From the Departments of Cell Biology and Anatomy and
§ Medicine, Mount Sinai School of Medicine, New York,
New York 10029 and the ¶ Department of Human Genetics, University
of Michigan Medical School, Ann Arbor, Michigan 48109
Received for publication, August 14, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Cdc37 is a molecular chaperone closely associated
with the folding of protein kinases. Results from studies using a yeast model system showed that it was also important for activation of the
human androgen receptor (AR). Based on results from the yeast model
system (Fliss, A. E., Fang, Y., Boschelli, F., and Caplan, A. J. (1997) Mol. Biol. Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in
animal cell systems. Our results show that Cdc37 binds to AR but not to
glucocorticoid receptors (GR) synthesized in rabbit reticulocyte
lysates. This binding occurs via the ligand-binding domain of the AR in
a manner that is partially dependent on Hsp90 and the presence of
hormone. Further studies using the yeast system showed that Cdc37 is
not interchangeable with Hsp90, suggesting that it functions at a
distinct step in the activation pathway. Expression of a dominant
negative form of Cdc37 in animal cells down-regulates full-length AR
but has very little effect on an AR truncation lacking the
ligand-binding domain or full-length GR. These results reveal
differences in the mechanisms by which AR and GR become active
transcription factors and strengthen the notion that Cdc37 has a wider
range of polypeptide clients than was realized previously.
Steroid hormone receptors comprise a family of proteins that bind
their steroid ligands inside cells, in either the cytosol or the
nucleus. Their conformation is stabilized prior to ligand binding by a
group of proteins known collectively as molecular chaperones. In
addition to maintaining apo-receptors in a stable conformation,
molecular chaperones also appear to prevent ligand-independent activation (1).
Many studies performed over the last 10 years have led to the
identification of molecular chaperones that have a general function in
the activation of steroid hormone receptors. Hsp90, for example, interacts with the androgen receptor
(AR),1 aryl hydrocarbon
receptor, estrogen receptor, glucocorticoid receptor (GR),
mineralocorticoid receptor, and the progesterone receptor (PR) and is
important for ligand binding by all of these receptors (reviewed in
Ref. 1). The pathway leading to Hsp90 association involves the action
of several other chaperones and co-chaperones (or helpers). The minimal
complement of chaperones and co-chaperones necessary for efficient
folding includes Hsp70, Hsp40, Hop, and p23 in addition to Hsp90 (2,
3). The pathway by which these chaperones act has common requirements
among several receptors, but there are also some differences. Common
requirements include the actions of Hsp70 and Hsp90 (4, 5). The
co-chaperone called Hop (Hsp organizing
protein; Ref. 6) binds to both Hsp90 and Hsp70, forming a
bridge between them. A complex containing the receptor, Hsp70, Hop, and
Hsp90 may be replaced by complexes containing just Hsp90, p23, and
immunophilins such as cyp40 or FKBP52. The function of p23 is unknown;
it is important for hormone binding by GR and PR (7) (3) yet has been
shown recently to inhibit the activity of AR (8). In a second case, AR
activity was shown to be stimulated by the long form of Bag-1 (Bag1-L), a co-chaperone of Hsp70, whereas this same isoform inhibited
hormone-dependent signaling by GR (9, 10). The AR and GR
also respond differently to deletion of yeast YDJ1, which
encodes an Hsp40 co-chaperone. In a Another example of a differential chaperone requirement for steroid
receptor activation involves Cdc37. Cdc37 is required for the folding
of several protein kinases and was proposed to be kinase specific based
on its failure to interact with aryl hydrocarbon receptor, estrogen
receptor, GR, or PR in vitro (15-17). On the other hand,
genetic studies using the yeast Saccharomyces cerevisiae as
a model system showed that Cdc37 was important for hormone-dependent activity by AR but not by GR (18). These
studies also revealed that Cdc37 functions at a later stage in the
folding process compared with Hsp90 or Ydj1p. This was implied from the results of experiments showing that mutation in CDC37
affected transactivation by AR but not hormone binding, whereas in
hsp82 and ydj1 mutants there was a strong
correlation between loss of activity and decreased affinity for hormone
(14, 19).
Several studies have shown that Cdc37 can bind to protein kinases, both
in association with and independently of Hsp90. The protein kinase
catalytic domain itself appears to be the target for interaction with
Cdc37 (20), and it can bind to or affect the activity of a broad range
of kinase subtypes, including casein kinase II (21),
cyclin-dependent kinases (22) (23), the mitogen-activated
protein kinase Ste11 (24), Mps1, a kinase required for spindle pole
body duplication (25), the nonreceptor tyrosine kinase v-Src (26), and
Raf (27). The role of Cdc37 in the folding process remains unclear,
although recent studies have begun to address its mechanism of action.
One suggestion was that Cdc37 targets Hsp90 for subsequent chaperone
action (23). On the other hand, overexpression of yeast Cdc37 can
suppress the defect in v-Src activity in an hsp82 mutant
strain, suggesting that it can act independently as a molecular
chaperone (28). Perhaps the most likely scenario is that Cdc37 has
chaperone activity that is closely tied to the presence of Hsp90 and
other co-chaperones. Recent studies have shown that the normally salt
labile interaction between Hsp90 and Cdc37 is made salt-resistant in
the presence of a client kinase. Furthermore, Cdc37 is present in
multi-chaperone complexes containing p23 and immunophilins (29). This
suggests that Cdc37 functions late in the folding pathway compared with other chaperones such as Hop/Hsp70/Hsp40, which may function further upstream, as they do in steroid hormone receptor folding.
Given that Cdc37 functions in association with protein kinases, it
remained possible that the defect in AR transactivation that we
observed in a yeast cdc37 mutant could have resulted from defective kinase activity. Support for this possibility was
strengthened by the finding that the AR is regulated in mammalian
cells, although only in part, by phosphorylation (30-32). We therefore
initiated a biochemical approach to assay for Cdc37 binding to the AR.
The rationale for this approach was based on the hypothesis that
interaction between these proteins would only occur if Cdc37 had a
direct effect on the ability of AR to adopt the active state, as it
does with v-Src but not with the GR, which were used as controls for the experiments described below.
Materials--
Dihydrotestosterone (DHT) was from Sigma and was
stored in ethanol at Cloning, Expression, and Purification of human
His6Cdc37--
Human Cdc37 with an N-terminal
six-histidine tag was prepared from human Cdc37 cDNA (Ref. 23; kind
gift of Dr. J. Wade Harper). The Cdc37 gene was amplified by PCR using
the primers: 5'-CATATGGTGGACTACAGCGTG-3' and
5'-GGATCCGCAGGTGGCGGTGGTAGC-3'. The recombinant Cdc37 gene was
amplified by 20 cycles at 55 °C annealing temperature and 72 °C
elongation temperature using Pfu polymerase. The 1140-base pair product was gel purified before subcloning into pcrScript. The
Cdc37 gene was excised from pcrScript with NdeI and
BamHI and ligated into similarly digested pET15b, which
contains an in-frame six-histidine tag. His6Cdc37 was
expressed in Escherichia coli strain BL21DE3. The strain was
grown overnight in 100 ml of LB medium (with 100 µg/ml ampicillin) at
37 °C. The culture was diluted 1:10 with fresh LB and incubated at
37 °C for an additional hour, and Cdc37 expression was induced by
addition of isopropyl- In Vitro Transcription, Translation, and His6Cdc37
Binding Reactions--
A plasmid containing the human AR (pSP72hAR-1)
used for in vitro transcription was the kind gift of Dr. E. Wilson. Plasmids encoding GR and v-Src used for in vitro
transcription were constructed by subcloning each gene into
pBluescript. The parent plasmid for rat GR was the kind gift of Dr. K. Yamamoto (pGN795), and that for v-Src was the kind gift of Dr. F. Boschelli (pRS316.v-Src). Plasmids encoding truncated versions of the
AR were synthesized by PCR amplification from pSP72hAR-1. pJR20 (AR
1-625; TAD-DBD) was constructed using the primers:
5'-ATGGAAGTGCAGTTAGGG-3' and 5'-TCAAGTCATCCCTGCTTCATA-3'. The product
was subcloned into pPCR-Script and mRNA synthesized using
BamHI linearized plasmid and T7 RNA polymerase. pJR21 (AR
624-919; LBD) was synthesized using 5'-ATGACTCTGGGAGCCCGG-3' and
5'-TCACTGGGTGTGGAAATA-3'. The product was subcloned into pPCR-Script and mRNA synthesized using EagI linearized plasmid and
T3 RNA polymerase. Messenger RNAs for AR, GR, and v-Src were
synthesized using T3 or T7 RNA polymerases. mRNAs for AR, AR
truncations, GR, and v-Src were translated in rabbit reticulocyte
lysates (RRL) according to instructions provided by the supplier
(Promega). Geldanamycin (100 µg/ml) or DHT (100 nM) were
added prior to the start of translation reactions. Control reactions
containing solvent alone (Me2SO for geldanamycin and
ethanol for DHT) were performed at the same time. After 2-h translation
reactions (typically 25 µl), the lysates were diluted to 400 µl
with extraction buffer (20 mM Hepes pH 7.5, 100 mM KCl, 0.1 mM EDTA pH 8) and incubated with 80 µg of His6Cdc37 that was prebound to Ni-NTA resin (15 µl of packed beads/reaction). The binding reaction was incubated at
4 °C for 1 h. The resin was pelleted, washed three times with extraction buffer containing 10 mM imidazole, and eluted
with 0.5 ml extraction buffer containing 150 mM imidazole
on ice for 10 min. Eluted proteins were precipitated with 10%
trichloroacetic acid for 20 min on ice. Precipitates were resuspended
in SDS-polyacrylamide gel electrophoresis sample buffer. The samples
were resolved by denaturing gel electrophoresis. The gels were fixed in
20% methanol and 10% acetic acid, washed three times in water, and
incubated with 1 M sodium salicylate for 20 min. The gels
were dried and exposed to x-ray film. In each case, binding of
35S-labeled proteins to His6Cdc37 was compared
with the level of labeled protein contained in 1% of the translation reaction.
Expression of Yeast His6yCdc37 and Isolation of
Complexes--
An N-terminal six-histidine-tagged version of yeast
Cdc37 was constructed by PCR amplification. The template for the
reaction was pRSS2 (26), which contained wild type CDC37. This was
amplified over 10 cycles at 37 °C annealing temperature and 72 °C
elongation temperature using Taq polymerase. The primers
were: 5'-AAGCTTATGCACCACCACCACCACCACGCCATTGATTACTCTAAG-3' and
5'-GGTACCGCTACATAAATTTCTA-3'. The 1.55-kilobase product was gel
purified and ligated into pcrScript. The His6yCDC37 gene was ligated
into a 2-µm plasmid (pRS424; TRP1) that also contained the ADH1
promoter to form pPL2. This plasmid and pARH were transformed into wild
type yeast strain W3031b. Transformants were grown in selective media,
and extracts were prepared as described previously (33).
His6yCdc37 was isolated after incubation of Ni-NTA resin with 0.5 ml of whole cell extract at 3 mg/ml for 1 h at 4 °C on a nutator. The resin was washed three times in extraction buffer (as above) containing 10 mM imidazole, and bound proteins
were eluted with extraction buffer containing 150 mM
imidazole as described above. Eluted proteins were precipitated with
10% trichloroacetic acid and redissolved in SDS-polyacrylamide gel
electrophoresis sample buffer. These samples were resolved by
denaturing gel electrophoresis and transferred to polyvinylidene
difluoride membranes. The membranes were processed for Western blot as
described below.
In Vivo Hormone Binding Assays--
Wild type yeast strain p82a
(34) and the isogenic Hsp90 mutant strain G313N (33) were transformed
by pARH, which constitutively expresses the human AR and pGalCDC37 (2 µm, URA3; Ref. 18). The cells were grown in glucose or galactose
containing selective medium to A600 = 0.2, and
1-ml aliquots were incubated with 100 nM
3H-R1881 (diluted 1:5 with unlabeled R1881 of the same
concentration), plus or minus 100-fold excess of cold R1881. The cells
were incubated at 30 °C for 1.5 h with shaking. The cells were
pelleted and washed three times with ice-cold water. The cells were
counted in 5 ml of scintillation fluid. Specific binding was calculated
by subtracting the counts retained by cells incubated with 100-fold
excess of unlabeled R1881.
Transfection--
A plasmid encoding Cdc37 for expression in
animal cells was constructed by subcloning the full-length Cdc37 gene
as an EcoRI/XbaI fragment into pCMV5. The
dominant negative Cdc37 (Cdc371-173) was isolated as a
500-base pair fragment by EcoI/RsaI digestion of
the EcoRI/XbaI fragment and subcloned into pCMV5.
CV1 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Hyclone). Cells were plated at
50% confluence for transfection by the calcium phosphate precipitation method, in Dulbecco's modified Eagle's medium containing 5%
charcoal-treated NuSerum IV (Collaborative Research. The 3xHREtkCAT
reporter and AR and GR expression vectors have been described (35, 36). After overnight incubation with the DNA precipitate (1 µg), cells were washed and refed media with or without hormone (10 Western Blot--
Proteins were transferred to nitrocellulose or
PDVF membranes using a semi-dry Transblot apparatus. Filters were
washed briefly with TTBS (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) and blocked overnight with TTBS
containing 5% nonfat dry milk. Filters were incubated with primary
antibodies (usually diluted 1:1000 in antibody dilution buffer; 1×
phosphate-buffered saline, 3% bovine serum albumin, 0.05% Tween 20, and 0.1% thimerosal) for 2 h. Filters were washed three times for
10 min each in TTBS. Filters were incubated with secondary antibody
(horseradish peroxidase-conjugated goat anti-mouse IgG, diluted
1:10,000 in antibody dilution buffer) for 1 h and were
subsequently washed as for the primary antibody. Filters were then
treated with the chemiluminescence reagent (Pierce) and exposed to
x-ray film for detection.
Interaction of Human Cdc37 with AR--
We initiated biochemical
studies to address whether Cdc37 could interact with the AR. An
N-terminal His6-tagged version of human Cdc37
(His6Cdc37) was constructed and overexpressed in E. coli. The recombinant protein was purified by one step affinity chromatography on Ni-NTA resin (Fig.
1A). Binding reactions were performed with 35S-labeled AR translated in rabbit
reticulocyte lysatesRRL after addition of recombinant
His6Cdc37. Interaction between AR and His6Cdc37
was monitored after reisolation of His6Cdc37 on Ni-NTA resin, washing the complex in buffers containing a low concentration of
imidazole, and elution of bound proteins with a high concentration of
imidazole. Eluted proteins were resolved by denaturing gel electrophoresis, and labeled binding partners of Cdc37 were detected by
fluorography. As shown in Fig. 1B, the translated AR appears primarily as a single band of ~110 kDa, and this was pulled-down on
Ni-NTA resin that was preadsorbed with His6Cdc37 (Fig.
1B, lane 3). There was very little binding of AR
to Ni-NTA resin in the absence of preadsorbed His6Cdc37
(Fig. 1B, lane 2). The amount of AR that was
captured in these assays was ~1% of the total amount translated (1%
of the translation was loaded in lane 1 of Fig. 1B as a standard). Similar experiments were performed with
v-Src, a well characterized Cdc37 binding protein (37), and ~1% of the translation product was also captured on
His6Cdc37:Ni-NTA resin using the same conditions (Fig.
1B, lanes 7-9). In other experiments we tested
whether the AR was binding to contaminants from E. coli that
copurified with His6Cdc37. However, no AR was pulled-down
on Ni-NTA resin that was preadsorbed with E. coli lysates
not containing His6Cdc37 (not shown). The specificity with
which Cdc37 interacted with the AR was examined further by testing
whether His6Cdc37 could also interact with the GR. In vitro translation of rat GR resulted in the appearance of several bands, the largest of which was of the size expected for the
full-length product (94 kDa). However, the GR did not associate with
His6Cdc37 when assayed by itself (Fig. 1B) nor
when mixed with the AR (data not shown). The results shown here for the
GR and v-Src are consistent with those described previously by Whitelaw
et al. (17), where Cdc37 was found to coimmunoprecipitate
with v-Src but not GR in lysates from cultured animal cells (17).
The binding site for Cdc37 on the AR was examined next. Based on
results using a yeast model system, we had predicted that Cdc37 would
function via the LBD. The rationale for this was 2-fold. First was that
defects in AR activity only occurred with the full-length molecule; AR
truncations containing just the transactivation and DNA-binding domains
(TAD-DBD) were fully functional in the cdc37 mutant
(18). This suggested that Cdc37 was not involved in folding of the
TAD-DBD truncation. Second, Hsp90 is known to interact with the AR-LBD,
and if Hsp90 and Cdc37 functioned together (as discussed in more detail
below), it seemed likely that this would occur via the LBD. We
therefore tested whether His6Cdc37 could interact with two
truncated forms of the AR: one containing the TAD-DBD () and a
second containing the ligand-binding domain and hinge regions (LBD;
624-919). These were translated in RRL and assayed for binding to
His6Cdc37 as described above. The results of these
experiments (Fig. 2) showed that
His6Cdc37 does indeed interact with the LBD to a similar
extent as that observed with full-length AR. Much lower, but
reproducible, levels of His6Cdc37 bound to the TAD-DBD,
although these were not much above the background levels.
The role of Hsp90 in His6Cdc37 binding to the LBD was
analyzed by performing AR translation in the presence of geldanamycin, an Hsp90 inhibitor. Geldanamycin is known to prevent association of
Hsp90·Cdc37 complexes with protein kinases and inhibit their maturation (27, 38, 39). Geldanamycin also inhibits maturation of
steroid hormone receptors including the AR, although to a lesser extent
than for estrogen receptor or GR (40). In the experiment shown in Fig.
3, geldanamycin addition (100 µg/ml) to
RRL did not affect the level of AR translation but did affect the
ability of AR to interact with His6Cdc37 by 2-3 fold (Fig.
3A). Similar results were obtained for v-Src (Fig.
3B), which also migrated slightly faster in the gel after
geldanamycin treatment. Further studies revealed that v-Src synthesized
in the presence of 100 µg/ml geldanamycin is catalytically
inactive,2 suggesting that
the geldanamycin treatment is completely effective at the concentration
used. The similarity with which geldanamycin treatment disrupted
interaction of His6Cdc37 with AR and v-Src suggested that
both complexes formed by a similar mechanism. In the case of v-Src and
other kinases, Cdc37 associates in a complex with Hsp90 and
independently of it (20) (27). Thus, the partial disruption of such
complexes by geldanamycin suggested that His6Cdc37 interacted with the AR in a manner that is partially dependent on Hsp90
but also partially independent.
Further investigation of Cdc37 interaction with the AR was performed in
the yeast system. For these studies, we constructed a His-tagged
version of yeast Cdc37 (His6yCdc37). The
His6yCdc37 protein was fully functional because it
suppressed the temperature-sensitive growth phenotype of a
cdc37 mutant strain (18) when constitutively expressed from
a low copy number CEN plasmid (not shown). To assay for binding,
His6yCdc37 was constitutively expressed from a multi-copy plasmid in a wild type yeast strain also expressing the AR (Fig. 4). Incubation of lysates from this
strain with Ni-NTA resin led to adsorption of His6yCdc37,
as detected by Western blot. Western blot analysis also revealed that
AR was coadsorbed with His6yCdc37, although Hsp90 was not
(Fig. 4A, middle and lower panels).
There was very little nonspecific adsorption of AR to the Ni-NTA resin in experiments using lysates from strains expressing the AR but not
His6yCdc37 (Fig. 4A, lanes 1 and
3), suggesting that AR was specifically bound to
His6yCdc37. In similar experiments the GR did not bind to
His6yCdc37 (Fig. 4B). The lack of stable
association of Hsp90 with His6yCdc37 was somewhat
surprising because we and others have observed some yeast Cdc37 to be
coadsorbed with His-tagged Hsp90 (28) (18). As pointed out by Kimura
et al. (28), however, yeast Cdc37 and Hsp90 proteins do not
form the same stable association as do their mammalian homologs.
Association of Hsp90 with steroid hormone receptors is important for
generating a high affinity ligand binding conformation. Hormone binding
facilitates subsequent steps in the activation pathway, such as
coactivator binding, dimerization, and association with chromatin. The
change from inactive to active states is therefore dependent on the
hormone itself, which stimulates Hsp90 dissociation from the AR (41) or
prohibits further interaction. We found that His6Cdc37
interaction with the AR was also hormone sensitive, because
His6Cdc37 binding was reduced after DHT was added to RRL prior to AR translation (Fig. 5).
However, this inhibition was relatively weak for full-length receptors,
where a reduction in complex formation by ~2-fold was commonly
observed. In experiments with the isolated LBD, however, the presence
of hormone almost completely inhibited complex formation. Similar
results were obtained by incubating the hormone with reactions after
translation but prior to incubation with His6Cdc37. Thus,
hormone binding appears to alter AR conformation in a manner that
affects Cdc37 interaction but does not necessarily inhibit its
association.
Cdc37 Does Not Substitute for Hsp90 Function in Hormone Binding by
AR--
Recent studies have shown that overexpressed Cdc37 can
partially compensate for loss of Hsp90 function in protein kinase
folding (28). We therefore tested for AR function in an
hsp82 yeast mutant in the presence or absence of
overexpressed yeast Cdc37. Previous studies have shown that the AR has
a reduced affinity for hormone in an hsp82 mutant yeast
strain (19). For these studies, AR was expressed in wild type and
hsp82G313N mutant strains. The G313N mutant was
chosen because its growth rate was reported to be modestly increased by
Cdc37 overexpression (28). Western blot analysis revealed that reduced
amounts of AR were recovered in the mutant strain compared with the
wild type (Fig. 6A). This
reduction, although quite variable in different experiments, was
largely suppressed by overexpression of Cdc37 (compare Fig.
6A, lanes 2 and 4). Results of hormone
binding studies correlated with the reduced AR levels in this strain
(Fig. 6B) and were ~10-fold lower in the mutant compared
with the wild type. These levels did not change in the hsp82
mutant upon overexpression of Cdc37, although increased amounts of AR
protein were recovered from the strain. It is possible that
overexpressed Cdc37 can stabilize AR in this hsp82 mutant,
although it apparently fails to facilitate a folding reaction in the
absence of functional Hsp90. Thus, binding of Hsp90 and Cdc37 to the
same domain of the AR has different functional consequences.
A Dominant Negative Form of Cdc37 Inhibits AR Activity in Mammalian
Cells--
Previous studies (27) showed that a truncated version of
Cdc37, containing the N-terminal half of the protein, acted in a
dominant negative manner for Raf activation. We prepared a similar construct (Cdc371-173) and coexpressed it in CV1 cells in
the presence of full-length AR, the TAD-DBD form of AR and the GR. A
reporter construct containing consensus hormone response elements for
both AR and GR regulating the CAT gene was also coexpressed to assay
receptor dependent gene expression. As shown in Fig.
7, coexpression of
Cdc371-173 led to a 2-fold decrease in
hormone-dependent reporter gene expression by AR. By
contrast, the same mutant had no inhibitory effect on the ability of a
TAD-DBD construct to stimulate hormone-independent gene expression and
was only mildly inhibitory on the activity of GR. These results are
analogous to those previously described from the yeast system (16)
where mutation in CDC37 affected hormone-dependent gene expression by full-length AR but had
no effect on the ability of the TAD-DBD form to function as a
constitutive activator. Overexpression of wild type Cdc37 had no effect
on the ability of AR to function as a transcriptional activator
(data not shown).
The paradigm for molecular chaperone function in steroid hormone
receptor activation evolved from studies on GR and PR. In this
paradigm, Hsp90 is recruited to the receptor ligand-binding domain via
the actions of an Hsp70/Hsp40 chaperone pair, working in association
with Hop, a co-chaperone that indirectly binds Hsp90 to Hsp70.
Hsp70/Hsp40 and Hop are subsequently replaced on the receptor by Hsp90
in a complex with p23 and one of several immunophilins, such as cyp40
or FKBP52 (4) (5). This paradigm does not include Cdc37 because it
appears not to interact with the GR or PR. On the other hand, there is
strong experimental support for Cdc37 function, in association with
Hsp90, in protein kinase folding; Cdc37 binds to protein kinases
in vitro and prevents them from denaturing. Mutation in
yeast Cdc37 affects the folding and activity of several different
protein kinases, and expression of a dominant negative form of Cdc37
inhibits Raf activity in animal cells (27, 42). We used similar
criteria to judge whether Cdc37 was also important for activation of
the human AR. Mutation in yeast CDC37, for example, inhibits
hormone-dependent activation of AR (18). AR also binds to
Cdc37 in vitro (Figs. 1 and 2), and a dominant negative form
of Cdc37 partially inhibits AR activity in animal cells (Fig. 6).
Although it is unknown what makes the AR require Cdc37 for activity,
there are some instances where it behaves quite differently from other
steroid receptors. One relates to the interaction of AR with
coactivators and its AF2 transcriptional activity. AR has a very weak
AF2 activity compared with GR, although both interact with similar
coactivator proteins. Recent studies suggest that interaction of AR
with coactivators such as SRC1 occurs in a relatively unique manner
(43, 44). Previous studies characterized coactivator binding to steroid
receptors as being via the C-terminal ligand-binding domain in a
hormone-dependent manner (45). The change in conformation that occurs upon ligand binding creates a site of interaction for
coactivators that stimulate AF2 function. In the case of AR, coactivators such as SRC1 bind to the N-terminal domain instead and in
a manner that requires interaction between the N terminus and the C
terminus (43) (44). Such conformational changes between the AR N and C
termini occur in a hormone-dependent manner (46, 47) and
may provide a basis for the Cdc37 requirement. This would be consistent
with a function for Cdc37 that is downstream of hormone binding (18).
Furthermore, Cdc37 association with full-length AR but not the LBD
persisted in the presence of hormone. This could be interpreted in
terms of Cdc37 association with ligand-bound AR requiring an intact
molecule containing both N- and C-terminal domains. We propose,
therefore, that Cdc37 facilitates conformational changes in the AR that
occur upon hormone binding, as the N terminus interacts with the C terminus.
The possibility that chaperones function downstream of ligand binding
in steroid hormone receptor activation has never been fully explored.
There is evidence for persistent Hsp70 association with ligand bound
receptors (48), although the functional significance of this
interaction remains unknown. A new study on the Drosophila Ecdysone receptor, however, showed that Hsp90 and several other co-chaperones facilitate heterodimerization with RXR and DNA binding (49). Chaperone involvement with Ecdysone receptor occurs only at this
late stage in the activation process, and they are not required for
hormone binding. Cdc37 may have an analogous function in AR activation.
In this case, however, Hsp90/Hsp70/Hsp40 are required for generating
and maintaining the high affinity hormone binding state (14, 19), and
Cdc37 would function at a later step to facilitate
ligand-dependent changes in conformation. It seems likely
that Hsp90 plays a role in complex formation between the AR and Cdc37,
because geldanamycin treatment compromised this interaction (Fig.
3A). This effect may be a direct consequence of the
interaction of Hsp90 with Cdc37, providing a targeting function.
Alternatively, Hsp90-dependent folding may be affected in
geldanamycin-treated lysates, and the AR might fail to adopt the
conformation that accepts Cdc37. It also appears likely, however, that
Cdc37 can stabilize misfolded AR, at least when overexpressed in yeast
cells. As shown in Fig. 6, overexpression of CDC37 in the
hsp82G313N mutant strain had a stabilizing
effect AR protein levels, which were substantially reduced in the
mutant alone. This effect was restricted to AR protein levels, however,
and CDC37 did not suppress the folding defect as measured by
the ability of AR to bind hormone. This means that whereas Hsp90 and
Cdc37 have a functional relationship, there exists circumstances where
they function independently of each other.
The interaction of Cdc37 with the AR has implications for some human
diseases, including prostate cancer and benign prostatic hyperplasia.
Prostate gland growth and differentiation depend on androgens, and
antiandrogens or drugs that affect DHT synthesis are effective
therapeutic agents against both cancer and benign prostatic
hyperplasia. Recent studies also indicate that Cdc37 is up-regulated in
prostate tumors and induces benign prostatic hyperplasia after
overexpression in prostate glands of transgenic mice (50). The effect
of Cdc37 in these diseases probably reflects its action on several
different signaling pathways involving protein kinases that regulate
cell cycle progression. Whether similar changes occur in AR signaling
is unknown, although our results suggest that Cdc37 plays a role in AR
activation in mammalian cells because a dominant negative form of Cdc37
partially down-regulated hormone-dependent activation of AR
(Fig. 7). Although fairly weak, the 2-fold down-regulation of
full-length AR by Cdc371-173 was not observed for the
TAD-DBD truncated form of AR. Furthermore, although a similar mutant
effectively inhibited activation of Raf (27), it was ineffective
against another Cdc37-dependent kinase, Hck, suggesting
that the action of this mutant is complex (51). Regardless of how well
the mutant inhibited AR, however, the finding that
Cdc371-173 can affect full-length AR but not TAD-DBD
further supports the argument that Cdc37 functions via the AR-LBD, as
indicated by the data shown in Fig. 2.
In summary, Cdc37 appears to function in AR activation in animal cells.
Cdc37 interacts specifically with the AR but not the GR ligand-binding
domain in a manner that is at least partially dependent on Hsp90. Our
results suggest that Cdc37 is not an exclusively kinase-specific
chaperone, although kinases may represent the majority of its clients.
This is reminiscent of the proteins that interact with cytosolic
chaperonins (Tric/CCT) of eukaryotes. Tric/CCT is required for actin
and tubulin folding and was believed initially to be a chaperone
specific for these proteins. Recent studies (52) now show that Tric/CCT
interacts with many newly synthesized proteins shortly after
translation. In this manner, some chaperones may have a broad
specificity that is partially obscured if their client base is
represented by protein families with a large or abundant membership,
such as cytoskeletal elements or protein kinases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ydj1 yeast
strain, GR exhibits increased levels of hormone-independent reporter
gene expression (11, 12), whereas AR does not (13, 14). These examples
suggest that different receptors, even those so closely related that
they can bind to the same hormone-responsive DNA elements, have
different molecular chaperone requirements for activation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. 3H-R1881
(methyltrienelone) and [35S]methionine were obtained from
PerkinElmer Life Sciences. Geldanamycin was obtained from the Drug
Synthesis and Chemistry Branch, Developmental Therapeutics Program,
Division of Cancer Treatment, National Cancer Institute, and stored in
dimethyl sulfoxide at
20 °C. Ni-NTA resin was obtained from
Qiagen. Pfu polymerase and the pPCR-Script vector was from
Stratagene. Isopropyl-
-D-thiogalactopyranoside was from
Roche Molecular Biochemicals and stored as a 100 mM
solution in water at
20 °C. Imidazole was obtained from Sigma. T3,
T7, and Taq polymerases were obtained from Promega. Rabbit
reticulocyte lysates (treated) were obtained from Promega. Antisera to
Hsp90, Cdc37, and the AR were described previously (18, 19).
Nitrocellulose membranes were from Immobilon and polyvinylidene
difluoride membranes were from Millipore.
-D-thiogalactopyranoside to a
final concentration of 1 mM for 2 h. The cells were
harvested by centrifugation and washed once with water and once with
extraction buffer (20 mM Hepes pH 7.5, 100 mM KCl, 0.1 mM EDTA pH 8). The cells were
resuspended in 5 ml of extraction buffer containing a protease
inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, aprotinin, chymostatin, leupeptin, and pepstatin each at 1 µg/ml). The cells were lysed by sonication with 5-s bursts (and 1 min rests at
4 °C). Cell debris was pelleted, and the supernatant was transferred
to a fresh tube and spun at 100,000 × g for 20 min at
4 °C. The supernatant was transferred to a fresh tube and incubated
with Ni-NTA resin at a ratio of 1 ml of packed resin/5 ml of extract.
This was incubated for 30 min at 4 °C on a nutator. The resin
was washed three times in extraction buffer containing 10 mM imidazole before elution of bound His6Cdc37
with extraction buffer containing 150 mM imidazole (at 2:1
ratio over packed resin) for 10 min at 4 °C. The elution was
performed three times, and eluates were dialyzed overnight at 4 °C
against 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 25 mM NaCl, and 0.5 mM phenylmethylsulfonyl fluoride. The dialyzed protein was stored at
80 °C. The
His6Cdc37 was typically purified to ~90% by this
procedure. Protein concentration was determined by the Bradford assay
using bovine serum albumin as a standard.
7
M dihydrotestosterone for AR and dexamethasone for GR).
Cells were harvested after 2 days, and CAT activity was measured as described previously (35).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Binding of His6Cdc37 to AR and
v-Src. A, Coomassie Blue-stained denaturing polyacrylamide
gel of E. coli extract containing His6Cdc37
(lane 1) and His6Cdc37 after one step
purification (lane 2). B, binding reactions of
AR, GR, and v-Src after in vitro translation in rabbit
reticulocyte lysates. Translation reactions (T; lanes
1, 4, and 6; 1% of reaction) were incubated
with Ni-NTA resin without ( ; lanes 2, 5, and
7) or with (+; lanes 3, 6,
and 8) preadsorbed His6Cdc37 and processed as
described under "Experimental Procedures." Arrowhead in
the middle panel in B refers to full-length GR.
Molecular size standards are shown at left in A and
B.
View larger version (14K):
[in a new window]
Fig. 2.
His6Cdc37 interacts
with AR ligand-binding domain. A, representation of
full-length and truncated versions of the AR. TAD-DBD refers
to the truncation containing the transactivation and DNA-binding
domains; LBD refers to the isolated ligand-binding domain.
B, binding reactions containing full-length AR, TAD-DBD and
LBD. Translations (T, 1%) and binding reactions
( and + lanes) labeled as in Fig. 1.
View larger version (25K):
[in a new window]
Fig. 3.
Role of Hsp90 in His6Cdc37
interaction with AR. A, AR translation reactions were
performed in the presence of Me2SO ( ) or 100 µg/ml
geldanamycin (GA; +) and subsequently assayed for
binding to His6Cdc37. Data are from nonconsequtive lanes
from the same gel. B, v-Src translation reactions treated as
in A.
View larger version (31K):
[in a new window]
Fig. 4.
Binding of AR to yeast Cdc37. A,
His6Cdc37 (His6yCdc37) was expressed in wild type yeast and
extracts from this strain (lanes 2 and 4) and a
control strain not expressing His6yCdc37 (lanes
1 and 3) were incubated with Ni-NTA resin (lanes
3 and 4). Western blot analysis was used to assay for
the presence of yeast Cdc37, AR, and Hsp90 as labeled (B).
Expression and affinity chromatography of His6yCdc37 in
strains expressing GR are shown.
View larger version (21K):
[in a new window]
Fig. 5.
Effect of DHT on His6Cdc37
binding to AR. Translation reactions containing full-length AR
(upper panels, lanes 1 and 2) or the
just the LBD (lower panels, lanes 1 and
2) were incubated in the presence of DHT (+, lanes
2 and 4) or ethanol ( , lanes 1 and
3). Binding reactions with His6Cdc37 were
performed as described (lanes 3 and 4).
View larger version (18K):
[in a new window]
Fig. 6.
Cdc37 does not substitute for Hsp90 in
yeast. A, Western blot analysis of wild type
(WT) and hsp82G313N mutant yeast
strains after growth in glucose (lanes 1 and 2)
or galactose (lanes 3 and 4) containing medium to
induce expression of Cdc37 from a multi-copy plasmid. Duplicate filters
were probed with antisera to AR, yeast Cdc37, Hsp90, and
phosphoglycerate kinase (PGK, as a loading control).
B, in vivo hormone binding assay with
3H-R1881 in wild type and hsp82G313N
mutant yeast strains expressing the AR in the absence or presence of
overexpressed Cdc37. Data are the means of three independent
assays ± S.D.
View larger version (15K):
[in a new window]
Fig. 7.
A dominant negative Cdc37 partially inhibits
transactivation by AR. CV1 cells were transfected with plasmids
containing the AR (A), TAD-DBD (B), or the GR
(C) and a reporter construct containing the CAT gene.
Reporter gene activity was determined after cotransfection with the
vector pCMV or pCMV containing Cdc371-173. Results are
plotted as a percentage of CAT activity of cells not transfected by
pCMV or pCMV containing Cdc371-173. The results are the
mean of n = 10 for A, n = 7 for B, and n = 9 for C. Bars indicate standard error.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Wade Harper for the gift of cDNA for human Cdc37 and Dr. E. Wilson for the gift of plasmids containing the AR.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK49065 (to A. J. C.) and DK56356 (to D. M. R.) and by funds from the Hirschl Foundation (to A. J. C.).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.
To whom correspondence should be addressed: Dept. of Cell
Biology and Anatomy, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-6563; Fax:
212-860-1174; E-mail: avrom.caplan@mssm.edu.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M007385200
2 P. Lee and A. J. Caplan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AR, androgen receptor; GR, glucocorticoid receptor; PR, progesterone receptor; DHT, dihydrotestosterone; Ni-NTA, nickel-nitrilotriacetic acid; PCR, polymerase chain reaction; RRL, rabbit reticulocyte lysate(s); CAT, chloramphenicol acetyltransferase; TAD, transactivation domain; DBD, DNA-binding domain; LBD, ligand-binding domain.
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