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Review article
Role of hsp90 and the hsp90-binding immunophilins
in signalling protein movement
William B. Pratt
a,
*, Mario D. Galigniana
a
, Jennifer M. Harrell
a
, Donald B. DeFranco
b
a
Department of Pharmacology, University of Michigan Medical School, 1301 Med. Sci. Res. Building III, Ann Arbor, MI 48109-0632, USA
b
Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
Received 20 December 2003; accepted 30 January 2004
Available online 21 March 2004
Abstract
The ubiquitous protein chaperone hsp90 has been shown to regulate more than 100 proteins involved in cellular signalling. These proteins
are called ‘client proteins’ for hsp90, and a multiprotein hsp90/hsp70-based chaperone machinery forms client proteinhsp90
heterocomplexes in the cytoplasm and the nucleus. In the case of signalling proteins that act as transcription factors, the client
proteinhsp90 complexes also contain one of several TPR domain immunophilins or immunophilin homologs that bind to a TPR domain
binding site on hsp90. Using several intracellular receptors and the tumor suppressor p53 as examples, we review evidence that dynamic
assembly of heterocomplexes with hsp90 is required for rapid movement through the cytoplasm to the nucleus along microtubular tracks. The
role of the immunophilin in this system is to connect the client proteinhsp90 complex to cytoplasmic dynein, the motor protein for
retrograde movement toward the nucleus. Upon arrival at the nuclear pores, the receptorhsp90immunophilin complexes are transferred to
the nuclear interior by importin-dependent facilitated diffusion. The unliganded receptors then distribute within the nucleus to diffuse patches
from which they procede in a ligand-dependent manner to discrete nuclear foci where chromatin binding occurs. We review evidence that
dynamic assembly of heterocomplexes with hsp90 is required for movement to these foci and for the dynamic exchange of transcription
factors between chromatin and the nucleoplasm.
D2004 Elsevier Inc. All rights reserved.
Keywords: Immunophilins; Steroid receptors; Hsp90; Dynein; Microtubules; Signal protein trafficking
1. Introduction
After ligand-dependent activation through receptors in
the plasma membrane or after direct receptor activation in
the cytoplasm, signalling proteins that affect gene transcrip-
tion must move to their sites of action within the nucleus.
This movement can be divided into four general steps: (1)
movement through the cytoplasm to the nuclear pores, (2)
transport across the nuclear pore complex, (3) movement
within the nucleus to loci for transcriptional activation, and
(4) subsequent dynamic exchange of transcription factors
between chromatin and the nucleoplasmic compartment.
The steroid receptors have proven to be useful models for
studying the movement of transcription factors in each of
these movement phases. These receptors form complexes
with the ubiquitous and essential chaperone hsp90, and these
complexes also contain tetratricopeptide repeat (TPR) do-
main immunophilins. There is a growing body of evidence
that both hsp90 and the hsp90-binding immunophilins
participate in various phases of receptor movement.
Since 1990, over 100 protein kinases and transcription
factors involved in cellular signalling have been reported to
be regulated by hsp90 [1]. Regulation is achieved in multiple
ways. In some cases, hsp90 is required for signalling protein
function. For example, binding of hsp90 to glucocorticoid
(GR), mineralocorticoid and aryl hydrocarbon (AHR) recep-
tors is required for ligand binding activity. In several cases to
0898-6568/$ - see front matter D2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2004.02.004
Abbreviations: hsp, heat shock protein; SR, steroid receptor; GR,
glucocorticoid receptor; AR, androgen receptor; AHR, aryl hydrocarbon
receptor; GFP, green fluorescent protein; HBD, hormone binding domain;
FKBP, FK506-binding protein; CyP, cyclosporine A-binding protein;
PPIase, peptidylprolyl isomerase; TPR, tetratricopeptide repeat; ARA9,
AHR-associated protein 9; XAP2, hepatitis B virus X-activating protein 2;
AIP, AHR-interacting protein; Arnt, AHR nuclear translocator; PPAR,
peroxisome proliferator-activated receptor; NLS, nuclear localization
signal; NES, nuclear export signal; Hop, hsp organizing protein.
* Corresponding author. Tel.: +1-734-764-5414; fax: +1-734-763-4450.
E-mail address: mgali@umich.edu (W.B. Pratt).
www.elsevier.com/locate/cellsig
Cellular Signalling 16 (2004) 857 – 872
be reviewed here, dynamic binding to hsp90 is required for
rapid signalling protein movement. There is some evidence
that binding of hsp90 to transcription factors may play a role
in the termination of transcriptional activation. Finally, in
many and perhaps all cases where there is a persistent
complex with hsp90, the signalling protein is stabilized to
rapid degradation by the ubiquitin-proteasome pathway. In
this way, hsp90 serves to maintain the abundance of a key
signalling protein, usually a protein kinase, such that normal
signal transduction through a pathway occurs.
In this review, we will focus on the roles of hsp90 and the
hsp90-binding immunophilins in signalling protein move-
ment, emphasizing the considerable recent progress in
understanding cytoplasmic – nuclear shuttling of three tran-
scription factors—the GR, the AHR, and the tumor sup-
pressor p53. We also review studies indicating that dynamic
assembly of heterocomplexes with hsp90 is required for the
high mobility of transcription factors within the nucleus.
The trafficking of signalling proteins was addressed in a
1999 review in Cellular Signalling [2], and this review will
emphasize work published in the past five years.
2. Assembly of signalling proteinhsp90immunophilin
complexes
Signalling proteinhsp90 complexes are formed by a
multiprotein hsp90/hsp70-based chaperone machinery, and
the assembly of these so-called ‘client protein’hsp90 com-
plexes has been the subject of a recent detailed review [1].
Briefly, the common pathway for complex assembly involves
an initial ATP-dependent interaction of the client protein with
the essential chaperone hsp70 and its nonessential cochaper-
one hsp40 to form a client proteinhsp70 complex that is now
‘primed’ to bind hsp90 and the nonessential cochaperone
Hop. A second ATP-dependent reaction then occurs, produc-
ing a client proteinhsp90 complex in which the bound hsp90
is converted to its ATP-dependent conformation. The small,
ubiquitous cochaperone p23 then binds dynamically to the
bound hsp90 to maintain it in the ATP-dependent conforma-
tion, thus stabilizing the client proteinhsp90 complex. This
assembly machinery is ubiquitous and conserved among
animal and plant cells, indicating that it performs essential
housekeeping functions [3].
After their formation, the client proteinhsp90p23 com-
plexes diverge from the common pathway in that protein
kinasehsp90 complexes quite selectively bind p50
cdc37
whereas transcription factorhsp90 complexes bind primar-
ily TPR domain immunophilins and immunophilin homo-
logs (reviewed in Ref. [2]). Both p50
cdc37
and the TPR
domain immunophilins bind directly to hsp90 but at differ-
ent sites, and the protein kinase complexes select for the
presence of p50
cdc37
because it also binds directly to the
catalytic domain of the kinase [4,5]. Similarly, transcription
factors, such as the steroid receptors (SR) and the aryl
hydrocarbon receptor, form hsp90 heterocomplexes that
contain a TPR domain immunophilin because the immuno-
philin binds directly to the transcription factor [6,7].
2.1. The TPR domain immunophilins
Several TPR domain proteins (Table 1) bind to a com-
mon TPR acceptor site located at the C-terminus of hsp90
[15– 19]. Tetratricopeptide repeats are degenerate sequences
of 34 amino acids that are involved in a variety of protein –
protein interactions [20]. The core of the TPR binding site
on hsp90 is the MEEVD sequence [21,22], and although the
Table 1
Mammalian TPR proteins that bind to hsp90
Protein Proposed role in SR action
Hop
(Hsp Organizing
Protein)
Binds via independent TPR domains to form
the hsp70Hophsp90 machinery for client
proteinhsp90 heterocomplex assembly; Hop
promotes rate of assembly but is not essential
for assembly
Immunophilins
FKBP52 Found in SRhsp90 heterocomplexes: Targets
retrograde SR movement by binding via PPIase
domain to cytoplasmic dynein; effect is
independent of PPIase activity
Increases GR hormone binding affinity in vivo;
effect requires both hsp90 binding and PPIase
activity [8]
FKBP51 Found in SRhsp90 heterocomplexes; FKBP51
expression blocks effect of FKBP52 on GR
steroid binding activity [8]; dynein binding
status unknown
CyP-40 Found in SRhsp90 heterocomplexes and binds
to cytoplasmic dynein
Immunophilin homologs
PP5 An okadaic acid-sensitive protein-serine phos
phatase with a TPR domain and a PPIase
homology domain [9]; PP5 is found in
SRhsp90 heterocomplexes, and it binds to
cytoplasmic dynein; its phosphatase activity
may be important for cytoplasmic – nuclear
trafficking
ARA9 (XAP2, AIP) Found in aryl hydrocarbon receptor (AHR)
hsp90 heterocomplexes; mediates the cyto-
plasmic localization of the AHR; has a PPIase
homology domain that does not interact with,
or interacts only very weakly with, cytoplasmic
dynein [56]
CHIP
(C-terminus of Hsc70
Interacting Protein)
Binds via TPR domain to hsc70, hsp70, or
hsp90 [10]; CHIP interaction with hsc70/hsp70
causes proteasome-dependent degradation of
many hsp90 client proteins; CHIP is an E3
ubiquitin ligase for the GR [11]
UNC-45 Binds via N-terminal TPR to hsp90 and via
C-terminal regions to myosin [12]
Tom 70 Mitochondrial import receptor with a TPR
domain that binds hsp90 [13]
Tpr2 A TPR domain protein that recognizes hsp90
and hsp70; it contains a J domain and
stimulates ATP hydrolysis by hsp70; its
expression affects GR activity negatively [14]
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872858
TPR proteins listed in Table 1 compete with each other for
binding to this site, there are some differences in their
binding determinants [23–25] (reviewed in Ref. [26]).
Hsp90 forms a homodimer, the TPR binding site lies within
the dimerization domain, and the number of TPR acceptor
sites per dimer has been controversial. Studies of saturation
binding of Hop to hsp90 dimer [16] and cross-linking of
hsp90FKBP52 complexes [7] are consistent with one TPR
binding site per dimer. In contrast, isothermal titration
calorimetry studies are consistent with binding of two
molecules of TPR protein to an hsp90 dimer [27,28].In
cross-linking studies, Gehring and his colleagues deter-
mined a heteromeric structure of 1 receptor:2 hsp90:1
immunophilin for several steroid receptor heterocomplexes
(reviewed in Ref. [29]), and that is the stoichiometry that we
will assume in this review.
The immunophilins are conserved proteins that bind
immunossuppressant drugs, such as FK506, rapamycin
and cyclosporine A. All members of the immunophilin
family have peptidylprolyl isomerase (PPIase) activity, and
they are divided into two classes: the FKBPs bind FK506
and rapamycin, and the cyclophilins (CyPs) bind cyclospor-
ine A. The immunosuppressant drugs occupy the PPIase site
on the immunophilin, blocking its ability to direct cis –trans
isomerization of peptidyl – prolyl bonds. Three high molec-
ular weight immunophilins with TPR domains—FKBP52,
FKBP51, CyP40—have been found in steroid receptor
hsp90 complexes (Fig. 1) [1]. A fourth SRhsp90 complex
protein, protein phosphatase 5 (PP5), is a protein-serine
phosphatase with three TPRs and a PPIase homology
domain with weak FK506 binding activity but no isomerase
activity [9]. Because these TPR proteins can exchange for
binding to hsp90, any single SRhsp90 complex can theo-
retically be associated over time with more than one
immunophilin. However, it has been shown that, at any
point in time, the immunophilins exist in separate GRhsp90
hsp90 heterocomplexes [30,31]. The relative amounts of
FKBP52, FKBP51, CyP40 and PP5 may vary somewhat
among the different steroid receptor heterocomplexes [26]
according to immunophilin interaction with the receptor
itself. There is a clear difference between the steroid receptor
complexes, which do not contain ARA9, and the AHR,
which appears to be bound exclusively to ARA9 as a result
of direct ARA9 binding to the AHR itself [32]. The TPR
domain immunophilins are distributed widely among animal
and plant cells and TPR domain binding to hsp90 is
conserved [33– 35]. This suggests that immunophilin bind-
ing to hsp90 is essential for both the action of the TPR
domain immunophilins and for major functions of hsp90.
2.2. The immunophilin PPIase activity
The broad distribution of the TPR domain immunophi-
lins and the presence of more than one member of the family
in most cells suggest that their function(s) is (are) important
for cell homeostasis and that there may be redundancy in
their action(s). The presence of the PPIase domain leads
naturally to the proposal that the action of the hsp90-binding
immunophilins is due to isomerization of prolyl peptide
bonds. Early experiments in intact cells demonstrated that
the isomerase inhibitors FK506 [36,37] and cyclosporine A
[31] could enhance dexamethasone-induced expression
from a reporter plasmid. Subsequent experiments in cytosol
preparations showed that addition FK506 at 25 jC stabilized
both progesterone [181] and glucocorticoid [182] recep-
torhsp90 complexes, and heterocomplex stabilization was
accompanied by a twofold decrease in the K
D
for ligand
binding. It is unclear whether these direct effects reflect
inhibition of the PPIase activity of hsp90-binding immuno-
philins or a physical stabilization. In addition, there are
indirect effects. It is known that both dexamethasone and the
immunosuppressant drugs are transported out of the cell by
the multidrug transporter Mdr1 (reviewed in Ref. [29]), and
Kralli and Yamamoto [38] showed that FK506 potentiates
dexamethasone responsiveness in L cells by increasing
dexamethasone accumulation without altering the hormone
binding properties of the GR.
Subsequent studies with squirrel monkey cells suggested
that immunophilins may be responsible for the relative
glucocorticoid insensitivity of these New World primates.
Squirrel monkeys have very high levels of circulating
corticosteroid and require much higher levels of hormone
for GR-dependent transactivation. However, the cloned
Bolivian squirrel monkey GR expressed in vitro was found
to bind dexamethasone with the same affinity as the human
GR [39]. Interest then focused on the high ratio of FKBP51
to FKBP52 found in squirrel monkey cells [40 – 42]. Human
FKBP51 and squirrel monkey FKBP51 are 94% identical
and have similar X-ray structures [43], but at similar levels
of expression, squirrel monkey FKBP51 is much more
Fig. 1. Functional domain structure of the hsp90-binding, TPR domain
immunophilins and immunophilin homologs. FKBP51 and FKBP52 have
an FK506-binding type of PPIase domain with isomerase activity. ARA9
and PP5 have domains homologous to the FK506-binding domain but have
no isomerase activity. CyP40 has a cyclosporine A-binding type of PPIase
domain with isomerase activity. PPIase and PPIase homology domains are
shaded, TPRs are slashed boxes. The numbering is for the human proteins.
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872 859
effective at reducing GR hormone binding affinity and
reporter gene expression. In contrast to these observations,
Patel et al. [44] found that the human GR has a normal dose
response for transactivation regardless of whether it is
expressed in New World or Old World primate cells. They
cloned the Guyanese squirrel monkey GR, and showed that
it had the same high affinity binding activity as the human
GR when expressed in COS-1 cells, but it had an order of
magnitude higher EC50 at transactivation than the human
GR in both squirrel monkey (New World) and COS-1 (Old
World) cells. In this case, the conclusion was that gluco-
corticoid resistance in the Guyanese squirrel monkey is at
least partly attributable to a naturally occurring mutation in
the GR gene that impairs GR transactivating activity.
To determine directly whether the hsp90-binding FKBPs
affect GR steroid binding activity, Riggs et al. [8] expressed
human FKBP51 and FKBP52 in Saccharomyces cerevisiae,
which does not contain any TPR domain FKBPs of its own,
and they showed that FKBP52 selectively potentiates GR-
dependent reporter gene activation. The potentiation was
due to an increase in GR hormone binding affinity that
required both the hsp90 binding activity and the PPIase
activity of FKBP52. Co-expression of FKBP51 with
FKBP52 blocked the potentiation but coexpression of PP5
did not affect the potentiation [8]. This work provides the
first evidence that an hsp90-binding immunophilin can
affect a client protein function (i.e. steroid binding) through
its peptidylprolyl isomerase activity. Presumably, the fold-
ing change due to isomerization occurs in the client protein
itself, although that remains to be demonstrated. It should
be noted that GR that has been assembled into GRhsp90
heterocomplexes by the purified heterocomplex assembly
system in the absence of immunophilins has normal high
affinity steroid binding activity. However, that does not in
any way argue against this model, because the GR that is
the client protein in those assays is immunopurified from
cell lysates where it was properly folded and in high affinity
binding state before it was stripped of its associated
chaperones.
The work of Riggs et al. [8] in yeast has raised the
important notion that a major function of the hsp90-bound
immunophilins is to modify the folding, and thus the
structure and function, of hsp90-bound client proteins. In
the future, it will be important to see if this model applies to
a variety of hsp90 client proteins and to other immunophi-
lins besides FKBP52. At present, the observation is specific
to the GR, as opposed to the estrogen receptor (also a client
protein), and is specific to FKBP52 versus other hsp90-
bound immunophilins [8]. It is curious that FKBP51 and
FKBP52 have highly conserved domain structures (Fig. 1)
and possess active isomerase domains, yet FKBP52
increases GR steroid binding activity and FKBP51 counter-
vails this effect. Such a yin and yang action is not consistent
with the notion of redundancy in action of TPR domain
immunophilins, but it could be of considerable regulatory
importance. In subsequent sections of this review, we will
not be concerned with the isomerase activity of the hsp90-
bound immunophilins. Rather, we will focus on the PPIase
domains as protein – protein interaction domains that serve
to link transcription factorhsp90 complexes to cytoplasmic
dynein, the motor protein responsible for retrograde move-
ment toward the nucleus along microtubular tracks.
3. Involvement of hsp90, immunophilins, and dynein in
receptor movement through the cytoplasm
The GR is an excellent model for studying the cytoplas-
mic– nuclear movement of a transcription factor, because it
is normally located in the cytoplasm of hormone-free cells
and its rapid movement to the nucleus is hormone-depen-
dent, placing movement under the control of the investiga-
tor. It is well established that the steroid receptors constantly
shuttle between the cytoplasm and the nucleus, with ligand-
dependent transformation of the GRhsp90 complex favor-
ing a change in this flux to a nuclear accumulation
(reviewed in Ref. [45]). On the basis of biochemical
observations, it was thought for many years that ligand-
dependent transformation of the GR resulted in release from
hsp90, leaving the chaperone-free receptor to move to the
nucleus. However, it is now clear that GR translocation
occurs in association with hsp90 [46,47], and it is thought
that ligand-dependent transformation converts the receptor
from a state that forms ‘persistent’ complexes with hsp90 to
a state that is in a much more dynamic receptorhsp90
complex assembly/disassembly cycle. The proposal that
dynamic GRhsp90 complex assembly is required for rapid
receptor movement is supported by a variety of observations
that have been previously reviewed [2,48].
The model for retrograde GR movement by dynein
motors along cytoskeletal tracks that is diagrammed in
Fig. 2 was first proposed in 1993 [49]. By that time, it
was well established that vesicles moved in both neurites
and cell bodies along cytoskeletal tracks in a process
requiring molecular motors [50], with cytoplasmic dynein
being the motor protein responsible for movement in the
retrograde direction toward the nucleus (reviewed in Refs.
[51,52]). We reasoned that protein solutes that were not
associated with vesicles might move in a similar manner. In
1993, the only immunophilin known to be in GRhsp90
complexes was FKBP52, which, although it localizes pre-
dominantly to the nucleus, has a cytoplasmic component
that localizes to microtubules [53– 55]. Accordingly, we
asked if cytosolic FKBP52 is in complexes with cytoplasmic
dynein and we showed by coimmunoadsorption that it is
[54]. Thus, we proposed that one function of FKBP52 was
to link the receptorhsp90 complex to the motor protein.
This model has been advanced considerably in the past
few years. It is now known that several of the hsp90-binding
immunophilins are linked via their PPIase domains to
cytoplasmic dynein [56], and the entire complex shown in
Fig. 2 has been isolated from cells and has been recon-
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872860
structed in a cell-free system [57 –59]. The cytoplasmic –
nuclear movement of the GR and some other transcription
factors has been impeded or blocked by inhibitors of hsp90,
by coexpression of a PPIase domain fragment that blocks
immunophilin binding to dynein, and by coexpression of
dynamitin, which dissociates dynein from its cargoes. Thus,
there is strong in vivo evidence for the validity of the GR
movement model of Fig. 2.
3.1. Inhibition of signalling protein movement with hsp90
inhibitors
Hsp90 is a member of a small family of proteins, the
GHKL ( Gyrase, Hsp90, Histidine Kinase, Mut L) family,
which possess a unique binding pocket for ATP [60]. This
nucleotide binding site near the N-terminus of hsp90 is the
site of action for the hsp90 inhibitors geldanamycin and
radicicol. In binding to this site, geldanamycin and radicicol
prevent hsp90 from assuming its ATP-dependent conforma-
tion, thus blocking client proteinhsp90 assembly and
hsp90 action. The discovery by the Neckers laboratory, in
1994, that geldanamycin acts as a quite specific inhibitor of
hsp90 [61] provided a rapid means of screening for hsp90-
regulated targets, initiating a rapid escalation in the identi-
fication of hsp90-regulated signalling pathways [1].
As shown first with the endogenous GR [62] and then
with a transfected green fluorescent protein fusion with the
GR (GFP-GR) [63], treatment of cells with geldanamycin
slows the rate of receptor translocation to the nucleus by an
order of magnitude (from t
1/2
f4.5 min to t
1/2
f45 min).
The rapid hsp90-dependent movement requires intact cyto-
skeleton [63], and when hsp90 is inhibited there is slow
movement that appears to occur by diffusion (Fig. 3). Axons
and dendrites are specialized cytoplasmic extensions where
proteins cannot move by random diffusion alone, and
ligand-dependent retrograde movement of the GFP-GR in
neurites is blocked by geldanamycin [64], indicating that the
hsp90-dependent movement machinery is obligatory in
these structures. Geldanamycin and radicicol also impede
ligand-dependent movement of the androgen receptor to the
nucleus [65,66], suggesting that all of the members of the
nuclear receptor family that form persistent complexes with
Fig. 3. Geldanamycin treatment or cotransfection of the PPIase domain
fragment of FKBP52 inhibits steroid-dependent GFP-GR translocation to
the nucleus. 3T3 cells were transfected with a plasmid expressing GFP-GR
and in the case of D cotransfected with a plasmid expressing the PPIase
domain fragment of FKBP52. Two days after transfection, 1 AM
dexamethasone or ethanol was added, the incubation was continued for
20 min, and cells were fixed and fluorescence was visualized. (A),
Untreated cells; (B), cells treated with dexamethasone; (C), cells treated
with geldanamycin and dexamethasone; (D), cells cotransfected with
PPIase domain treated with dexamethasone. The graph shows data
compiled from >100 cells scored for nuclear translocation on a scale from
0 for nuclear fluorescence much less than cytoplasmic fluorescence to 4 for
nuclear fluorescence much greater than cytoplasmic fluorescence. See Ref.
[58] for details of methods. (.) Cells treated with dexamethasone; (E)
cells treated with dexamethasone and geldanamycin; (n) cells cotransfected
with the PPIase domain and treated with dexamethasone.
Fig. 2. TPR domain immunophilins link the GRhsp90 heterocomplex to
dynein for retrograde movement along microtubules. Cytoplasmic dynein is
the motor protein that processes along microtubules in a retrograde
movement to the nucleus. Dynein is a large multisubunit complex (f1.2
MDa) comprised of two heavy chains (HC) that have the processive motor
activity, three intermediate chains (IC), and some light chains that are not
shown. Also not shown is the dynein-associated dynactin complex, of
which dynamitin is a component. The immunophilin (IMM) links to GR-
bound hsp90 via its TPR domain (solid black crescent) and it links to
dynein or a component of the dynactin complex via its PPIase domain
(dotted crescent).
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872 861
hsp90 and are localized to the cytoplasm of hormone-free
cells may require dynamic heterocomplex assembly with
hsp90 for rapid movement through the cytoplasm to nuclear
pores.
The AHR is different from steroid receptors in that its
DNA binding domain is a basic helix-loop-helix rather than
a double zinc finger structure, and rather than forming
homodimers in the nucleus it forms heterodimers with the
Arnt (Aryl hydrocarbon receptor nuclear translocator) pro-
tein. However, like the steroid receptors, the AHR shuttles
in and out of the nucleus, it forms persistent complexes with
hsp90, and ligand-induced nuclear accumulation of the
AHR is inhibited by geldanamycin [67,68].
The tumor suppressor protein p53 is a transcription factor
that can induce cell growth arrest, apoptosis, cell differen-
tiation and DNA repair in response to DNA strand breakage
and other types of cell stress (reviewed in Ref. [69]). P53
mutations occur in more than half of all human tumors and
inactivation of p53 is the most common alteration found in
human cancer. One mechanism of inactivation is exclusion
from the nucleus, and some p53 mutants retained in the
cytoplasm are in complex with hsp90 [70,71]. Using a
temperature-sensitive mutant of p53 where cytoplasmic–
nuclear movement occurs upon shift to permissive temper-
ature, we have shown that p53 movement is impeded by
radicicol [72].
Although almost all of the work to date has examined the
role of hsp90 in trafficking of signalling proteins that are
protein solutes in the cytoplasm, hsp90 interacts with the
cytoplasmic portions of a number of plasma membrane
receptors and ion channels. Recent evidence suggests that
hsp90 is involved in the trafficking as well as the turnover of
some of these membrane associated signalling proteins.
Thus, in addition to accelerating their proteasomal degrada-
tion, geldanamycin has been found to inhibit the intracellu-
lar trafficking of two receptor tyrosine kinases, epidermal
growth factor [73] and ErbB2 [74], and it inhibits the
maturation and targeting of two ion channels, the CFTR
chloride channel [75] and the hERG cardiac potassium
channel [76]. Radicicol treatment of brain slices inhibits
the constitutive trafficking of AMPA-type glutamate recep-
tors back into synapses during their continuous cycling
between synaptic and non-synaptic sites [77]. Interestingly,
the synaptic cycling of AMPA receptors was also impeded
by expression of the hsp90-binding TPR domain fragment
of PP5 but not by a TPR domain mutant that does not bind
hsp90, suggesting that the cycling may be mediated by an
hsp90-binding TPR domain protein. As another example,
hsp90 is required for signalling by the asubunit of the
heteromeric G
12
protein [78], and geldanamycin inhibits the
targeting of Ga
12
into lipid rafts, thus inhibiting its move-
ment into discrete membrane domains [79].
At this time, we are clearly at an early stage of under-
standing the role of hsp90 in signalling protein trafficking
through the cytoplasm to the nucleus, from the Golgi to the
plasma membrane, and signalling protein movement at the
inner surface of the plasma membrane. Whether hsp90 plays
a general role in the targeted movement of proteins or a
more specific role in the movement of a limited number of
signalling proteins is not known. What we propose is that
one function of the hsp90/hsp70-based chaperone machin-
ery in forming client proteinhsp90 complexes is to ‘cap-
ture’ proteins into multichaperone complexes that, through
the hsp90-bound immunophilins, can link them to motor
systems for their movement along cytoskeleton. An impor-
tant concept is that the chaperone machinery can interact
with proteins in their native, least energy state without
regard to a protein’s size, shape, amino acid sequence or
function [1]. This ability to interact with a wide variety of
client proteins combined with the diversity that arises from
the various TPR-domain proteins that associate with the
client protein and hsp90 may provide an integrated system
for targeted movement of proteins to diverse sites of action
within the cell.
3.2. The immunophilin role in movement
Steroid receptorhsp90 complexes were first formed in
vitro by incubating immunoadsorbed receptors stripped of
their associated chaperones with rabbit reticulocyte lysate.
In addition to containing the hsp90 heterocomplex assembly
machinery (hsp90, hsp70, Hop, hsp40, p23), reticulocyte
lysate contains immunophilins and cytoplasmic dynein.
GRhsp90 heterocomplexes prepared in this manner con-
tain TPR domain immunophilins and cytoplasmic dynein
[58]. The linkages shown in Fig. 2 are known because
addition of a TPR fragment of PP5 to reticulocyte lysate
yields GRhsp90 complexes without immunophilins or
dynein, and addition of an expressed PPIase domain frag-
ment of FKBP52 yields GRhsp90immunophilin com-
plexes without dynein [58]. The fragment of FKBP52
comprising the segment between the PPIase domain and
the first TPR (Fig. 1) has less homology with FKBP12
(28%) than the PPIase domain fragment (49%) [80], and
neither it nor FKBP12 compete for immunophilin binding to
dynein [57]. Also, the presence of FK506 has no effect on
the immunophilin association with dynein [57]. Immunoad-
sorption of immunophilins from brain cytosol showed that
FKBP52, CyP40 and PP5 were in native complexes with
dynein that could be disrupted by incubation with the
PPIase domain fragment of FKBP52 [56]. In contrast to
these three immunophilins (FKBP51 was not tested), ARA9
was not recovered in cytosolic complexes with dynein [56].
Thus, the PPIase domains of FKBP52, CyP40 and PP5
determine immunophilin binding in a heterocomplex with
cytoplasmic dynein. All PPIase or PPIase homology
domains do not bind dynein (e.g. FKBP12, ARA9), and
binding occurs even when isomerase activity is blocked
with FK506. It is unclear whether it is dynein itself or
perhaps a component of the dynein-associated dynactin
complex (e.g. dynamitin) that interacts with the PPIase
domain. We have identified a weak binding of purified
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872862
FKBP52, PP5 and a PPIase domain fragment with purified,
expressed mouse cytoplasmic dynein intermediate chain
[56]. But, it should be noted that similar weak interactions
occur with peptidyl prolines [81], and direct binding of
PPIase to dynein intermediate chain could be nonspecific in
this way. Cytoplasmic dynein is thought to link to vesicles
and organelles indirectly through dynactin (reviewed in Ref.
[51]), and immunophilin interaction with cytoplasmic dy-
nein could reflect such an indirect linkage.
Davies et al. [59] have developed an intriguing model
in which steroid binding to the LBD of the GR induces an
exchange of immunophilins and increased amounts of
dynein in the GRhsp90immunophilin complexes. Focus-
ing on just the hsp90-binding FKBPs, they note that prior
to introducing dexamethasone, cytosolic GRhsp90 com-
plexes contain primarily FKBP51, but after cytosol is
incubated with dexamethasone at 4 jC, the complexes
contain primarily FKBP52. This ligand-induced exchange
of FKBP51 for FKBP52 is accompanied by a threefold
increase in receptor-associated cytoplasmic dynein. The
same switching of FKBPs and increased dynein was seen
when receptors were exposed to dexamethasone in intact
cells at 4 jC, and this switching was accompanied by
receptor movement to the nucleus. Here again, we see the
notion of FKBP51 and FKBP52 having opposing yin and
yang actions, much as was seen in the data of Riggs et al.
[8] for the effect of these two immunophilins on steroid
binding affinity.
It is known that the NL1 nuclear localization signal is
occluded in the unliganded GR and becomes accessible to
NL1-specific antibody upon steroid-dependent transforma-
tion [82], and it is clear that the NL1 site is blocked or
conformationally altered by hsp90 and opened by its
removal [83]. Binding of steroid deep within the ligand
binding cleft promotes closing of the cleft with comcomi-
tant loss of the GR’s ability to form ‘persistent’ complexes
with hsp90. This change in hsp90 binding by the LBD
may open the positively charged NL1 as a potential
binding site for FKBP52, which possesses a short nega-
tively charged hinge segment that lies just C-terminal to its
PPIase domain. This negatively charged segment of
FKBP52 is electrostatically complementary to the NL1
region of the GR (reviewed in Ref. [84]), which is required
for direct binding of the FKBP52 to hsp90-free receptor
[7]. Opening up the NL1 upon steroid binding to the LBD
may thus favor the binding of FKBP52 over FKBP51 and
account for the ligand-induced change. Because the pres-
ence of dynein in the GRhsp90immunophilin complex
increases with the exchange for FKBP52, it is inherent to
the model that FKBP51 does not bind dynein or binds it
very poorly compared to FKBP52. Although this would
be predicted, the dynein binding status of FKBP51 is
unknown.
Like the PPIase domains themselves, the interactions of
hsp90-binding immunophilins with dynein are conserved. It
has been shown, for example, that the wheat TPR domain
immunophilins wFKBP73 and wFKBP77 bind through their
PPIase domains to mammalian cytoplasmic dynein [35].
Indeed, the entire assembly system is conserved, in that
immunopurified mouse GR incubated with wheat germ
lysate forms GRwheat hsp90wheat immunophilin com-
plexes that bind rabbit cytoplasmic dynein [35].This
suggests that that ability to form the ‘transportosome’
complex [49] shown in Fig. 2 is fundamental in the biology
of the eukaryotic cell.
Like FKBP52, the portion of PP5 that is cytoplasmic
colocalizes with microtubules, and in cells transfected with
a plasmid encoding the PPIase domain fragment of
FKBP52, the microtubular localization of PP5 is disrupted
[56]. This is consistent with the microtubular localization of
PP5 being determined by binding through its PPIase domain
to the dynein/dynactin complex. In cells where the PPIase
domain fragment is coexpressed with the GFP-GR, steroid-
dependent translocation of the receptor to the nucleus is
slowed in the same manner as it is when cells are treated
with geldanamycin (Fig. 3) [58]. The tumor suppressor p53
is in hsp90 heterocomplexes that contain the same immu-
nophilins as the GR, and expression of the PPIase domain
also inhibits translocation of a temperature-sensitive mutant
of p53 to the nucleus [72]. Coexpression of FKBP12, which
does not compete for immunophilin binding to dynein in
vitro, does not affect GFP-GR translocation in vivo. Thus,
like hsp90 itself, the hsp90-binding immunophilins are
required for rapid movement to the nucleus, consistent with
their role in linking the GRhsp90 complex to the dynein
motor for such retrograde movement (Fig. 2).
The immunophilin homolog PP5 is of particular interest
with regard to another role in movement relating to its
phosphatase domain. PP5 (reviewed in Ref. [85])isan
okadaic acid-sensitive protein-serine phosphatase that in its
basal state has a low activity because of autoinhibition by
its TPR domain. When the TPR domain is removed by
partial proteolysis, the phosphatase activity increases sev-
eralfold [86,87]. The interaction of PP5 through its TPR
domain with the TPR acceptor site on hsp90 also relieves
the autoinhibition, increasing its phosphatase activity [88].
PP5 is of special interest because an okadaic acid-sensitive
phosphatase activity is required for GR shuttling [47,89].
However, suppression of PP5 expression was found to
cause both nuclear accumulation of GFP-GR [90] and to
activate GR-dependent expression from a reporter in the
absence of hormone [91]. These studies led to the conclu-
sion that PP5 affects the cytoplasmic – nuclear shuttling of
the GR by suppressing nuclear accumulation [91]. This is
precisely the opposite of what would be predicted if PP5
were the target of the okadaic acid effect in cytoplasmic –
nuclear shuttling experiments [47,89]. Despite the current
confusion, given that PP5 is the only TPR domain, okadaic
acid-sensitive protein phosphatase found in GRhsp90
heterocomplexes, it is likely that its phosphatase activity
will be found to play an important role in receptor
translocation.
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872 863
3.3. Roles of other hsp90-binding TPR domain proteins in
movement
In addition to a role in targeting protein movement
through the cytoplasm by linking hsp90 client proteins to
dynein, it has been proposed that some hsp90-binding TPR
domain proteins may act at targeted organelles to accept
proteinhsp90 complexes [92]. For proteins with mitochon-
drial localization signals, the Tom70 component of the
mitochondrial import receptor may serve this purpose. The
association of the hsp90/hsp70-based chaperone machinery
with mitochondrial import started with the partial purifica-
tion of a large mass complex (200 –250 kDa) containing
hsp70 that maintained proteins to be imported into mito-
chondria in an import competent state [93]. This complex
was shown to contain hsp90 as well as hsp70 and to
assemble GRhsp90 heterocomplexes in which the receptor
had steroid binding affinity [94]. Indeed, this was the first
demonstration that hsp90 and hsp70 worked together in a
multi-chaperone assembly machinery. In a subsequent study
of TPR proteins binding to hsp90, it was shown that Tom70
(known then as Mas70p) bound to hsp90 in a manner that
was competed by Hop [92]. It was proposed at that time that
proteins being moved to the mitochondria might undergo a
‘‘hand off’’ from the movement system to Tom70. In a
recent definitive study, Young et al. [13] demonstrated that
hsp90 and hsp70 dock onto a Tom70 TPR domain at the
outer mitochondrial membrane to deliver a set of preproteins
through the membrane.
A similar system may have evolved for import of
peroxisomal proteins. A variety of proteins termed peroxins
(Pex) are required for peroxisome assembly and protein
import. Peroxisomal proteins destined for the peroxisomal
matrix are targeted to the organelle by peroxisomal targeting
signals (PTSs). Most peroxisomal matrix proteins are tar-
geted by PTS1 and their import is determined by Pex5p
[95], which may not only bind PTS1-containing proteins but
participate in their entry into the matrix and export into
cytosol [96]. Pex5p contains seven TPRs in the C-terminus,
and an intact TPR domain is necessary but not sufficient for
interactions with PTS1-containing proteins [97,98]. Both
hsp70 and ATP are involved in the binding of Pex5p to
PTS1 protein [99], and import of isocitrate lyase into
peroxisomes is inhibited by antibodies against wheat germ
hsp70 and Escherichia coli hsp90 [100]. Pex5p has also
been reported to coimmunoadsorb with hsp90 [101]. Pex5p
may be involved in moving chaperone-bound PTS1 proteins
to the peroxisome where it interacts with other peroxins to
effect their entry.
ARA9 (also called XAP2 and AIP) was isolated in yeast
two-hybrid screens for proteins interacting with the AHR
[102– 104]. ARA9 contains three TPRs in its C-terminus
and a PPIase homology domain (50% similarity and 27%
identity with human FKBP52 PPIase domain) without
PPIase activity in its N-terminus [102–104]. The TPR
domain binds hsp90 and the N-terminal region is essential
for ARA9 to regulate intracellular localization of the AHR
[105]. By immunofluorescence, the unliganded AHR is
located in both the cytoplasm and nucleus, but when
ARA9 is overexpressed, the AHR is redistributed to the
cytoplasm [67,106– 108]. This redistribution of AHR to the
cytoplasm is inhibited by geldanamycin [67], suggesting
that both hsp90 and ARA9 are required for anterograde
movement from the nucleus or for trapping of the AHR in
the cytoplasm. The ARA9-mediated cytoplasmic localiza-
tion of the AHR is inhibited by cytochalasin B, which
inhibits polymerization of actin filaments [109]. Thus, it is
currently thought that ARA9/XAP2/AIP anchors the ligand-
free AHR to actin filaments to maintain its cytoplasmic
localization [109]. Although ARA9/XAP2/AIP has not been
found in steroid receptorhsp90 heterocomplexes, it has
beenfoundincomplexwithPPARa, which is also a
member of the nuclear receptor superfamily [110].PPARa
mediates the carcinogenic effects of peroxisome prolifera-
tors in rodents and has been recovered from cytosols as a
PPARahsp90ARA9 complex [110].
3.4. Role of cytoplasmic dynein in movement
The tumor suppressor p53 was the first transcription
factor shown to be moved to the nucleus by cytoplasmic
dynein [111]. p53 was found to colocalize with micro-
tubules in several human carcinoma cell lines and to be in
cytosolic heterocomplexes with microtubules. Both over-
expression of dynamitin and microinjection of anti-dynein
antibody before DNA damage abrogated subsequent nuclear
accumulation of p53 [111]. Dynamitin is a 50-kDa subunit
of the dynein-associated dynactin complex, and its over-
expression blocks dynein function by dissociating the motor
protein from its cargoes [51,112]. Inhibition of movement
through expression of dynamitin is powerful evidence that
movement occurs through attachment to cytoplasmic dy-
nein. Microtubule-perturbing drugs also inhibit nuclear
accumulation of p53 [112], consistent with dynein-depen-
dent movement along microtubular tracks.
Although the linkage between p53 and dynein was not
worked out when these experiments were performed, it is
now clear that p53hsp90 complexes are linked to cyto-
plasmic dynein by immunophilins in the same manner as
shown for the GR in Fig. 2 [72].InDLD-1human
colorectal adenocarcinoma cells, which contain a mutant
p53 that is located in the cytoplasm in complex with hsp90,
both the GR and p53 are in complexes with the same
hsp90-binding immunophilins, which are present in the
similar relative amounts in each complex. Thus, it is not
surprising that p53 movement to the nucleus in human
colorectal carcinoma cells expressing a mouse p53 temper-
ature-sensitive mutant is inhibited by geldanamycin treat-
ment and by expression of a PPIase domain fragment in the
same manner as the GR [72].
In contrast to p53, the hormone-free GR has usually been
found to be diffusely located throughout the cytoplasm by
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872864
immunofluorescence, although there are some reports of
colocalization with microtubules (reviewed in Ref. [113]).
Like p53, however, GRhsp90immunophilindynein het-
erocomplexes immunoadsorbed from cytosol of taxol-trea-
ted cells contain tubulin, and GR translocation to the nucleus
is inhibited by coexpression of dynamitin (JM Harrell, work
in progress). Now that techniques for implicating dynein in
movement have been worked out, it is likely that other
transcription factors will be found to move rapidly through
the cytoplasm in a dynein-dependent manner.
4. Transport across the nuclear pores
Once they arrive at the nuclear membrane, signalling
proteins undergo a facilitated diffusion through nuclear
pores, which allow the selective inward and outward passage
of transport receptors called importins and exportins
(reviewed in Refs. [114–116]). In the case of proteins with
a classical nuclear localization signal (NLS), such as SV40 T
antigen, nucleoplasmin and steroid receptors, two importin
proteins are involved in nuclear entry. Importin-ais the NLS
receptor, and it also binds importin-h, which is the unit of the
complex that interacts with motifs in the nucleoporins to
facilitate passage through the pore. Passage is very rapid, and
it is unidirectional because of cycling of Ran. Ran is a Ras-
related small GTPase that switches between a GDP- and a
GTP-bound state. A nucleotide exchange factor in the
nucleus generates RanGTP, and a GTPase-activating protein
(GAP) that is excluded from the nucleus converts RanGTP to
RanGDP at the cytoplasmic face of the nuclear pore. After
the cargo-importin complex traverses the pore, RanGTP
binds to importin and the cargo is released. The RanGTP
importin-hcomplex passes back through the pore without
cargo to the cytoplasmic face where RanGAP converts
RanGTP to RanGDP, releasing free importin to participate
in another cycle of cargo entry. To shuttle in and out of the
nucleus, signalling proteins that are transcription factors
(e.g., steroid receptors, AHR, p53) possess nuclear export
signals (NES) that determine movement in the reverse
direction through the pores to the cytoplasm. NES proteins
bind exportins, and passage is unidirectional in the same
Ran-regulated manner. CRM1 is an exportin for NES pro-
teins that is inhibited by the drug leptomycin B, which has
been a useful tool for studying signalling protein movement.
Unlike the passage of proteins into mitochondria and
other organelles where proteins must be unfolded to pass,
proteins pass through the nuclear pores intact, and the pores
are large enough to permit passage of multimolecular
complexes up to 1 –3 10
6
Da [114]. Given the dynamic
nature of the client proteinhsp90 heterocomplex assembly/
disassembly cycle, it is possible that heterocomplexes could
be disassembled before passage and reassembled on the
nuclear side of the pore. It is clear, however, that the GR can
pass both into [117] and out of [118] the nucleus as a
GRhsp90 heterocomplex; thus, there is no requirement for
heterocomplex disassembly prior to pore passage. At this
time, it seems likely that the dominant mode of passage
through nuclear pores is as receptorhsp90immunophilin
heterocomplexes (see discussion in Ref. [118]). Indeed,
because neither hsp90 nor the hsp90-binding immunophilins
have NLSs of their own, it seems likely that their presence
in the nucleus must be due to the fact that they are carried in
by multiple NLS-containing client proteins.
The factors that determine the cytoplasmic versus nuclear
localization of ligand-free steroid receptors are not known.
Some hormone-free receptors (e.g. glucocorticoid and min-
eralocorticoid receptors) are predominantly localized to the
cytoplasm while others (e.g. estrogen and progesterone
receptors) are located in the nucleus. This difference in
localization exists despite the fact that all of the receptors
are shuttling in and out of the nucleus and all are in
receptorhsp90immunophilin complexes [29,45].One
contribution to the localization might be that the NLS in
the case of the receptors that are in the cytoplasm is
repressed in the unliganded-receptorhsp90immunophilin
complex whereas the NLS is accessible in unliganded-
receptor complexes that are nuclear in localization. In that
case, the localization should be entirely receptor-specific,
but that is not the case. The unliganded mouse GR in mouse
L cells, for example, is cytoplasmic, whereas the mouse
receptor expressed in Chinese hamster ovary cells is nuclear,
and in both cases it is present as GRhsp90 heterocom-
plexes and the receptors react equivalently with antibody
directed against the NL1 [119]. Preferential accumulation of
steroid receptors in cytoplasm or nucleus would result if one
direction of passage through the nuclear pore were favored
over the other (see Ref. [48] for discussion), but differences
in receptor localization due to factors affecting the intrinsic
rate of receptor passage through the nuclear pore in either
direction have not been established. One can of course
change localization by deletion or addition of an NLS or
NES, but the factors determining the localization of the
ligand-free wild-type receptors remain unclear.
The involvement of importins in receptor entry into the
nucleus is buttressed by several observations. For example,
the unliganded GRhsp90 complex has been shown to bind
importin-ain vitro, with the NL1 being critical for binding
[46], and fluorescence resonance energy transfer data indi-
cate that the GR interacts directly with importin-ain vivo
[120]. The importance of Ran in GR import is supported by
the observation that expression of some Ran mutants mark-
edly reduces GFP-GR accumulation in the nucleus [121].
The AHR has an NLS in its N-terminus that binds importin-
a, which targets the receptor to the nuclear rim [122].
Binding of the AHR to importin-ais ligand-dependent and
inhibited by geldanamycin, implying a role for hsp90 [68].
One study focused on binding of AHR to importin-h, and
coexpression of ARA9 was found to decrease recovery of
importin-hwith the receptor [123], an effect that could
account for redistribution of the AHR to the cytoplasm upon
ARA9 expression [67,106 – 108,123]. The steroid and xeno-
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872 865
biotic receptor (SXR) is an orphan nuclear receptor that
activates the expression of some drug metabolizing enzymes
(e.g., CYP3A4) and the drug efflux transporter ABCB1.
Import of GFP-SXR was promoted by addition of importin-a
in an in vitro nuclear transport assay [124].
In contrast to the consistent data for a role for importins
in receptor import into nuclei, conflicting data have been
published regarding a requirement for CRM1 in nuclear
export. GR export from nuclei of Cos7 cells was inhibited
by leptomycin B [46], and leptomycin B promoted nuclear
accumulation of the progesterone B receptor in T47D
human breast cancer cells [125], implying a role for
CRM1 in export of these receptors. Leptomycin B induces
nuclear accumulation of AHR, and immunoprecipitation of
the AHR is accompanied by coimmunoprecipitation of
CRM1 [109]. On the other hand, nuclear export of the GR
in BHK cells was found to be insensitive to leptomycin B
and to be regulated by calreticulin instead of CRM1 [126].
Calreticulin is normally considered to be a chaperone
protein of the endoplasmic reticulum, but in this case it
mediated Ran-dependent GR nuclear export, with the DNA
binding domain of the receptor functioning as an NES
[126]. Liu and DeFranco [127] found that slow nuclear
export of wild-type GR following hormone withdrawal in
Cos1 cells was not inhibited by leptomycin B, but rapid
export of a GR chimera containing the NES from InB
proceeds through the CRM1 pathway. Interestingly, a lep-
tomycin B-insensitive NES has been identified in the ligand
binding domain of the androgen receptor [128].This
CRM1-independent NES is active in the absence of andro-
gen and repressed upon ligand-binding, leading the authors
to suggest this provides a mechanism by which androgen
regulates the nuclear-cytoplasmic shuttling of its receptor
[128]. Lee and Bai [129] showed that mutation of a
phosphorylated threonine in the NES of estrogen receptor
ato alanine converts the receptor from leptomycin B-
insensitivity to leptomycin B-sensitivity. Thus, it is conceiv-
able that nuclear receptors have the capacity to utilize
multiple distinct pathways for nuclear export. This could
generate an additional level of control for receptor traffick-
ing as each distinct nuclear export pathway could be
subjected to unique regulatory influences.
In addition to importins and exportins, the chaperone
hsc70/hsp70 has been reported to play a role in passage of
some classical NLS proteins (SV40 T antigen, nucleoplas-
min) through nuclear pores [130,131]. It has been suggested
that hsp70 is somehow involved in NLS recognition [131],
but no clear and consistent role for hsp70 has been defined.
Yang and DeFranco [132] directly compared the nuclear
import of SV40 T antigen and the GR in permeabilized
cells. They demonstrated that depletion of hsp70 did not
affect GR import under the same conditions where SV40 T
antigen import was inhibited. If hsp70 acts to facilitate NLS
interactions with transport receptors, its role in nuclear
import may be restricted to substrates whose NLSs are
relatively inaccessible or not configured appropriately.
Many signalling responses cause the nuclear localization
of transcription factors or of protein kinases that are trans-
located and then activate transcription factors located in the
nucleus. Here, we have touched on factors regulating
nuclear localization only of hsp90 client proteins. The
factors regulating nuclear localization of several families
of signalling proteins that are not client proteins for hsp90
have been reviewed by Cyr [133].
5. Movement within the nucleus
The early studies of steroid receptor localization in nuclei
by indirect immunofluorescence showed them to be dis-
persed throughout the nucleus and to be excluded from
nucleoli. Subsequent examination of endogenous glucocor-
ticoid [134,135] and mineralocorticoid [136] receptors by
confocal laser scanning microscopy revealed that they were
present in multiple discrete foci located throughout the
nonnucleolar space. The development of receptor fusions
with jellyfish fluorescent proteins permitted examination of
receptor localization and movement in nuclei of living cells.
Within a 4-year period, multiple papers were published
showing that glucocorticoid [137,138], mineralocorticoid
[138,139], progesterone [140],estrogen[141],androgen
[142,143], thyroid hormone [144], vitamin D [145] and
retinoid X [145] receptor fusion proteins accumulated in
punctuate foci throughout the nucleus excluding nucleoli
(reviewed in Ref. [146]).
In several of the receptor fusion protein papers, it is noted
that in the absence of ligand, the localization of nuclear
fluorescence is less defined and formation of the discrete
foci is agonist-dependent [137,139– 141,143,145].Thus,
there is the impression that receptors that have passed
through the nuclear pores move to ‘staging areas’ from
which they can proceed to discrete foci if they have
undergone ligand-dependent transformation [146]. There is
some evidence that the cycling of receptor into recep-
torhsp90 complexes may be involved in the ligand-depen-
dent movement of receptor to the discrete nuclear foci where
chromatin binding is thought to occur. Georget et al. [65]
constructed a GFP-NLS-AR, which contained three nuclear
localization signals between GFP and the AR and was
localized diffusely in the nucleus in the absence of hormone.
When these receptors were bound with steroid at 4 jC and
then the temperature was jumped to 37 jC in the presence of
geldanamycin, the ligand-dependent formation of discrete
nuclear foci was inhibited [65].
Virtually nothing is known about how molecules move
within the nucleus. Protein mobility within the nucleus can
be diffusion-limited [183], but this may apply to a limited
fraction of resident nuclear proteins. Furthermore, interac-
tions of nuclear proteins with either soluble or solid state
partners clearly retard their mobility [183].Asnuclear
protein function is often directed to specific subnuclear
compartments, a movement system may exist to facilitate
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872866
such targeted delivery. For example, movement of proteins
from nucleoli along tracks through the nucleus to the
nuclear pores has been demonstrated [147], but the nature
of the filaments (nuclear actin?) and potential motor proteins
(nuclear myosin?) is unknown. However, it is possible that
movement of steroid receptors within the nucleus may occur
on a movement system with some of the features of
movement through the cytoplasm as depicted in Fig. 2.It
should be emphasized again here that unliganded receptors
that are nuclear are recovered in the cytosol after cell rupture
where they are present as receptorhsp90immunophilin
heterocomplexes [29]. Indeed, hsp90-binding immunophi-
lins may be involved in targeting movement within the
nucleus. It has been shown, for example, that the nuclear
FKBP52 in Chinese hamster ovary cells expressing mouse
GR is located in the same nonrandom loci throughout the
nucleus as the hormone-free GR [54]. Such a localization is
consistent with the notion that FKBP52 might target recep-
tor movement, at least to the proposed ‘staging areas’ within
the nucleus.
5.1. Hsp90 and receptor cycling within the nucleus
In 1976, Munk and Foley [148] observed that steroid
dissociated from nuclear GRs upon hormone withdrawal
much faster than receptors returned to the cytoplasm and
that these nuclear GRs could rebind hormone. Given the
subsequent demonstration that GRhsp90 complex is nec-
essary for hormone binding [29], this suggested that the
receptor could recycle to the GRhsp90 complex within the
nucleus. In several important experiments, the DeFranco
laboratory has demonstrated such a trafficking cycle within
the nucleus (reviewed in Ref. [48,149 – 152]]). Using digi-
tonin-permeabilized cells to examine in vitro nuclear export
of the GR, they showed that GR released from chromatin
could recycle to chromatin upon rebinding hormone without
exiting the nucleus [153]. Geldanamycin inhibits recycling
of these hormone-withdrawn GRs to the hormone binding
state, and it inhibits GR release from chromatin during
hormone withdrawal [154]. This is consistent with a role
for the hsp90/hsp70-based chaperone machinery in the
termination of transcriptional activation as free hormone
levels decline. Furthermore, in recent experiments using a
novel in situ system to assess GR nuclear mobility, receptor
movement within the nucleus was reconstituted with the
purified hsp90/hsp70-based chaperone machinery [180].
Thus, as illustrated in Fig. 4, molecular chaperones may
participate in various stages of GR trafficking within the
nucleus and facilitate both receptor exchange from specific
binding sites within chromatin and overall mobility as the
receptor navigates the nuclear space in search of high
affinity target sites.
Fig. 4. Model for the recycling of GRs in the nucleus by the hsp90/hsp70-based chaperone machinery. After dissociation of hormone (H), the GRs are released
from high-affinity chromatin binding sites by the chaperone machinery. The chaperone machinery is depicted here as acting in two ATP-dependent steps, the
first involving hsp70 and the second hsp90, as discussed by Pratt and Toft [1]. This is the sequence of events determined for GRhsp90 heterocomplex
assembly by reticulocyte lysate and by the purified chaperone machinery. Although it has not been demonstrated that hsp70/hsp40 is the first component of the
chaperone machinery to interact with chromatin-bound GR, it is likely that the same mechanism applies. It is also not known whether GR dimer to monomer
conversion occurs while the receptor is bound to chromatin or while it is assembling into a complex with hsp90, but the stoichiometry of the final
heterocomplex is one molecule of GR bound to a dimer of hsp90 [29]. The nonessential cochaperone Hop, which brings the chaperones together into an
hsp90Hophsp70hsp40 complex, has been omitted to simplify presentation. When the GR-bound hsp90 achieves the ATP-dependent conformation, the GR
has returned to its hormone binding state and p23 can bind to hsp90 to stabilize the complex. Nuclear GR that has been recycled into GRhsp90p23
complexes can bind hormone without exiting the nucleus and be recycled to the chromatin-bound state.
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872 867
5.2. Effect of p23 on transcription complex disassembly
The p23 component of the heterocomplex assembly
system is not essential for receptorhsp90 heterocomplex
assembly in vitro [155] or in vivo [156,157], but it has
become an important tool for examining the role of hsp90 in
transcription complex disassembly and receptor recycling in
the nucleus. In studies in vitro, two mechanisms of p23
action have been demonstrated. In GRhsp90 assembly
experiments, p23 has been shown to act as an hsp90
cochaperone and bind to GRhsp90 complexes once they
are formed, stabilizing them to disassembly [158]. p23 also
has a direct chaperone action of its own in that it inhibits
aggregation of denatured proteins, maintaining them in a
folding-competent state [159,160]. Both of these mecha-
nisms have been invoked to explain in vivo effects of p23
on nuclear receptor cycling.
The first in vivo observations were made by Knoblauch
and Garabedian [161] who performed a screen in yeast to
identify factors that would improve function of a mutant
estrogen receptor with decreased hormone binding capacity.
The yeast homolog of p23 (yhp23) was isolated, and its
overexpression was shown to increase estrogen receptor-
dependent transcriptional activation by increasing estradiol
binding in vivo. When the estrogen receptor and GFP-yhp23
were coexpressed, the GFP-yhp23 moved with the receptor
to the nucleus and was released back into the cytoplasm
when yeast were treated with estradiol. Expression of
human p23 in MCF-7 cells also increased transcriptional
activation by the estrogen receptor [161]. These observa-
tions were interpreted in terms of p23 acting as an hsp90
cochaperone to support a role for the hsp90/hsp70-based
chaperone machinery in estrogen receptor signal transduc-
tion [161].
Freeman et al. [162] examined the effect of p23 on
several intracellular receptors in yeast and found that
glucocorticoid and progesterone receptor-dependent tran-
scription was increased, whereas transcription from miner-
alocorticoid, estrogen, androgen, thyroid hormone, and
retinoic acid receptors was decreased. It was determined
that p23 interacted preferentially with thyroid hormone-
receptor-response element ternary complexes in vitro to
stimulate receptor dissociation from DNA. p23 appeared
to compete with the coactivator GRIP1 in that a fragment of
GRIP1 inhibited p23-dependent dissociation of thyroid
hormone receptor from DNA. The interpretation here was
that p23 effects are different from those of hsp90, with p23
directly interacting with the receptor at a late step in
receptor-mediated signal transduction [162]. Freeman and
Yamamoto [163] targeted p23 by fusion to the Gal4 DNA
binding domain to localize in vivo at GAL4 binding sites
neighboring thyroid hormone or glucocorticoid response
elements in a reporter. Expression of Gal4-p23 reduced
thyroid hormone receptor-dependent transcriptional activa-
tion by 100-fold and GR-dependent activation by 35-fold.
In contrast, Gal4-hsp90 produced only a slight inhibition
(twofold). Less extensive p23 inhibition was observed with
the non-receptor transcription factors NF-nBandAP1
expressing from similar linked promoters after their activa-
tion by tumor necrosis factor aand phorbol myristate
acetate, respectively. Importantly, it was shown in chromatin
immunoprecipitation assays that both p23 and hsp90 local-
ized to glucocorticoid response elements in a hormone-
dependent manner. In experiments in vitro, p23 inhibited
transcriptional activation by preformed regulatory com-
plexes, consistent with a role of p23 in promoting disas-
sembly of the complexes [163].
Because transcriptional inhibition by forced localization
of p23 to a hormone response element abrogated receptor-
induced activation and hsp90 inhibited activation less, the
notion that p23 causes disassembly of transcriptional regu-
latory complexes via a direct chaperone effect has been
seriously considered [163,164]. However, as the authors
note, it was not determined whether disassembly of intact
complexes requires energy [163]. Although it was demon-
strated under very different conditions, GR release from
nuclear matrix has been found to be ATP-dependent [165],
and it is likely that p23 is exerting its action through hsp90.
It has been shown that p23 is the limiting component of the
multiprotein hsp90/hsp70-based chaperone system in vivo,
and that it acts in vivo to stabilize client proteinhsp90
complexes [166]. The Garabedian laboratory has shown that
the ability of yeast p23 mutants to increase or decrease
estrogen receptor-dependent signal transduction correlates
with their ability to bind hsp90 [167]. Taken together, these
observations argue rather strongly that p23 effects in vivo
do not reflect a direct chaperoning interaction with the client
protein of hsp90. Rather, they reflect a direct interaction
with hsp90 as a cochaperone to stabilize its association with
the client protein, thus increasing the efficiency of the
hsp90/hsp70-based chaperone machinery [166].
5.3. Rapid exchange of receptors with regulatory sites
The availability of GFP-receptor fusion proteins has
permitted the study of real-time movement of receptors in
subregions of the nucleus using photobleaching techniques,
such as fluorescence recovery after photobleaching (FRAP).
The observations have led to the realization that a number of
transcription factors are in highly dynamic interactions with
their regulatory sites (reviewed in Ref. [168]). The Hager
laboratory examined the binding of GFP-GR to an artificial
amplified array of mouse mammary tumor virus reporter
elements on chromosome 4 in a mouse cell line [169]. This
array includes 800 to 1200 binding sites for the GR, and it
displays as a single patch of bright fluorescence in cells
expressing GFP-GR treated with dexamethasone. Photo-
bleaching experiments showed that the hormone-bound
GR exchanges rapidly with the chromosomal regulatory
sites with a half maximal time for fluorescence recovery
of f5s[169]. The work of Hager and his colleagues is
consistent with a dynamic ‘‘hit and run’’ model in which the
W.B. Pratt et al. / Cellular Signalling 16 (2004) 857–872868
ligand-activated GR binds to chromatin, recruits a remodel-
ing activity, facilitates transcription factor binding and is
then lost from the template [169 – 171]. The GR coactivator
GRIP-1 undergoes the same rapid exchange as the GR
[172].
Stenoien et al. [173] examined the nuclear localization of
bioluminescent derivatives of estrogen receptor aand
steroid receptor coactivator 1 (SRC-1). Estradiol caused
the receptor to change from a diffuse nucleoplasmic pattern
to localize in discrete foci and the coactivator colocalized to
the same foci in an estradiol-dependent manner. Subsequent
photobleaching experiments [174] revealed a particularly
high mobility for the unliganded estrogen receptor chimera
in the nucleus (fluorescence recovery t
1/2
< 1 s) whereas the
mobility of the hormone-bound receptor was somewhat
slower (t
1/2
f5– 6 s), with SRC-1 showing a similar mo-
bility. Thus, the agonist-bound estrogen receptor procedes to
intranuclear foci where it is matrix-bound, but the receptors
within the foci undergo rapid exchange with the nucleo-
plasm. Under conditions of ATP depletion, the unliganded
ER was immobilized, whereas there was little effect on
receptor mobility in estradiol-treated cells [174].The
authors note that the ATP dependency of the mobility of
the unliganded receptor could reflect ATP-dependent hsp90
heterocomplex assembly.
At this time, the mechanism of signalling protein move-
ment within the nucleus is unknown. Strong arguments have
been advanced that protein movement within the nucleus is
diffusional and that stochastic mechanisms are involved in
the regulation of gene expression [175,176]. While there
may be some subset of nuclear proteins whose mobility is
strictly limited by diffusion, it seems likely that the suc-
cessful orchestration of complex regulated biochemical
pathways in the nucleus would require some mechanism
for direct delivery of transcription factors and assembly of
regulatory complexes. Here, we have summarized evidence
that the hsp90/hsp70-based assembly machinery may be
involved in movement of steroid receptors to discrete foci
within the nucleus where transcriptional regulation is
thought to occur as well as in disassembly of the regulatory
complexes. However, other chaperones and cochaperones as
well as a potential karyoskeleton may be involved in this
process in addition to the components of the hsp90/hsp70-
based assembly machinery. The demonstration of the high
mobility of receptors in the nucleus and the effects of
geldanamycin and p23 on receptor release from chromatin
have stimulated a number of perspective and opinion pieces
[150,152,164,168,177 – 179] that attest to the broad interest
in this topic.
6. Summary
Inherent to an understanding of cellular signalling is the
ultimate understanding of how signalling proteins travel to
their sites of action in various cell compartments. Initially,
interest focused on the nature of the signals (e.g. NLSs,
NESs, MLSs) that target the movement of protein solutes to
specific cellular compartments. Recently, several laborato-
ries in the signal transduction field have begun to focus on
the mechanisms of signalling protein movement within the
cytoplasm and nucleus. Although there is considerable
evidence that the dynamic assembly of heterocomplexes
with hsp90 is involved in the movement of transcription
factors that are hsp90 client proteins within the cytoplasm
and within the nucleus, the notion that hsp90 is involved in
signalling protein movement is at an early stage of devel-
opment. We do not yet know whether dynamic hsp90
heterocomplex assembly is involved in the movement of
just a few transcription factors, or whether the hsp90/hsp70-
based chaperone machinery is involved in the long-range
movement and local mobility of a wide range of signalling
protein solutes, including some of the many protein kinases
whose activity and/or turnover are regulated by hsp90 [1].
Acknowledgements
Preparation of this review and the authors’ work reported
herein were supported by the National Institutes of Health
Grants CA28010 and DK31573 to W.B.P. and CA43037 to
D.B.D. The authors would like to thank Ed Sanchez for his
helpful comments on the manuscript.
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