 |
INTRODUCTION |
Although targeted movement of certain signaling proteins such as
the steroid receptors, signal transducers and activators of
transcription, and mitogen-activated protein kinases through the
cytoplasm to the nucleus is necessary for signal transduction, virtually nothing is known about the machinery that enables these protein solutes to traverse the cytoplasm. Steroid receptors undergo a
constant shuttling into and out of the nucleus (1-4) (for review, see
Ref. 5) and, depending upon the receptor and the cell type, the
hormone-free, untransformed receptor may be predominantly nuclear or
cytoplasmic in location. In most cells, the untransformed glucocorticoid receptor (GR)1
is predominantly localized in the cytoplasm, and upon hormone binding
and transformation, the receptor translocates to the nucleus (6-8) in
a manner that is determined by nuclear localization signal (NLS)
sequences in the receptor (6). Because movement of the receptor can be
initiated by addition of steroid, the GR is a particularly useful model
for studying targeted protein movement through the cytoplasm.
Studies performed with inhibitors suggest that the protein chaperone
hsp90 and an okadaic acid-sensitive protein phosphatase may each play a
role in GR translocation from the cytoplasm to the nucleus. The
involvement of hsp90 in receptor movement is likely to involve dynamic
assembly and disassembly of GR·hsp90 heterocomplexes. For example,
Yang and DeFranco (9) showed that molybdate, which binds to hsp90 and
stabilizes GR·hsp90 heterocomplexes in vivo (10), trapped
the GR in the cytoplasm of cells chronically exposed to hormone. It was
suggested that the receptors can be exported from nuclei after
hormone-dependent translocation, but they cannot be
reimported into nuclei in the presence of molybdate (10). Association
of receptors with hsp90 is a dynamic process (11), and it has been
shown that GR and hsp90 can move together from the cytoplasm to the
nucleus (12). It has also been shown that treatment of cells with
geldanamycin, an antibiotic that binds to hsp90 (13) at its nucleotide
binding site (14) and prevents formation of normal receptor·hsp90
heterocomplexes (15), impedes steroid-induced movement of the GR from
the cytoplasm to the nucleus (16). Taken together, these observations
are consistent with the notion that hsp90 plays some role in GR movement.
The notion that a phosphatase may be required for receptor shuttling
evolved from the work of Qi et al. (7, 17), who found that
GRs in cells transformed with v-Mos, an oncoprotein that is a
cytoplasmic serine/threonine protein kinase, undergo hormone-dependent transfer to the nucleus but that they are
inefficiently retained, and they cycle back to the cytoplasm, where
they do not regain the capacity for hormone-dependent
translocation. Subsequently, DeFranco et al. (18) showed
that okadaic acid, a serine/threonine protein phosphatase inhibitor,
acts like v-Mos to trap GR that has cycled out of the nucleus into the
cytoplasm in a form that cannot undergo hormone-dependent
recycling to the nucleus. These observations suggest that a cytoplasmic
phosphatase is required for recycling of GR that has passed through
stages of nuclear import, transcriptional activation, and nuclear
export (18). It is not known whether the phosphatase activity is
required for GR transformation to a state that can be translocated or
whether it is required for subsequent movement of the transformed
receptor (18).
Recently, we have used cytoskeletal disrupting agents, such as colcemid
and cytochalasin D, and a fusion protein of murine GR with
Aequorea green fluorescent protein (GFP-GR) to determine whether there is any linkage between hsp90-dependent
receptor movement and the cytoskeleton (19). As previously reported by Perrot-Applanat et al. (20), for the progesterone receptor, we found that GFP-GR underwent rapid (t1/2 ~5 min)
steroid-dependent translocation to the nucleus both in cells with intact cytoskeleton and in cells with completely disrupted cytoskeletal networks. However, in cells with a normal cytoskeleton, the hsp90 inhibitor geldanamycin slowed translocation of the GFP-GR by
close to an order of magnitude (t1/2 ~45 min),
whereas in cells with disrupted cytoskeletal networks, 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 activity of hsp90
plays a role. In cells where the cytoskeletal networks have been
artifactually disrupted, movement is still
steroid-dependent, but the transformed GFP-GR moves through
the cytoplasm by diffusion and is not affected by geldanamycin.
In this work, we show that treatment of cells with okadaic acid
inhibits GR transformation as defined by loss of its high affinity
association with hsp90, and we show that disruption of microtubules
with colcemid eliminates the okadaic acid block of hormone-dependent recycling of GFP-GR to the nucleus. We
suggest that under physiological conditions where the cytoskeleton is intact, the GR shuttles between the cytoplasm and the nucleus with the
help of a machinery that utilizes cytoskeletal tracts, involves the
dynamic activity of hsp90, and requires the activity of an okadaic
acid-sensitive phosphatase. However, when the cytoskeleton is
disrupted, the GR moves through the cytoplasm by diffusion in a manner
that is still steroid-dependent but does not require the
okadaic acid-sensitive phosphatase activity and hsp90 dissociation.
 |
EXPERIMENTAL PROCEDURES |
Materials
NIH/3T3 mouse embryo fibroblasts were purchased from the
American Type Culture Collection (Rockville, MD). Phenol red-free Dulbecco's modified Eagle's medium (DMEM) and LipofectAMINE were from
Life Technologies, Inc. Geldanamycin was obtained from the Drug
Synthesis and Chemistry branch of the Developmental Therapeutics Program, National Cancer Institute. Colcemid, colchicine,
-lumicolchicine, and charcoal-stripped, delipidated calf serum were
from Sigma. BuGR2 monoclonal anti-glucocorticoid receptor IgG was from
Affinity BioReagents (Golden, CO), and the AC88 monoclonal IgG
anti-hsp90 was from StressGen (Victoria, BC, Canada). Hybridoma cells
producing the FiGR monoclonal IgG anti-GR (21) were generously provided by Jack Bodwell (Dartmouth Medical School). Construction of the GFP-GR
expression plasmid was described previously (19).
Methods
Cell Culture and Transfection--
NIH/3T3 cells were grown on
22 × 22-mm coverslips in DMEM supplemented with 10% bovine calf
serum in 35-mm tissue culture dishes. When cells were ~60%
confluent, they were rinsed three times with serum-free medium and then
incubated for an additional hour in fresh medium. For each transfection
of GFP-GR cDNA, a solution containing 2.5 µg of DNA and 10 µl
of LipofectAMINE in 0.8 ml of Opti-MEM I medium was added to culture
dishes containing 2 ml of DMEM and mixed gently to ensure uniform
distribution. Cells were incubated with the transfection mixture for
6 h at 37 °C, and the medium was then replaced by complete
growth medium for an additional 18 h of incubation. At the end of
this incubation, the coverslips were washed extensively with and then
incubated overnight in phenol red-free DMEM supplemented with 10%
charcoal-stripped, delipidated bovine calf serum. The cells were used
for GFP-GR translocation experiments.
GFP-GR Translocation--
For receptor translocation and steroid
withdrawal experiments, cells transfected as described above were
incubated for 1 h with 0.1 µM corticosterone to
permit GFP-GR translocation to the nucleus. The medium was then removed
and cells were washed 3-4 times and incubated for 20 h in phenol
red-free DMEM with charcoal-stripped serum with or without 50 nM okadaic acid. After 20 h, 0.1 µM
corticosterone was readded for 1 h to permit GFP-GR recycling into
the nucleus. For microtubule disruption, 0.6 µg/ml colcemid was added
1 h prior to readdition of corticosterone. For geldanamycin
inhibition of translocation, 20-h withdrawn cells were incubated for
1.5 h at 0 °C with corticosterone to permit receptor binding,
and geldanamycin was added during the last 0.5 h at 0 °C to
permit equilibration with the cells. At the end of the 1.5 h at
0 °C, cells were incubated at 37 °C for 20 min to permit
steroid-dependent GFP-GR translocation.
Fluorescence Visualization--
At the end of the 1-h incubation
with corticosterone, the coverslips were rinsed with phosphate-buffered
saline at room temperature and simultaneously fixed and permeabilized
by immersion in cold methanol (
25 °C) for 30 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 photographed with a Leitz Aristoplan
epiillumination microscope and a Leitz Vario-Orthomat camera using
T-Max 3200 film. The bar in Fig. 1 represents 10 µm.
Scoring of GFP-GR Translocation--
Cells were scored for
GFP-GR translocation as we have described previously (19), 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 mean ± 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 a Bonferroni t test.
Assay of GR-associated hsp90--
Untransfected 3T3 cells
treated with corticosterone and okadaic acid, as indicated in Fig. 6,
were suspended in 1.5 volumes of HEM buffer (10 mM HEPES
(pH 7.4), 1 mM EDTA, 20 mM sodium molybdate) and ruptured by Dounce homogenization. Cell homogenates were
centrifuged for 30 min at 100,000 × g, with the
supernatant being the cytosol. The GR was immunoadsorbed from replicate
aliquots (500 µl) of cytosol with FiGR ascites as described
previously (16). The immune pellets were washed 4 times by suspension
in 1 ml of TEGM buffer (10 mM TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic
acid (pH 7.6), 50 mM NaCl, 4 M EDTA, 10%
glycerol, 20 mM molybdate), and boiled in SDS sample buffer
with 10%
-mercaptoethanol, and proteins were resolved on 8%
SDS-polyacrylamide gels. After transfer to Immobilon-P membranes, the
GR was probed with 2 µg/ml BuGR and hsp90 with 1 µg/ml AC88, and
the immunoblots were incubated a second time with
125I-labeled goat anti-mouse IgG.
 |
RESULTS |
Okadaic Acid Inhibition of GFP-GR Recycling from Cytoplasm to
Nucleus--
To determine whether okadaic acid blocks
agonist-dependent recycling of the GFP-GR in the same
manner as previously reported for endogenous GR in rat fibroblasts
(18), we performed the experiments summarized in the legend to Fig.
1. In 3T3 cells expressing GFP-GR, the
fusion protein is localized to the cytoplasm in the absence of steroid
(Fig. 1A) and is translocated to the nucleus when cells are
incubated with the agonist corticosterone (Fig. 1C). The
same agonist-dependent translocation is seen in cells incubated simultaneously with okadaic acid (Fig. 1, B and
D). Under both conditions (i.e. with and without
okadaic acid), withdrawal of corticosterone for 20 h is
accompanied by return of the GFP-GR to the cytoplasm (Fig. 1,
E and F). In the absence of okadaic acid,
retreatment with corticosterone causes recycling of the GFP-GR to the
nucleus (Fig. 1G). However, in the presence of okadaic acid,
agonist-dependent recycling to the nucleus is inhibited (Fig. 1H).

View larger version (103K):
[in this window]
[in a new window]
|
Fig. 1.
Okadaic acid inhibits
steroid-dependent GFP-GR recycling from cytoplasm to
nucleus. 3T3 cells expressing GFP-GR were incubated with CORT
and/or 50 nM OA as indicated below, and fluorescence was
visualized and scored as described under "Methods." A,
no treatment; B, OA for 1 h; C, CORT for
1 h; D, CORT and OA for 1 h; E, CORT
for 1 h followed by steroid withdrawal for 20 h;
F, treated as in E but OA was present during
withdrawal; G, treated as in E but reincubated
for 1 h with CORT after withdrawal; H, treated as in
G but OA was present during withdrawal and reincubation with
CORT. The bar graphs present the nuclear translocation
scores in the absence (hatched bar) or presence (solid
bar) of OA, determined as described under "Methods." Condition
G differs from H at a significance of
p < 0.006.
|
|
Fig. 2A shows the time course
of the return of GFP-GR to the cytoplasm upon withdrawal of
corticosterone. In all of the experiments in this work, we use a 20-h
withdrawal period before retreatment with agonist. Fig. 2B
presents the concentration dependence of okadaic acid inhibition of
agonist-dependent GFP-GR recycling to the nucleus. In this
case, cells were treated with corticosterone to translocate the GFP-GR
to the nucleus and then withdrawn in the presence of various
concentrations of okadaic acid. At the end of the 20-h withdrawal
period, corticosterone was added to each culture, and the incubations
were continued for an additional 1 h to permit the GFP-GR to
reenter the nucleus. At concentrations below 25 nM okadaic
acid, complete recycling to the nucleus is achieved, but at higher
concentrations, increasing inhibition is observed. In the rest of the
experiments in this work, we use 50 nM okadaic acid, which
is the minimum concentration required for maximum inhibition of GFP-GR
recycling to the nucleus.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration dependence of okadaic acid
inhibition. A, time course of corticosterone withdrawal
is shown. 3T3 cells expressing GFP-GR were incubated for 1 h with
0.1 µM corticosterone, the cells were washed several
times, and the incubation was continued in the presence ( ) or
absence ( ) of corticosterone. At the indicated times, GFP-GR
fluorescence was examined and is expressed as the nuclear translocation
score. B, okadaic acid concentration dependence is shown.
Cells were incubated for 1 h with corticosterone, followed by
steroid withdrawal for 20 h in the presence of various
concentrations of okadaic acid. At the end of the withdrawal period,
corticosterone was readded for 1 h and GFP-GR nuclear
translocation scores were assayed. The dotted line
represents the score of cells that were never treated with
corticosterone.
|
|
Cytoskeletal Disruption with Colcemid Restores Agonist-induced
GFP-GR Recycling in Okadaic Acid-treated Cells--
To determine the
effect of cytoskeletal disruption on okadaic acid inhibition of GFP-GR
recycling, corticosterone-treated cells were withdrawn in the presence
or absence of okadaic acid and then treated for 1 h with colcemid.
We have shown previously that a 1-h treatment with 0.6 µg/ml colcemid
completely eliminates microtubules in 3T3 cells (19). As shown in Fig.
3, the GFP-GR is predominantly
cytoplasmic in cells that have been withdrawn in the presence of
okadaic acid and then treated with colcemid (Fig. 3B,
upper right). However, when these cells with disrupted cytoskeleton are treated again with corticosterone, there is complete translocation of the GFP-GR to the nucleus, even in the presence of
okadaic acid (Fig. 3B, lower right). In control
cells that were treated identically but not exposed to colcemid, GFP-GR
translocation to the nucleus was inhibited by okadaic acid (Fig.
3A, lower right). Thus, the colcemid treatment
restored agonist-induced GFP-GR recycling to okadaic acid-treated
cells.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 3.
Colcemid abolishes okadaic acid inhibition of
GFP-GR reentry into the nucleus. 3T3 cells expressing GFP-GR were
incubated for 1 h with corticosterone, followed by steroid
withdrawal for 20 h in the presence (+OA) or absence
( OA) of okadaic acid as indicated. Cells were then treated
for 1 h in the absence (A) or presence (B)
of colcemid to disrupt microtubules and then incubated an additional 20 min without (upper panels) or with (lower panels)
corticosterone to effect GFP-GR nuclear transfer. The bar
graphs show the nuclear translocation scores (mean ± S.E.)
of three independent experiments. The effect of okadaic acid without
colcemid (A, lower right panel) is significantly
different from the effect with colcemid (B, lower
right panel) at p < 0.001. Dotted
lines represent cells never treated with corticosterone.
|
|
The effect of cytoskeletal disruption on the rate of agonist-induced
nuclear reentry of the GFP-GR is shown in Fig.
4. Reentry occurs at a similar rate
(t1/2 ~5 min) in okadaic acid-free cells with
disrupted cytoskeleton (open circles) as in cells with an
intact cytoskeletal system (closed circles). In cells
withdrawn in the presence of okadaic acid and not treated with colcemid
(closed squares), about one-third of the cytoplasmic GFP-GRs
move to the nucleus within the first few minutes after readdition of
corticosterone, but a plateau of movement is achieved, and the
remainder of the GFP-GRs stays in the cytoplasm. The GFP-GR that moves
to the nucleus under this condition may be GFP-GR that was synthesized
during the withdrawal period, and because it has not cycled through the
nucleus, it is not affected by okadaic acid, as shown in Fig.
1D. In contrast, the GFP-GRs in okadaic acid-treated cells
where the cytoskeleton has been disrupted by colcemid (open
squares) undergo essentially complete agonist-induced nuclear
reentry at the same rapid rate as in cells not treated with okadaic
acid.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of okadaic acid and colcemid on the
rate of GFP-GR nuclear reentry. 3T3 cells expressing GFP-GR were
treated with corticosterone and withdrawn in the presence
(squares) or absence (circles) of okadaic acid
and then treated for 1 h with (open symbols) or without
(closed symbols) colcemid. Corticosterone was then added to
all conditions, and nuclear translocation was assayed at the indicated
times.
|
|
The Effects of Both Okadaic Acid and Cytoskeletal Disruption Are
Reversible--
In Fig. 5A,
cells withdrawn in the presence of okadaic acid (condition 3) were
either treated with corticosterone (condition 4) or washed and
incubated for 1 h in okadaic acid-free medium prior to treatment
with corticosterone (condition 5). It is clear that the okadaic acid
inhibition of GFP-GR reentry shown in condition 4 is eliminated when
cells are washed free of okadaic acid, as shown in condition 5. As
shown in condition 6, the agonist-dependent nuclear reentry
of about one-third of the cytoplasmic GFP-GRs that occurs in okadaic
acid-treated cells (cf. condition 4 with 3) is blocked if
the hsp90 inhibitor geldanamycin is present during the 20-min
incubation with corticosterone. Like the inhibition of reentry with
okadaic acid alone, the total inhibition of reentry achieved by
geldanamycin and okadaic acid together is reversed by washing the cells
(condition 7).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
The effects of okadaic acid and colcemid are
reversible. A, reversal of okadaic acid inhibition of
GFP-GR reentry is shown. 3T3 cells expressing GFP-GR were treated with
corticosterone and withdrawn in the presence or absence of okadaic
acid. Withdrawn cells were then treated for 20 min with CORT and OA or
washed and incubated for 1 h without OA and then treated with
CORT, as indicated. Conditions are: 1, withdrawn without OA;
2, withdrawn without OA and treated with CORT; 3,
withdrawn with OA; 4, withdrawn with OA and treated with
CORT and OA; 5, withdrawn with OA, washed and incubated
1 h without OA, and then treated with CORT; 6,
withdrawn with OA and treated for 20 min with CORT and 10 µM geldanamycin; 7, withdrawn with OA,
incubated for 20 min with geldanamycin and OA, washed and incubated for
1 h without OA or geldanamycin, and then treated for 20 min with
CORT. B, reversal of colcemid effect. 3T3 cells withdrawn in
the presence of OA were incubated for 1 h in the presence of OA
and either colcemid, colchicine, or lumicolchicine. Some cells were
then treated for 20 min with CORT, and others were washed, incubated
for 3 h in colcemid-free medium with OA, and then treated with
CORT. Conditions are: 1, withdrawn in the absence of OA and
treated with CORT; 2, withdrawn in the presence of OA and
treated with CORT; 3, withdrawn with OA, incubated with
colcemid and OA; 4, withdrawn with OA, incubated with
colcemid and OA, and treated with CORT; 5, withdrawn with
OA, incubated with colcemid and OA, washed and incubated in
colcemid-free medium with OA, and then treated with CORT; 6,
withdrawn with OA, incubated with 1 µM colchicine and OA,
and treated with CORT; 7, withdrawn with OA, incubated with
1 µM -lumicolchicine and OA, and treated with
CORT.
|
|
We have shown previously that the cytoskeletal network returns to
normal when 3T3 cells with completely disrupted microtubules are
incubated for a short time in colcemid-free medium (19). Fig.
5B shows that incubation of 3T3 cells in colcemid-free
medium restores okadaic acid inhibition of GFP-GR nuclear reentry to colcemid-treated cells (cf. condition 5 with 4). The
proposal that it is disruption of the cytoskeleton that permits the
GFP-GR in colcemid-treated cells to travel through the cytoplasm to the nucleus despite the action of okadaic acid is supported by the data of
conditions 6 and 7 in Fig. 5B. In this case, the 3T3 cells that were withdrawn in the presence of okadaic acid were incubated for
1 h either with colchicine or with
-lumicolchicine, a
biologically inactive isomer of colchicine that does not interact with
tubulin (22). We have previously reported that 3T3 cells treated with 1 µM colchicine lose their microtubules, whereas cells
treated with 1 µM lumicolchicine do not (19). As shown in
Fig. 5B, okadaic acid did not inhibit GFP-GR nuclear reentry
in colchicine-treated cells (condition 6), but it inhibited reentry in
-lumicolchicine-treated cells (condition 7).
In Okadaic Acid-treated Cells, the GR Does Not Undergo
Agonist-dependent Dissociation from hsp90--
The GR is
recovered from hormone-free cells as a 9 S GR·hsp90 heterocomplex,
and shortly after treatment of cells with steroid, it is recovered from
cytosol as the 4 S hsp90-free GR (for review, see Ref. 23). This
steroid-dependent transformation of the receptor has been
regarded as a prerequisite to translocation of the GR from the
cytoplasm to the nucleus. In the experiment corresponding to Fig.
6A, we asked whether okadaic
acid blocked receptor transformation. In this case, untransfected 3T3
cells were treated with corticosterone and okadaic acid as above, the
cells were then ruptured, and the endogenous 3T3 cell GR was
immunoadsorbed and assayed for receptor-associated hsp90 by Western
blotting. As shown in Fig. 6A, receptors that have undergone
agonist-dependent translocation to the nucleus and been
withdrawn in either the presence or absence of okadaic acid are in
heterocomplexes with hsp90. Corticosterone retreatment of cells
withdrawn in the absence of okadaic acid yielded hsp90-free GR, whereas
corticosterone treatment of cells withdrawn in the presence of okadaic
acid yielded GR·hsp90 heterocomplexes. In cells withdrawn in the
presence of okadaic acid, there is no further nuclear movement after
10-15 min of retreatment with corticosterone (Fig. 4), and even after
1 h of corticosterone retreatment, receptors in these cells are
recovered as GR·hsp90 heterocomplexes (data not shown). These data
suggest that an okadaic acid-sensitive phosphatase activity is required
for transformation of glucocorticoid receptors that have been cycled
into and out of the nucleus.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6.
Okadaic acid inhibits GR transformation.
A, inhibition of GR transformation is shown. Untransfected
3T3 cells were incubated for 1 h with CORT, withdrawn
(WD) in the absence or presence (+OA) of okadaic
acid, and reincubated for 15 min in the presence of CORT, as indicated.
Cytosols were prepared and aliquots were immunoadsorbed with nonimmune
IgG (NI) or with the FiGR antibody anti-GR (I).
The immune pellets were washed and assayed for GR and GR-associated
hsp90 by SDS-polyacrylamide gel electrophoresis and Western blotting.
B, colcemid does not affect okadaic acid inhibition of GR
transformation. Cells treated as above were treated with colcemid for
1 h at the end of withdrawal and prior to retreatment with CORT
for 10 min. The control cells were simply exposed to colcemid for
1 h.
|
|
We showed in Fig. 3B that cytoskeletal disruption with
colcemid restores agonist-induced nuclear transfer of receptors in cells withdrawn in the presence of okadaic acid, and under these conditions, GFP-GR recycles to the nucleus within 10-15 min (Fig. 4).
We have also shown that the endogenous cellular GR transfers to the
nucleus within 10-15 min in colcemid-treated cells (8). Thus, we asked
whether receptors that were recycled into the nucleus in cells that
were withdrawn in the presence of okadaic acid and colcemid were
recovered in the hsp90-free or hsp90-bound form. The 15-min interval
after steroid treatment was chosen for the experiments corresponding to
Fig. 6, because the GR that has transferred to the nucleus is still
recovered in the cytosolic fraction after cell rupture. After 20 min of
steroid treatment, most of the GR is retained in the nuclear fraction
of colcemid-treated cells (data not shown). As shown in Fig.
6B, receptors that have been withdrawn in either the
presence or absence of okadaic acid and then treated with colcemid are
in heterocomplexes with hsp90. Corticosterone treatment of cells with a
disrupted cytoskeleton that were withdrawn in the absence of okadaic
acid resulted in hsp90-free GR, but in cells withdrawn in the presence
of okadaic acid, receptors remain in heterocomplex with hsp90. Thus,
the receptors in okadaic acid-withdrawn cells where the cytoskeleton is
disrupted undergo agonist-dependent translocation to the
nucleus (Fig. 3B) without undergoing transformation as
defined by agonist-dependent dissociation of the GR·hsp90
heterocomplex (Fig. 6B).
 |
DISCUSSION |
Two mechanistically different inhibitors of
steroid-dependent GR cytoplasmic nuclear translocation have
been identified as geldanamycin and okadaic acid. Geldanamycin markedly
inhibits the rate of receptor translocation, but in time, all of the
receptors eventually become localized in the nucleus (16, 19).
Geldanamycin inhibits both the initial transfer of the GR (16, 19) as
well as the recycling of GR that has exited the nucleus upon withdrawal of hormone (Fig. 5A and data not shown). Because
geldanamycin is an hsp90 inhibitor, we have suggested that dynamic
assembly of steroid-bound GR heterocomplexes with hsp90 and its
associated immunophilins, such as FK506 binding protein 52, by the
hsp90-based chaperone system plays a role in GR movement through the
cytoplasm. Because geldanamycin slows but does not block GR movement,
it seems that the role of the hsp90-based chaperone system is to facilitate movement. Perhaps it does so by facilitating the association of the receptor with a piece of movement machinery with which the
receptor associates less efficiently when assembly of mature GR·hsp90
heterocomplexes is inhibited by geldanamycin.
In contrast to geldanamycin, okadaic acid does not inhibit the initial
steroid-dependent transfer of the GR from cytoplasm to
nucleus; rather, it inhibits only the recycling of receptors that have
passed through the original cycle of nuclear import, transcriptional
activation, and nuclear export (18) (Fig. 1). Also, in contrast to
geldanamycin's inhibition of the rate of movement, okadaic acid blocks
recycling of the majority of receptors (Fig. 4). Okadaic acid appears
to block receptor recycling by inhibiting a dephosphorylation event
that is required for steroid-dependent receptor
transformation (Fig. 6). The GR is a phosphoprotein (for review, see
Ref. 24) that becomes hyperphosphorylated after cells are exposed to
agonist (25, 26). Dephosphorylation of the hyperphosphorylated GR
itself could be the event required for receptor transformation, but
other components of the untransformed receptor complex are also
phosphoproteins (e.g. hsp90 and FK506 binding protein 52),
and their dephosphorylation or the dephosphorylation of another protein
may be critical for receptor transformation in intact cells.
Here we have assayed receptor transformation by assaying the decrease
in the fraction of receptors recovered as GR·hsp90 heterocomplexes from steroid-withdrawn cells that were retreated with corticosterone (Fig. 6). This steroid-dependent transformation of the
receptor to a form that is no longer in stable heterocomplex with hsp90 is apparently required to permit the GR to associate with the movement
machinery in normal cells with intact cytoskeleton. In contrast to
hormone-free progesterone receptor·hsp90 or estrogen receptor·hsp90
heterocomplexes, which are constitutively localized to the nucleus (for
review, see Ref. 23), both of the GR NLSs appear to be blocked or
conformationally inactive when the unliganded GR is in stable
heterocomplex with hsp90. This may explain why the unliganded GR is
cytoplasmic. Consistent with the notion that the NL1 is activated upon
GR transformation, it has been shown that dissociation of GR from
stable heterocomplex with hsp90 permits access of an NL1-specific
antibody to the NLS (27, 28). In cells that have been withdrawn in the
presence of okadaic acid, the GR is in stable heterocomplex with hsp90
(Fig. 6), and it binds steroid normally (18), but receptor
transformation as assayed by conversion to hsp90-free GR is blocked
(Fig. 6).
Several years ago, it was shown that microtubule disruption does not
prevent cytoplasmic nuclear translocation of the progesterone receptor
(20) or the GR (8), and it does not affect steroid-mediated transcriptional activation by the GR (29). In cells treated with
colcemid, neither geldanamycin (19) nor okadaic acid (Figs. 3-5)
inhibits cytoplasmic nuclear translocation of the GR. Thus, when the
cytoskeleton is disrupted, the GR appears to move by a different
mechanism. A reasonable model for the normal cell would be that, like
vesicles, the GR moves through the cytoplasm on cytoskeletal tracts
requiring the participation of cytoskeleton-associated motor proteins.
When the cytoskeletal network is intact, diffusion of protein solutes
like the GR is limited, and in some way dynamic cycling of the
liganded, transformed GR with hsp90 facilitates a connection between
the NL1 and the movement system. In contrast, the comparatively stable
association of the unliganded, untransformed GR with hsp90 blocks
access of NLS recognition protein(s) to NL1. Okadaic acid appears to
block receptor transformation, which is required for movement along the
cytoskeleton-based movement system. We speculate that when the
cytoskeleton is disrupted, a major limitation on diffusion of protein
solutes has been removed, and the GR may diffuse through the cytoplasm,
with its penetration through the nuclear pores being determined by
interactions with nuclear uptake proteins such as importin (for review
of the nuclear import of steroid receptors, see Ref. 5). It is
interesting that recycling of the GR in okadaic acid-withdrawn cells
treated with colcemid is strictly steroid-dependent (Figs.
3B and 5B). Yet, under these conditions, the GR
appears to remain in stable association with hsp90 (Fig.
6B). Some steroid-dependent event other than
dissociation from hsp90 must have occurred such that GR transformation
is uncoupled from GR movement in the cell treated with both colcemid
and okadaic acid.
Because Perrot-Applanat et al. (20) demonstrated that the
progesterone receptor could move from the cytoplasm to the nucleus in
cells where the cytoskeletal networks were completely disrupted, it has
been assumed that the cytoskeleton is not involved in steroid receptor
movement through the cytoplasm of the physiologically normal cell. The
observations represented in this paper and those of Galigniana et
al. (19) suggest that there are two movement mechanisms and that
intact cytoskeleton is required for GR movement in the physiologically
normal cell.