1 to inhibit GR-dependent transcription.
It has been speculated that the serine- and threonine-rich
E2X4 domain is necessary for the
inhibition of GR function by BAG-1 (38). However, we demonstrate that
deletion of the entire E2X4 motif
domain does not affect the ability of BAG-1 to counteract GR-dependent transcription. This explains the recent
finding that mutations of phosphorylation sites within this domain
retain the activity of BAG-1 (40). Against expectation, we discovered
that a small DNA binding domain of only 8 amino acids is necessary for
inhibition of GR function. This DNA binding domain is uncommon for
transcriptional regulatory proteins, because DNA binding appears to be
nonspecific (39). The nonspecificity of DNA binding is in line with our
observation that spacing of the two positively charged stretches of
three lysines and three arginines is not important for DNA binding.
This suggests an electrostatic interaction of these positive charges
with the negatively charged phosphate backbone of DNA.
Another domain of BAG-1 essential for inhibition of
GR-dependent transcription is the hsp70 interaction domain
(38). By employing a point mutation that interferes with binding to
hsp70, we were able to prove that the importance of this domain is not merely structural stabilization of the protein but that interaction with hsp70 indeed is necessary. Moreover, we demonstrate that binding
to hsp70 and binding to DNA have to be linked on the same molecule,
i.e. a mixture of two BAG-1 proteins that either carried a
mutation in the one or the other domain showed no effect on GR-dependent transcription.
What are the structural requirements of BAG-1 for its effect on other
nuclear receptors? In the case of the androgen receptor BAG-1 enhances
transcription, but the domains required are similar at first sight,
because the N-terminal domain of the long isoform BAG-1L and the hsp70
interaction domain are both required (9). However, it appears that the
N-terminal domain of BAG-1L is necessary only for nuclear targeting,
because BAG-1M is inefficient unless it is forced to the nucleus by
adding a targeting sequence (9). Similarly, the vitamin D receptor also
has been reported to be enhanced by BAG-1L but not by BAG-1M. Moreover,
the hsp70 interaction domain is required (10). It should be noted
however, that more recently an inhibition of vitamin D receptor by
BAG-1L has been reported (11). In the case of the retinoic acid
receptor, an inhibition by BAG-1M has been demonstrated (12), but no
domain analysis was conducted so far.
Thus, taken these reports on the effects of BAG-1 on nuclear receptors
together, it seems clear that, although the effect of BAG-1 can be
either inhibitory or stimulatory, the N-terminal and C-terminal domains
are required. We could prove that the function of the C terminus is
indeed the interaction with hsp70. The N terminus of BAG-1L has been
suggested to be important for nuclear targeting of BAG-1. However, GR
and the retinoic acid receptor are inhibited also by BAG-1M. Moreover,
BAG-1M can be translocated into the nucleus upon activation of GR by
hormone (40). Thus, we propose that the additional N-terminal amino
acids of BAG-1L are necessary only for those receptors that do not
promote nuclear transport of BAG-1M. This is also supported by the
observation that BAG-1M is effective on vitamin D receptor upon forced
nuclear targeting (9). We discovered that the N-terminal 8 amino acids of BAG-1M, which probably represent the entire DNA binding domain, are
also required for its function on GR. It will be interesting to
evaluate the function of this domain for the effect of BAG-1M on other
nuclear receptors.
Regarding the function of the DNA binding domain of BAG-1M, it is of
note that BAG-1M binds nonsequence-specifically to DNA. Its
intracellular concentration can be estimated from careful analyses in a
variety of tumor cell lines (30, 35) to be one molecule per 10-100 kb
of DNA. Moreover, it is a predominantly cytosolic protein (30, 40).
Therefore, other factors must determine when and where BAG-1M binds to
DNA. We propose the following model: BAG-1 is transported into the
nucleus along with GR upon activation of the receptor with hormone
(40). GR interacts with the chromatin at glucocorticoid response
elements. The first step in GR-mediated transactivation presumably is
remodeling of the local chromatin structure by cofactors recruited by
GR (53). Once the chromatin is restructured, BAG-1 gains access to the DNA, which interferes with the further functions of GR for efficient transactivation. Access of BAG-1 to the DNA might be promoted by the
rapid exchange with regulatory sites of GR (54).
The guidance of a nonsequence-specific DNA-binding protein to specific
chromosomal loci as suggested here for BAG-1 is not without precedence;
Cdc6 is an essential protein in yeast which is recruited to replication
origins in the G1 cell cycle phase by another replication
factor, origin recognition complex protein 1 (55). It is intriguing not
only that cdc6 alone, like BAG-1, binds nonspecifically to DNA but also
that site-directed mutagenesis identified the basic protein motif KRKK
as essential for DNA binding and function of the protein (56).
Apparently, this motif is very similar to the basic motif of the N
terminus of BAG-1 that we demonstrate is not only essential for binding
to DNA but also for functional integrity with respect to the inhibition
of GR-dependent transcription.
Finally, because we demonstrate that DNA binding and interaction with
hsp70 are required in cis, it is likely that hsp70 is required during this process, i.e. hsp70 might be present
during chromatin remodeling or even promoting it. In general,
chaperones like hsp70 and hsp90 are considered to act in the cytosol.
However, nuclear translocation of hsp70 along with hormone-activated GR and BAG-1 has been reported (40). In addition, there is ample evidence
that hsp90 is involved in subcellular trafficking and chromatin
recycling of GR (57, 58), expanding the original observation that the
Hsp90/Hsp70-based chaperone machinery of reticulocyte lysate could
dissociate the hormone free GR from DNA and regenerate the
nonDNA-binding GR-Hsp90 heterocomplex (59). Moreover, a nuclear role in
regulating the chromatin assembly and activity of nuclear receptors has
been reported recently for the hsp90-interacting chaperone p23 (60,
61). An open question is whether the described disassembly of
transcriptional regulatory complexes by p23 requires ATP (61), as the
requirement of hsp90 or hsp70 for the effect of p23 remains to be elucidated.
In summary, overwhelming evidence has accumulated that molecular
chaperones and associated proteins like BAG-1 have distinct functions
in the nucleus. Obviously, DNA binding of BAG-1 is a clear indication
of a nuclear function of this protein. Our model proposed above also
implies that BAG-1 may contribute to promote disassembly of
transcriptional regulatory complexes by an Hsp70-based process,
similarly to p23, which may act via hsp90. This model is also
consistent with the observation that DNA-independent transrepression by
GR is not inhibited by BAG-1 (38). Future experiments will show whether
the DNA binding domain is important for other functions of BAG-1 and
whether the model proposed here can also be applied to other nuclear receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression vector
pCH110 (Amersham Biosciences) or 1 µg of SV40-driven
Renilla luciferase-driven expression vector (Promega) were
added. Transfection was performed using an electroporation system
(Biotechnologies & Experimental Research, San Diego) after determination of the optimal electrical field strength (47). Electroporated cells were replated and cultured for 24 h in fresh medium (containing 10% steroid-free FCS) supplemented with 100 nM dexamethasone (Sigma) or the solvent of dexamethasone
(i.e. ethanol).
-Galactosidase Assays--
Firefly luciferase
and
-galactosidase assays were as described before (48). Either
cells were scraped in 200 µl of lysis buffer (0.1 M
KHPO4, pH 7.8, 1 mM dithiothreitol) and
cytosolic extracts were made by three freeze and thaw cycles and
subsequent centrifugation. 50 µl of each supernatant (corresponding
to ~1-2 × 105 cells) were transferred to a 96-well
plate. 150 µl of 33 mM KHPO4, pH 7.8, 1.7 mM ATP, 3.3 mM MgCl2, 13 mM luciferin (Roche Molecular Biochemicals) was added to
each sample by the injector of an automatic luminometer (Luminat LB 96, Wallac GmbH, Freiburg, Germany), and light emission was measured for
10 s. To correct for variations in transfection efficiencies,
values of the luciferase assay were normalized to either
-galactosidase or Renilla luciferase activities. To
measure
-galactosidase activity, 50 µl of cell extract was added
to 100 µl of galactosidase buffer (60 mM
Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 50 mM
-mercaptoethanol) on a 96-well plate. 20 µl of 2 mg/ml
O-nitrophenyl
-D-galactopyranoside was added,
and the reaction was incubated at 37 °C. After 10-30 min,
absorption was measured at 405 nm in a multiphotometer (Dynatech MR5000).
/HindIII DNA fragment in binding buffer (39) for
30 min at room temperature. Protein DNA complexes were resolved on
native 5% acrylamide gels in TBE buffer and visualized after overnight
incubation on x-ray films (Eastman Kodak).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11-67, Table
I) or the putative DNA binding domain (BAG-1M
N10, Fig. 1). These mutants were analyzed in
transient transfection assays in two cell lines, COS-7 cells and the
neuroblastoma cell line SK-N-MC. Cells were expressed with a reporter
plasmid carrying the luciferase gene driven by the GR-sensitive mouse
mammary tumor virus (MMTV) promoter, a Renilla luciferase
reference plasmid, and either an empty expression vector or a vector
expressing one of the BAG-1M mutants. The light units measured for the
reporter enzyme were normalized by the light units of the control
enzyme (Renilla luciferase). Stimulation after incubation
with 100 nM dexamethasone was in the range of 200-300-fold
and was set to 100%.
Structure of BAG-1M, BAG-1S, and the mutants BAG-1M N10 and
BAG-1M
11-67
View larger version (14K):
[in a new window]
Fig. 1.
Effect of wild type BAG-1M,
N10, and
11-67 on
GR-dependent transcription. A, SK-N-MC
cells were transiently transfected with 3.5 µg of GR-responsive
MMTV-luciferase (MTV-Luc) reporter gene, 1 µg of an
internal control plasmid encoding Renilla luciferase under
the control of the SV40 promoter, and 4 µg of a human GR-encoding
plasmid (pRK7GRHA). In addition, 6 µg of either an empty expression
vector (C = control) or the mutants BAG-1M
N10,
BAG-1M
11-67, or the isoforms BAG-1M and BAG-1S were cotransfected.
Cells were transfected using electroporation and replated and cultured
for 24 h in fresh medium either with or without 100 nM
dexamethasone. Luciferase activities were corrected by
Renilla luciferase activities and are presented as percent
activity with the activity of control vector-transfected cells set as
100%. Results represent mean values ± S.E. of eight independent
experiments performed in sextuplicate. B and C,
representative Western blot of either GR or BAG protein. Whole cell
extracts used for luciferase assays were prepared for SDS-PAGE and
immunoblotting. Antibodies were directed against either the C terminus
of BAG-1 or the HA tag on GR.
9-67). Moreover, we changed the spacing between
lysines 2-4 and arginines 6-8 by either deleting threonine 5 (BAG-1M
Thr5) or inserting one alanine (BAG-1M
+Ala5) or two alanines (BAG-1M +2Ala5). After
expression in bacteria, proteins were purified using Ni-NTA-agarose
(Fig. 2, A and B, 1st lane of each
mutant), and the histidine tails were cleaved with tobacco etch virus
protease (Fig. 2, A and B, 2nd lane of
each mutant).
Structure of BAG-1 mutated in the DNA binding domain
View larger version (47K):
[in a new window]
Fig. 2.
A and B, bacterially
expressed BAG-1 and mutants. BAG-1 isoforms and mutants were cloned
into the bacterial expression vector pProexHTa (Invitrogen) that
delivered a histidine tail to the N terminus of the proteins. BAG-1M
isoforms and mutants were expressed in E. coli, purified
using Ni-NTA-agarose columns following the QIAexpressionist protocol
(Qiagen Inc., Hilden, Germany). The histidine tail was removed by
cleaving with tobacco edge virus protease, and proteins were analyzed
by SDS-PAGE. Staining was performed with Coomassie Blue R-250.
Noncleaved and cleaved proteins were loaded alternately onto gels.
M, BAG-1M; KA, BAG-1M KA;
T5, BAG-1M
Thr5; +A5,
BAG-1M +Ala5, +2A5,
BAG-1M +2Ala5,
9-67, BAG-1M
9-67,
11-67, BAG-1M
11-67; S, BAG-1S,
N10, BAG-1M
N10. C and D, DNA
binding of BAG-1M isoforms and mutants. Radiolabeled 125-bp fragment
from phage
-DNA/HindIII was incubated with either 1 (1st lane of each mutant) or 2 µg (2nd lane of
each mutant) of protein for 30 min at 25 °C, and complexes were
separated on an acrylamide gel under native conditions. Lane
0, labeled DNA without protein; other lanes = binding
reactions, coding as in A and B. Shown are
representative autoradiograms.
was chosen as template. Gel shift assays
(Fig. 2C) revealed that mutation of lysines 2-4 to alanines abolished DNA binding, consistent with a recent report (39). However,
deletion of the E2X4 domain
(BAG-1M
11-67; Fig. 2D) had no influence on
DNA binding. Even additional deletion of two N-terminal amino acids
retained DNA binding (BAG-1M
9-67; Fig. 2C).
BAG-1S, as expected, did not bind to DNA. Therefore, it appears that
the first 8 N-terminal amino acids are sufficient to confer the ability
of BAG-1 to bind to DNA. Moreover, spacing between the positively
charged amino acids lysines 2-4 and arginines 6-8 is not important
for DNA binding, because these mutants bind to DNA as efficiently as
BAG-1M (Fig. 2, C and D). Therefore, BAG-1M apparently contains a short, unusual DNA binding domain.
9-67, BAG-1M
Thr5, BAG-1M +Ala5, and BAG-1M
+2Ala5 all are able to inhibit GR-dependent
transcription (Fig. 3A). In
contrast, BAG-1S and BAG-1M KA lost the inhibitory effect on GR. Again,
the protein levels of GR were comparable throughout the experiments
(Fig. 3B), as were the levels of the different BAG-1 mutants
(Fig. 3C). Similar data were obtained in HeLa cells (data
not shown).
View larger version (14K):
[in a new window]
Fig. 3.
DNA binding-defective mutants are unable to
inhibit GR. A, SK-N-MC cells were transiently
transfected with 3.5 µg of GR-responsive MMTV-luciferase
(MTV-Luc) indicator gene, 1 µg of an internal control
encoding Renilla luciferase under the control of the SV40
promoter, and 4 µg of a human GR-encoding plasmid (pRK7GRHA). In
addition, 6 µg of either an empty expression vector (C,
control) or the mutants BAG-1M KA, BAG-1M 9-67, BAG-1M
+Ala5, BAG-1M +2Ala5, BAG-1M
Thr5, and the wild type BAG-1M were cotransfected. Cells
were transfected by electroporation, replated, and cultured for 24 h in fresh medium either with or without 100 nM
dexamethasone. Luciferase activities were corrected by
Renilla luciferase activities and are presented as percent
activity with the firefly luciferase activity of control
vector-transfected cells set as 100%. Results represent mean
values ± S.E. of six independent experiments performed in
sextuplicate. B and C, representative Western
blot of either GR or BAG protein. Whole cell extracts used for
luciferase assays were prepared for SDS-PAGE and immunoblotting.
Antibodies were directed against either the C terminus of BAG or the HA
tag on GR.
View larger version (22K):
[in a new window]
Fig. 4.
The DNA binding domain and the hsp70
interaction domain of BAG-1 need to be present in
cis. A, SK-N-MC cells were transiently
transfected with 3.5 µg of GR-responsive MMTV firefly luciferase
(MTV-Luc) indicator gene, 2.5 µg of an internal control
plasmid encoding -galactosidase (pCMV
Gal) under the control of
the cytomegalovirus promoter, and 4 µg of a human GR-encoding plasmid
(pRK7GRHA). In addition, an empty expression vector (C,
control), the mutants BAG-1M R237A (70mut) and BAG-1M KA,
and the wild type BAG-1M were cotransfected, either with 6 µg of the
respective plasmid alone (lanes 2-4) of 3 and 6 µg of
BAG-1M KA and BAG-1M 70mut together (lanes 5 and
6) or BAG-1M hsp70mut and BAG-1M together (lanes 7 and 8). Cells were transfected using
electroporation, replated, and cultured for 24 h in fresh medium
either with or without 100 nM dexamethasone. Luciferase
activities were corrected by
-galactosidase activities and are
presented as percent activity with the firefly luciferase activity of
control vector-transfected cells set as 100%. Results represent mean
values ± S.E. of four independent experiments performed in
sextuplicate. B and C, representative Western
blot of either GR or BAG protein. Whole cell extracts used for
luciferase assays were prepared for SDS-PAGE and immunoblotting.
Antibodies were directed against either the C terminus of BAG or the HA
tag on GR.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Andrea Jarzabek for excellent technical assistance; C. Behl for providing SK-N-MC cells and the plasmid MTV-Luc; H. Büning and M. Hallek for providing COS-7 cells; D. Spengler for providing the plasmids pRK7GR and pRK7GRHA; Cornel Babel for invaluable help with artwork; and H. Sondermann for the hsp70 interaction mutant of BAG-1.
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FOOTNOTES |
---|
* 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.
The on-line version of this article (available at
http://www.jbc.org) contains sequences of the oligonucleotides.
¶ To whom correspondence should be addressed: Max Planck Institute of Psychiatry, Kraepelinstraße 10, D-80804 Munich, Germany. Tel.: 49-89-30622531; Fax: 49-89-30622605; E-mail: theorein@mpipsykl.mpg.de.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M212000200
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ABBREVIATIONS |
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The abbreviations used are: GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus; HA, hemagglutinin; FCS, fetal calf serum; Ni-NTA, nickel-nitrilotriacetic acid; RAR, retinoic acid receptor; hsp, heat shock protein; hip, hsp70-interacting protein; hop, hsp70/hsp90 organizing protein.
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