 |
INTRODUCTION |
Steroid receptors move continuously into and out of the nucleus
(Refs. 1-4; for review, see Ref. 5), and, depending upon the receptor,
the hormone-free, untransformed receptor may have a predominantly
nuclear or cytoplasmic localization. The hormone-free glucocorticoid
receptor (GR)1 is localized
to the cytoplasm of most cells, and after steroid binding and
transformation, it translocates to the nucleus (6-8). Several studies
with inhibitors suggest that the multiprotein hsp90-based chaperone
system and the hsp90-binding immunophilin FKBP52 are involved in
movement of the GR along microtubular tracks to the nucleus (for
review, see Ref. 9).
Assembly of receptors into heterocomplexes with hsp90 is a dynamic
process (10), and it has been shown that the GR and hsp90 can move
together from the cytoplasm to the nucleus (11). A couple of
observations suggest that the role of hsp90 in receptor movement is
likely to involve dynamic assembly and disassembly of GR·hsp90
heterocomplexes. For example, Yang and DeFranco (12) showed that
molybdate, which binds to hsp90 and stabilizes GR·hsp90 heterocomplexes in vivo (13), traps the GR in the cytoplasm of cells continuously exposed to hormone. Molybdate in this case was
thought to inhibit reimport of the GR into the nucleus by inhibiting
the dynamic cycling of receptors into and out of their complexes with
the hsp90 chaperone. Also, geldanamycin, an antibiotic that binds to
the nucleotide binding site on hsp90 (14) and prevents formation of
normal receptor·hsp90 heterocomplexes (15), impedes steroid-induced
movement of the GR from the cytoplasm to the nucleus (16, 17).
Some localization studies have shown the untransformed GR to colocalize
with microtubules (for review, see Ref. 18), but the evidence
supporting movement along microtubular tracks is indirect. Although
microtubule disrupting agents, such as colcemid, do not affect the
overall rate of steroid-dependent receptor translocation to
the nucleus (8, 19), they eliminate the hsp90-dependent mode of receptor movement (17). Using a fusion protein of murine GR
with Aequorea green fluorescent protein (GFP), we found that steroid-dependent GFP-GR translocation to the nucleus is
rapid (t1/2 = ~5 min) both in cells with intact
cytoskeleton and in cells with disrupted cytoskeletal networks (17).
However, in cells with normal cytoskeleton, the hsp90 inhibitor
geldanamycin slowed translocation of the GFP-GR by an order of
magnitude (t1/2 = ~45 min), whereas in cells with
colcemid-disrupted microtubules, geldanamycin had no effect on the
translocation rate (t1/2 = ~5 min). This suggests
two mechanisms of GR movement. Under physiological conditions where the
cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a
movement machinery in which the hsp90 heterocomplex assembly machinery
plays a role. In cells where the microtubules are disrupted with
colcemid, movement is still steroid-dependent, but the
transformed GR moves through the cytoplasm by diffusion (17, 20).
In normal cells, the retrograde movement of vesicles toward the nucleus
is known to occur on cytoskeletal tracks in a process requiring the
molecular motor protein cytoplasmic dynein (for review, see Refs. 21
and 22). However, like GFP-GR translocation to the nucleus, short range
vesicle movement still occurs by diffusion when microtubules are
disrupted (23). Using retrograde vesicular movement as a model, we have
looked for potential links between the GR and cytoplasmic dynein. In
addition to hsp90, steroid receptor heterocomplexes contain
hsp90-binding immunophilins (for review, see Ref. 24), and we have
shown that cytoplasmic dynein is coimmunoadsorbed from cytosols with
FKBP52, one of the major immunophilins in GR·hsp90 heterocomplexes.
The immunophilins are proteins possessing peptidylprolyl isomerase
(PPIase) domains that bind immunosuppressant drugs of the FK506 group
(the FKBPs) or of the cyclosporin A group (the cyclosporin A-binding
proteins). Three high molecular weight immunophilins, FKBP52, FKBP51,
and cyclosporin A-binding protein-40, exist in receptor·hsp90
heterocomplexes (24). In addition to their PPIase domains, these high
molecular weight immunophilins contain three tetratricopeptide repeats
(TPRs), which are degenerative sequences of 34 amino acids that
determine their binding to a common TPR acceptor site on hsp90
(25-27). We have shown that FKBP52 also binds directly to the GR and
that a 35-amino acid segment spanning the proto-nuclear localization
signals that comprise the NL1 nuclear localization signal of the GR
(see Ref. 5 for NL1 composition) is sufficient for FKBP52 binding (28).
FKBP52 binds either directly or via a linker protein with cytoplasmic
dynein (29), and coimmunoadsorption of dynein is competed by a fragment
of FKBP52 comprising its PPIase domain (28).
These binding studies support the notion (30, 31) that FKBP52, which
itself colocalizes with microtubules (29, 32), may target receptor
movement toward the nucleus by determining its attachment to the
retrograde dynein motor protein. To date, the only in vivo
observation linking FKBP52 to GR movement is the demonstration that
microinjection of an antibody raised against a conserved negative
sequence (6 of 8 amino acids) between domains I (PPIase) and II of
FKBP52 (33) impedes steroid-dependent cytoplasmic-nuclear translocation of the endogenous GR in L cells (8).
In this work, we show that cotransfection of 3T3 fibroblasts with
GFP-GR and the PPIase domain of FKBP52 slows subsequent steroid-dependent translocation of the GFP-GR to the
nucleus (t1/2 = ~45 min) to the same extent as
treatment with the hsp90 inhibitor, geldanamycin. The effect is
specific, inasmuch as cotransfection with FKBP12 does not affect GFP-GR
movement. If cells are treated with colcemid to disrupt microtubules
prior to steroid addition, the presence of competing PPIase domain does
not inhibit movement. But, when colcemid is removed and microtubules
reform, GFP-GR translocation is again slowed by the presence of the
PPIase domain. The observations are consistent with an uncoupling of
the receptor from movement along microtubules by competition for the
binding of FKBP52 to the dynein motor.
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EXPERIMENTAL PROCEDURES |
Materials
NIH 3T3 mouse embryo fibroblasts were purchased from the
American Type Culture Collection (Manassas, VA). Phenol red-free Dulbecco's modified Eagle's medium (DMEM) was from Life Technologies, Inc. Charcoal-stripped calf serum and colcemid were from Sigma. The
Trans-Fast kit for cell transfection was from Promega (Madison, WI).
The mouse monoclonal IgG (MAB1618) against the 74-kDa intermediate chain subunit of mammalian (bovine brain) cytoplasmic dynein was purchased from Chemicon Intl. (Temecula, CA). Rabbit reticulocyte lysate was from Green Hectares (Oregon, WI), and geldanamycin was
obtained from the Drug Synthesis and Chemistry branch of the Developmental Therapeutics Program, National Cancer Institute. Complete-Mini protease inhibitor tablets were from Roche Molecular Biochemicals. The EC1 anti-FKBP52 mouse monoclonal IgG was kindly provided by Dr. Lee Faber (Medical College of Ohio, Toledo, OH), and
the UPJ56 antiserum against FKBP52 (34) was a gift from Dr, Karen Leach
(Pharmacia and Upjohn, Inc., Kalamazoo, MI). The BuGR2 mouse monoclonal
IgG used for immunoblotting the GR was from Affinity Bioreagents
(Golden, CO), and the FiGR monoclonal IgG used for GR immunoadsorption
was generously provided by Dr. Jack Bodwell (Dartmouth Medical School).
The AC88 mouse monoclonal IgG against hsp90 was from StressGen
(Victoria, Canada), and 125I-conjugated goat anti-mouse IgG
was from PerkinElmer Life Sciences. Construction of the GFP-GR was
described previously (17). The cDNAs for rabbit FKBP52, FKBP52
Met6-Gly148 (Domain I plus hinge), FKBP52
Gly32-Lys138 (Domain I core), and human FKBP12
were described previously (35, 36) and were subcloned into the pSG5PL
mammalian expression vector from Stratagene (La Jolla, CA). The
baculovirus for mouse GR was obtained from Dr. Edwin Sanchez (Medical
College of Ohio, Toledo, OH) and was described previously (37). The
baculovirus for the FLAG-tagged TPR domain of rat PP5 (38) was kindly
provided by Dr. Michael Chinkers (University of South Alabama, Mobile, AL).
Methods
Cell Culture and Transfection--
NIH 3T3 mouse fibroblasts
were grown on 11 × 22-mm coverslips placed in 35-mm culture
plates in 2 ml of DMEM containing 10% bovine calf serum, under a 5%
CO2 atmosphere at 37 °C. When the cells were ~60%
confluent, the medium was replaced by 0.75 ml of DMEM with 5% calf
serum, and the incubation was continued for 1 h. Cells were then
transfected with 2.4 µg of plasmid encoding GFP-GR and 16 µg of
pSG5PL-FKBP52 domain I plus hinge
(Met6-Gly148), or pSG5PL-FKBP52 core domain I
(Gly32-Lys138), or pSG5PL-FKBP12, or pSG5PL
vector alone. The cotransfection mixture was prepared by preincubating
plasmids with 4.5 µl of Trans-Fast reagent/µg of total DNA and 0.25 ml of DMEM for 10 min at room temperature. The mixture was then added
to the cell cultures and incubated for 1 h, prior to adding 4 ml
of DMEM with 10% calf serum without washing out the mixture used for
transfection. After 18 h, the medium was removed, cells were
washed four times, 2 ml of phenol red-free DMEM with 10%
charcoal-stripped calf serum were added, and cells were incubated for
an additional 24-30 h.
GFP-GR Translocation--
Cells transfected as described above
were incubated with 1 µM dexamethasone, and at various
times, coverslips were removed, and cells were rinsed with ice-cold
phosphate-buffered saline and fixed by immersion in
20 °C methanol
for at least 15 min. Cells were rinsed again with phosphate-buffered
saline, and the coverslips were inverted onto a slide with 5 µl of
mounting solution (1 mg/ml p-phenylenediamine in 10%
phosphate-buffered saline, 90% glycerol, pH 9.0). Cells were
visualized with a Leitz Aristoplan epiillumination microscope.
Cells were scored for GFP-GR translocation as we have described
previously (17), using a score of 4 for nuclear fluorescence much
greater than cytoplasmic fluorescence, 3 for nuclear fluorescence greater than cytoplasmic fluorescence, 2 for nuclear fluorescence equal
to cytoplasmic fluorescence, 1 for nuclear fluorescence less than
cytoplasmic fluorescence, and 0 for nuclear fluorescence much less than
cytoplasmic fluorescence. The translocation scores represent the
means ± S.E. from three experiments in which >100 cells per
condition per experiment were scored. Significance of differences was
measured by analysis of variance followed by Bonferroni t test.
Immunoadsorption of FKBP52--
For immunoadsorption of FKBP52,
aliquots (150 µl) of rabbit reticulocyte lysate were immunoadsorbed
for 3 h at 4 °C to 14 µl of protein A-Sepharose with 7 µl
of UPJ56 antiserum against FKBP52. Immune pellets were washed three
times by suspension in 1 ml of TEG buffer (10 mM TES, pH
7.6, 50 mM NaCl, 4 mM EDTA, 10% (w/v)
glycerol) with 20 mM sodium molybdate prior to gel
electrophoresis and immunoblotting using 0.1% EC1 to probe for FKBP52.
Glucocorticoid Receptor Heterocomplex Reconstitution--
Mouse
GR was overexpressed in Sf9 cells as described (37), and cytosol
was prepared by Dounce homogenization in 1.5 volumes of buffer (10 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 tablet of Complete-Mini protease
inhibitor mix/3 ml of buffer). The lysate was centrifuged at
100,000 × g for 30 min, and the supernatant was stored
at
70 °C. Receptors were immunoadsorbed from 60-µl aliquots of
cytosol by rotation for 2 h at 4 °C with 14 µl of protein
A-Sepharose and 7 µl of FiGR ascites, and the immune pellets were
washed once with 1 ml of TEG buffer. Prior to incubation with
reticulocyte lysate, immunoadsorbed receptors were stripped of hsp90 by
incubating for 2 h with 300 µl of TEG buffer containing 0.7 M NaCl and 0.2% Nonidet P-40. The immune pellets were then
washed three times with 1 ml of TEG buffer containing 0.7 M
NaCl and twice with 10 mM Hepes, pH 7.4. GR·hsp90
heterocomplexes were assembled by incubating these stripped GR immune
pellets with 50 µl of rabbit reticulocyte lysate and 5 µl of an
ATP-regenerating system (50 mM ATP, 250 mM
creatine phosphate, 20 mM magnesium acetate, and 100 units/ml of creatine phosphokinase). The assay mixtures were incubated
for 30 min at 30 °C, with suspension of the pellets by shaking the
tubes every 2 min. At the end of the incubation, the pellets were
washed three times with 1 ml of ice-cold TEGM buffer (TEG with 20 mM sodium molybdate), and boiled in SDS sample buffer.
Western Blotting--
To assay GR and associated proteins,
immune pellets were resolved on 10% SDS-polyacrylamide gels, and
proteins were transferred to Immobilon-P membranes. The membranes were
probed with 0.2 µg/ml BuGR for GR, 1 µg/ml AC88 for hsp90, 0.1%
UPJ56 for FKBP52, and 0.1% of MAB1618 for dynein. The immunoblots were
then incubated a second time with the appropriate
125I-conjugated counter antibody to visualize
immunoreactive bands.
Immunoadsorption of Native GR·hsp90
Heterocomplexes--
Cytosol was prepared from L929 mouse fibroblasts
by Dounce homogenization in 1.5 volumes of buffer (10 mM
Hepes, pH 7.4, 1 mM EDTA, 20 mM molybdate, and
1 tablet of Complete-Mini protease inhibitor mix/3 ml of buffer) and
centrifugation at 100,000 × g for 30 min. Receptors
were immunoadsorbed from 250-ml aliquots of cytosol by rotation for
2.5 h at 4 °C with 14 µl of protein A-Sepharose and 7 µl of
FiGR ascites, and the immune pellets were washed three times with 1 ml
of TEG buffer with 20 mM molybdate.
Purification of FKBP52 Domain I Core--
pGEX
T plasmid
expressing GST-FKBP52 Gly32-Lys138 was used to
transform Escherichia coli strain UT5600. The fusion protein
was purified by binding to GSH-agarose beads, and the core PPIase domain I of FKBP52 was released by incubation at 4 °C with thrombin.
 |
RESULTS |
FKBP52 Domain I Inhibits Steroid-dependent GFP-GR
Translocation--
Because the purified FKBP52 domain I
(Met6-Gly148) fragment competes for FKBP52
association with cytoplasmic dynein in vitro (28), we asked
whether coexpression of domain I would alter the rate of GFP-GR
movement from the cytoplasm to the nucleus in vivo. In the
absence of steroid, the GFP-GR expressed in 3T3 cells is predominantly
localized to the cytoplasm (Fig. 1,
A, C, and E), and upon exposure to
dexamethasone, it moves to the nucleus (Fig. 1B). As we
reported previously (17), nuclear translocation of the GFP-GR is
inhibited when hsp90 function is inhibited by geldanamycin (Fig.
1F). As shown in Fig. 1D, cotransfection with
FKBP52 domain I also inhibits GFP-GR translocation. The bar
graphs on the right in Fig. 1 summarize the
translocation scores from several hundred cells per condition, where a
value of zero reflects a completely cytoplasmic localization and a
value of four a completely nuclear localization, a scoring system we
have used previously with the GFP-GR (17, 20). After the 20-min
interval of steroid treatment, nuclear translocation of the GFP-GR is
inhibited to the same extent by coexpression of FKBP52 domain I as it
is by treatment with geldanamycin.

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Fig. 1.
Cotransfection of FKBP52 domain I inhibits
steroid-dependent GFP-GR translocation to the nucleus.
3T3 cells were transfected with plasmids expressing GFP-GR and either
FKBP52 domain I (Met6-Gly148) (C
and D) or pSG5PL vector (A, B,
E, and F). Two days after transfection, 1 µM dexamethasone (+DEX) or ethanol
( DEX) was added, the incubation was continued for 20 min,
and cells were fixed and fluorescence was visualized. Conditions are:
cells cotransfected with GFP-GR and empty vector incubated in the
absence (A) or presence (B) of DEX; cells
cotransfected with GFP-GR and FKBP52 domain I incubated in the absence
(C), or presence (D) of DEX; and cells
cotransfected with GFP-GR and empty vector were preincubated on ice for
90 min with ethanol (E) or DEX (F), 10 µM geldanamycin was added, and the incubation was
continued for an additional 30 min on ice prior to incubation for 20 min at 37 °C to permit GFP-GR translocation. The bar
graphs present the nuclear translocation scores in the absence
(hatched bar) or presence (solid bar) of steroid
as determined under "Methods." Solid bars of conditions
D and F are both significantly different from
B at p < 0.001. The bar in
F represents 10 µm.
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Inhibition of GFP-GR Translocation Requires
Microtubules--
Geldanamycin slows the rate of GFP-GR translocation,
but by 60 min of steroid exposure, essentially all of the receptors
have moved to the nucleus (Ref. 17 and Fig.
2A). As shown in Fig. 2A, cotransfection with FKBP52 domain I slows GFP-GR
translocation to the same extent as geldanamycin, and geldanamycin
treatment of cells coexpressing FKBP52 domain I does not yield a slower rate of translocation than that seen with either condition alone. As
shown in Fig. 2B, neither coexpression of FKBP52 domain I
nor treatment with geldanamycin slows GFP-GR translocation in cells that have been treated with colcemid to disrupt microtubules.

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Fig. 2.
Expression of FKBP52 domain I inhibits GFP-GR
movement only when microtubules are present. A,
inhibition of GFP-GR movement by domain I and geldanamycin. 3T3 cells
transfected with GFP-GR and either pSG5PL-FKBP52 domain I
(Met6-Gly148) (Domain I) or empty
pSG5PL vector (Vector). After 2 days, the cells were placed
in ice and incubated for 90 min with 1 µM DEX, 10 µM geldanamycin (GA) was then added, and the
incubation on ice was continued for 30 min. The temperature was then
shifted to 37 °C to permit nuclear translocation, and
immunofluorescence was assayed at various times. Each value represents
the translocation score (mean ± S.E.) of three independent
experiments. The values for vector + geldanamycin, Domain I, and Domain
I + geldanamycin are all significantly different from vector alone at
p < 0.01 from 5 min through 40 min and are not
different from each other. B, colcemid disruption of
microtubules eliminates inhibition of GFP-GR movement by both FKBP52
domain I and geldanamycin. 3T3 cells transfected as above were
incubated for 1 h with 0.6 µg/ml colcemid to disrupt
microtubules prior to incubating them on ice with DEX and geldanamycin
and shifting them to 37 °C to permit nuclear translocation. The
apparent slight lag in the kinetics at the first time point for two of
the conditions is unique and has not been observed with geldanamycin in
other experiments (17).
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We have shown previously, that treatment with colcemid under these
conditions yields complete disruption of microtubules in 3T3 cells
(17). When colcemid-treated cells are incubated in medium without
colcemid for 1 h, the microtubular network is restored, and
geldanamycin again inhibits GFP-GR movement (17). In the experiment of
Fig. 3A, cells that were
treated with colcemid, such that steroid-dependent GFP-GR
movement was not inhibited by FKBP52 domain I (condition 3)
or geldanamycin (condition 4), were withdrawn from colcemid
for 1 h prior to steroid addition. In the colcemid-withdrawn
cells, GFP-GR translocation was inhibited by FKBP52 domain I
(condition 5) and by geldanamycim (condition 6).
These observations strongly suggest that, under normal physiological conditions where the microtubular network is intact, the GFP-GR is
linked via an associated immunophilin, such as FKBP52, to a system for
movement along microtubular tracks.

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Fig. 3.
The colcemid blockade of inhibition is
reversible, and there is no inhibition of GFP-GR movement by
FKBP12. A, the effect of colcemid on the inhibition of
GFP-GR translocation rate is reversible. 3T3 cells transfected with
GFP-GR and either vector or FKBP52 domain I were treated for 1 h
with 0.6 µg/ml colcemid (conditions 1-6) prior to assay
of DEX-dependent translocation to the nucleus. Column
1, transfected with vector, no DEX treatment; column 2,
cells transfected with vector and treated with DEX; column
3, cells expressing FKBP52 core domain I treated with DEX;
column 4, cells transfected with vector treated with DEX and
geldanamycin; column 5, cells expressing FKBP52 core domain
I and treated with colcemid were incubated in colcemid-free medium for
1 h to permit microtubule repolymerization prior to treatment with
DEX; column 6, cells transfected with vector and treated
with colcemid were incubated in colcemid-free medium for 1 h prior
to treatment with DEX and geldanamycin. Conditions in columns
5 and 6 are significantly different from conditions in
columns 3 and 4, respectively, at
p < 0.001. B, domain I core inhibits GFP-GR
translocation but FKBP12 does not. 3T3 cells were transfected with
GFP-GR and pSG5PL-FKBP52 domain I plus hinge
(Met6-Gly148), or pSG5PL-FKBP52 core domain I
(Gly32-Lys138), or pSG5PL-FKBP12, or empty
pSG5PL vector. Two days later, DEX was added for 20 min, and
fluorescence localization was determined. Column 1,
transfected with vector, no DEX treatment; column 2, cells
transfected with vector and treated with DEX; column 3,
cells expressing FKBP52 domain I treated with DEX; column 4,
cells expressing FKBP52 core domain I treated with DEX; column
5, cells expressing FKBP12 treated with DEX; column 6,
cells transfected with vector and treated with DEX and
geldanamycin as described under Fig. 2. The data represent the
mean translocation score ± S.E. for 150 cells/condition.
Conditions in columns 3, 4, and 6 are
significantly different from conditions in column 2 at
p < 0.001.
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Specificity of Inhibition of GFP-GR Translocation--
The FKBP52
domain I (Met6-Gly148) expressed in the
experiments of Figs. 1, 2, and 3A contains all of domain I
and a short, negatively charged hinge region that connects domains I
and II. To be sure that the PPIase domain was responsible for
inhibition of receptor movement, we coexpressed the GFP-GR with FKBP52
core domain I (Gly32-Lys138), which does not
contain the hinge segment. As shown in Fig. 3B, GFP-GR
translocation is inhibited to the same extent in cells expressing
FKBP52 core domain I (condition 4) as in cells expressing FKBP52 domain I plus hinge (condition 3). We determined by
immunoblotting that the levels of both FKBP52 domain I
(Met6-Gly148) and core domain I
(Gly32-Lys138) were 35-fold higher than the
endogenous FKBP52 in nontransfected cells (data not shown).
Although domain I alone has the same PPIase activity as the full-length
FKBP52 (35), that enzymatic activity is not related to inhibition of
GFP-GR movement. This is deduced from the fact that the
immunosuppressant drug FK506, which binds in the active site and blocks
PPIase activity, does not affect either the rate of
steroid-dependent GFP-GR translocation or inhibition of the rate of translocation by FKBP52 domain I (data not shown). We have also
shown previously that FK506 does not affect FKBP52 binding to
cytoplasmic dynein (28).
Inhibition of GFP-GR movement by FKBP52 domain I does not reflect an
inhibition by PPIase domains in general. We have shown previously that
purified FKBP12 does not affect FKBP52 binding to cytoplasmic dynein
(28). In Fig. 3B (condition 5), it is shown that
cotransfection of 3T3 cells with human FKBP12 does not affect
steroid-dependent GFP-GR translocation. The level of FKBP12
in transfected cells was determined by [3H]FK506 binding
to be 40-fold higher than all endogenous [3H]FK506
binding activity in nontransfected cells.
GR·hsp90 Heterocomplexes Containing Cytoplasmic Dynein--
The
experiment of Fig. 4 presents three
treatments that selectively affect the binding of FKBP52 to hsp90 or
dynein. In this experiment, aliquots of rabbit reticulocyte lysate were
incubated with various additions at 30 °C, and then FKBP52 was
immunoadsorbed. Immunoadsorption of FKBP52 is accompanied by
coadsorption of both hsp90 and cytoplasmic dynein (lane 2).
The TPR domain fragment of rat PP5 markedly reduces FKBP52 binding to
hsp90 but not to dynein (lane 3), whereas FKBP52 core domain
I eliminates FKBP52 binding to dynein but does not affect its binding
to hsp90 (lane 4). FKBP52 binding to dynein is not affected
by FK506 (lane 6), which again suggests that the binding is
independent of PPIase activity (28). Importantly, pretreatment of
reticulocyte lysate with geldanamycin markedly reduces FKBP52 binding
to hsp90 (lane 8).

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Fig. 4.
Effects of geldanamycin and competition with
TPR and PPIase domain fragments on coimmunoadsorption of hsp90 and
dynein with FKBP52. Aliquots (150 µl) of reticulocyte lysate
supplemented with protease inhibitor mixture were incubated for 30 min
at 30 °C with the indicated additions in a final volume of 200 µl.
Samples were then immunoadsorbed at 4 °C with the UPJ56 antibody
against FKBP52, and proteins in the washed immune pellets were resolved
by SDS-polyacrylamide gel electrophoresis and Western blotting.
Lane 1, nonimmune pellet of reticulocyte lysate; lanes
2-8, FKBP52 pellets of reticulocyte lysate incubated with buffer
alone (lane 2), with lysate of Sf9 cells expressing
the TPR domain fragment of rat PP5 (lane 3), with 280 µg
of purified domain I core (Gly32-Lys138)
(lane 4); with nonexpressing Sf9 lysate (lane
5), with 1 µM FK506 (lane 6), with 0.5 µl of Me2SO (lane 7), with 12.5 µM geldanamycin in Me2SO (lane
8).
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Reticulocyte lysate contains the multi-protein hsp90/hsp70-based
chaperone machinery that assembles GR·hsp90 heterocomplexes (reviewed
in Ref. 24). In the experiment of Fig.
5A, we asked whether the
GR·hsp90 heterocomplex formed in reticulocyte contained dynein.
Immunoimmobilized mouse GR that was stripped of its associated hsp90
was incubated with rabbit reticulocyte lysate, and after washing the
immunopellet, the GR and associated rabbit proteins were resolved by
SDS-polyacrylamide gel electrophoresis and immunoblotting. As shown in
lane 3, the GR is assembled into heterocomplexes containing hsp90, FKBP52, and cytoplasmic dynein. If the lysate is preincubated with geldanamycin before it is incubated with the GR, then there is a
marked decrease in hsp90, and there is no FKBP52 or dynein (lane
4). FKBP52 is bound to hsp90 via its TPR domain (25), and
preincubation with a TPR domain fragment of PP5 yields a GR·hsp90 complex that does not contain FKBP52 or dynein (lane 5). As
shown in Fig. 5B, preincubation with FKBP52 core domain I,
eliminates dynein without affecting the amount of hsp90 or FKBP52 in
the GR heterocomplex. These observations are consistent with a model in
which the hsp90-bound GR is linked to the motor protein via an
immunophilin, such as FKBP52.

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Fig. 5.
GR hsp90 heterocomplexes assembled
in reticulocyte lysate contain cytoplasmic dynein. A, a
TPR domain protein, such as FKBP52, is required for dynein presence in
the GR·hsp90 heterocomplex. Stripped GR immune pellets were incubated
for 30 min at 30 °C with reticulocyte lysate that was preincubated
for 30 min at 30 °C under conditions indicated below. The pellets
were then washed, and the GR and associated proteins were assayed by
Western blotting as described under "Methods." Lane 1,
nonimmune pellet incubated with reticulocyte lysate; lanes
2-5, stripped GR immune pellets incubated with buffer (lane
2), with lysate preincubated with buffer (lane 3), with
lysate preincubated with 10 µM geldanamycin (lane
4), or with lysate preincubated with lysate of Sf9 cells
expressing the TPR domain fragment of rat PP5 (lane 5).
B, purified core domain I of FKBP52 competes for dynein
binding to GR·hsp90 heterocomplexes. Stripped GR immune pellets were
incubated with reticulocyte lysate preincubated with buffer or various
amounts of purified core domain I of rabbit FKBP52. Lane 1,
nonimmune pellet incubated with reticulocyte lysate; lane 2,
stripped GR immune pellet incubated with buffer; lanes 3-6,
stripped GR immune pellets incubated with reticulocyte lysate
preincubated with buffer (lane 3), 30 µg (lane
4), 60 µg (lane 5), or 90 µg (lane 6) of
purified FKBP52 core domain I. C, dynein is present in
native GR·hsp90 heterocomplexes. Aliquots (250 µl) of L cell
cytosol were immunoadsorbed with nonimmune IgG (lane 1) or
FiGR antibody against the GR (lane 2). The pellets were then
washed, and the GR and associated proteins were assayed by Western
blotting.
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In Fig. 5C, the GR was immunoadsorbed from cytosol prepared
from mouse L cells. Cytoplasmic dynein was coimmunoadsorbed, showing that it is a component of native GR·hsp90 heterocomplexes formed in
the cell.
 |
DISCUSSION |
Inhibition of GFP-GR translocation by expression of the PPIase
domain of FKBP52 is consistent with a model in which rapid receptor
movement in cells with intact cytoskeleton requires participation of
one or more of the high molecular weight, hsp90-binding immunophilins (8). We have previously reported that the rate of GR translocation is
inhibited by geldanamycin (16, 17), which blocks GR·hsp90 heterocomplex assembly, but the way in which dynamic GR·hsp90 heterocomplex assembly might promote GR movement along cytoskeletal tracts was unknown. FKBP52 provides a link between the receptor heterocomplex and cytoplasmic dynein, a motor protein for retrograde movement along microtubules. Treatment of reticulocyte lysate with
geldanamycin inhibits FKBP52 binding to hsp90 (Fig. 4) and inhibits
GR·hsp90 assembly, yielding receptors that are not associated with
FKBP52 and dynein (Fig. 5A). A similar effect in
vivo could explain why rapid GR movement in normal cells with
intact microtubules is inhibited by geldanamycin. The ability of both
geldanamycin treatment and FKBP52 domain I expression to disrupt GR
linkage to dynein could explain why the two manipulations slow the rate of GFP-GR movement to the same extent and why there is no further slowing with the two manipulations together (Fig. 2A).
In this work, we show that dynein is present in native GR·hsp90
heterocomplexes immunoadsorbed from L cell cytosol and that dynein-containing GR·hsp90 heterocomplexes are formed in reticulocyte lysate. These complexes contain FKBP52 (Fig. 5); however, complexes containing other immunophilins, such as FKBP51, are also formed (39).
Assembly of GR·hsp90 heterocomplexes in the presence of the TPR
domain fragment blocks the binding of all of the immunophilins (26).
Thus, at this time, we can only say that the presence of dynein in the
GR·hsp90 heterocomplex requires a TPR domain immunophilin, with
FKBP52 providing at least part, and perhaps all, of that linker function.
The common feature of immunophilins is the presence of their PPIase
domains, and the PPIase domain of FKBP52 possesses PPIase activity even
when it is expressed as a domain fragment (35). However, the PPIase
inhibitor FK506 does not affect FKBP52 binding to cytoplasmic dynein
(Fig. 4 and Ref. 28), or the rate of GFP-GR translocation, or
inhibition of translocation by the FKBP52 PPIase domain. Thus, the
PPIase activity of FKBP52 does not seem to be involved in the
interaction with cytoplasmic dynein or retrograde movement of GFP-GR.
The PPIase domain of FKBP52 functions as a dynein interaction domain
independent of binding by FK506. In this respect, it differs from the
interaction of FKBP12 with calcineurin, where binding requires the
FK506-bound immunophilin (40).
It is clear that the PPIase domains of immunophilins are involved in
different protein-protein interactions and effects. For example, the
low molecular weight immunophilin FKBP12 does not affect GFP-GR
movement, whereas the PPIase domain of FKBP52 does (Fig.
3B). FKBP12 also does not compete for FKBP52 binding to cytoplasmic dynein, whereas the PPIase domain of FKBP52 does (28). Although FK506-bound FKBP12 binds to and inhibits the calcineurin phosphatase (40), neither FK506-bound FKBP52 nor its expressed PPIase
domain fragment inhibits calcineurin activity in vitro (41).
Our impression is that the PPIase domains may constitute a family of
protein-protein interaction domains, with no overlap of binding targets
between FKBP12 and the high molecular weight, hsp90-binding imunophilins.
Because we see the same rate of GFP-GR nuclear translocation in the
presence and absence of microtubules (Fig. 2), it may be that nuclear
import and not receptor movement through the cytoplasm is rate-limiting
for nuclear accumulation under both conditions. When the microtubules
are intact, it seems that receptor movement occurs predominantly on a
microtubule-based movement system that requires dynamic interaction of
the receptor with hsp90 and the immunophilin. When cytoskeleton is
disrupted, the GR may move through the cytoplasm by diffusion. Although
movement of the receptor through the cytoplasm is not the rate-limiting
step in cytoplasmic-nuclear translocation under physiological
conditions in cells with intact cytoskeleton, it becomes rate-limiting
when geldanamycin or the PPIase domain of FKBP52 are present to
uncouple the receptor from the movement system. Thus, in the normal
cell, rapid movement of protein solutes by diffusion may be limited by
the organized cytoskeleton, and attachment to a movement system permits
more rapid delivery to their sites of action. Such a movement system may not be essential for targeted protein movement in non-neuronal cells. But in axons, movement solely by diffusion is not possible, and
a movement system, such as that described here, would be essential.
In this work, we have provided the first in vivo evidence
that the immunophilin PPIase domain links the glucocorticoid receptor to a retrograde movement system. We also show for the first time that
GR·hsp90·immunophilin heterocomplexes are bound to the retrograde motor protein cytoplasmic dynein via the PPIase domain of the immunophilin component.