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J Biol Chem, Vol. 274, Issue 52, 36980-36986, December 24, 1999
From the FKBP52 is a high molecular mass immunophilin
possessing peptidylprolyl isomerase (PPIase) activity that is inhibited
by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor·hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common
heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR
domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate
also yields co-immunoadsorption of cytoplasmic dynein, and we show that
co-immunoadsorption of dynein is competed by a fragment of FKBP52
containing its PPIase domain, but not by a TPR domain fragment that
blocks FKBP52 binding to hsp90. Using purified proteins, we also show
that FKBP52 binds directly to the hsp90-free glucocorticoid receptor.
Because neither the PPIase fragment nor the TPR fragment affects the
binding of FKBP52 to the glucocorticoid receptor under conditions in
which they block FKBP52 binding to dynein or hsp90, respectively,
different regions of FKBP52 must determine its association with these
three proteins.
More than a dozen transcription factors and a dozen protein
kinases are known to exist in heterocomplexes with the abundant, ubiquitous, and essential protein chaperone
hsp90 1 (for review, see
Refs. 1 and 2). In addition to hsp90, native steroid receptor
heterocomplexes contain one of several proteins that possess
tetratricopeptide repeats (TPRs), which are degenerative sequences of
34 amino acids involved in protein-protein interactions (3). The TPR
proteins in steroid receptor·hsp90 heterocomplexes include high
molecular mass immunophilins such as FKBP52 (4-7), FKBP51 (8-10), and
CyP-40 (11-13) as well as the protein serine/threonine phosphatase PP5
(14, 15). The TPR domains of these proteins are required for their
binding to hsp90 (14, 16-19), and the TPR proteins and protein
fragments containing only the TPR domains compete with each other for
binding (12, 15, 18, 20) to a TPR domain acceptor site (20) that is
located in the C-terminal 12-kDa domain of hsp90 (21). The
immunophilins have been shown to exist in independent
receptor·hsp90·FKBP52 and receptor·hsp90·CyP-40 heterocomplexes
(12, 13), and inasmuch as the TPR proteins compete for the binding of
each other, these complexes must be very dynamic in the sense that a
single receptor·hsp90 heterocomplex could be associated sequentially
with several different TPR proteins over a short time.
The aryl hydrocarbon receptor (AHR) is recovered from the cytosol in
AHR·hsp90 heterocomplexes that contain a 37-kDa FKBP homolog with
three TPRs that has been called ARA9, AIP, or XAP2 (22-24).
ARA9/AIP/XAP2 is specifically associated with AHR·hsp90 heterocomplexes and not glucocorticoid receptor (GR)·hsp90
heterocomplexes, whereas the reverse is the case for FKBP52 (25). Thus,
there are differences in heterocomplex composition between AHR·hsp90 heterocomplexes and steroid receptor·hsp90 heterocomplexes, and there
may well be differences in the relative amount of one immunophilin versus another recovered in hsp90 heterocomplexes with
different steroid receptors (19).
In contrast to the nuclear receptors, the dominant protein recovered in
protein kinase·hsp90 heterocomplexes is a 50-kDa phosphoprotein originally identified as a component of the
pp60v-src·hsp90 heterocomplex (for review, see
Refs. 26 and 27). This 50-kDa protein has been cloned and identified as
the vertebrate homolog of the yeast cell cycle control protein Cdc37
(28-30), and it is called p50cdc37. Genetic evidence suggests
that this protein is required for Src function (31) and for signaling
via the sevenless receptor, a protein-tyrosine kinase of
Drosophila (32). p50cdc37 does not contain TPRs, and
it binds to a site on hsp90 that is different from the TPR acceptor
site, but the two sites must be topologically adjacent to each other
because TPR proteins compete for the binding of p50cdc37 to
hsp90 and p50cdc37 is not recovered in the same hsp90
heterocomplexes as the TPR proteins (33). Both the
cyclin-dependent protein kinase Cdk4 and the
serine/threonine kinase v-Raf exist in heterocomplexes containing hsp90
and p50cdc37, and p50cdc37 has been shown to bind
directly to both Cdk4 (29) and Raf (33) as well as to hsp90. The
combination of exclusive binding of p50cdc37 versus
a TPR domain protein to hsp90 plus direct binding of p50cdc37
to the kinase appears to account for selection of the dominant hsp90·p50cdc37 composition that is observed with a variety of
protein kinase heterocomplexes immunoadsorbed from the cytosol
(33).
Although native steroid receptor·hsp90 heterocomplexes contain one of
the TPR domain proteins, they do not contain p50cdc37 (34, 35);
however, no direct binding of TPR protein to a steroid receptor has
been detected that would account for selection of an immunophilin
versus p50cdc37 as a partner in the heterocomplex.
Cross-linking studies of steroid receptor·hsp90 heterocomplexes have
revealed a common heterotetrameric structure containing 1 molecule of
receptor, 2 molecules of hsp90, and 1 molecule of immunophilin
(36-38). However, it is not known whether there are two TPR acceptor
sites/hsp90 dimer and one is blocked when the receptor is bound or
whether there is one TPR acceptor site/hsp90 dimer irrespective of the
presence of the chaperoned protein. In this paper, we examine three
protein-protein interactions of the abundant immunophilin FKBP52. By
cross-linking of complexes formed with purified proteins, we show that
there is one FKBP52-binding site/dimer of hsp90. We also demonstrate direct binding of FKBP52 to the GR and provide evidence for a contact
site residing between amino acids 465 and 500 of the human GR (hGR). We
have previously reported that cytoplasmic FKBP52 is localized to
microtubules (39, 40) and that immunoadsorption of FKBP52 from the
cytosol is accompanied by co-immunoadsorption of cytoplasmic dynein
(39). Here we show that co-immunoadsorption of dynein is blocked by a
bacterially expressed fragment of FKBP52 that contains its
peptidylprolyl isomerase (PPIase) domain, suggesting that this
conserved region of FKBP may be involved directly or indirectly in the
association with dynein.
The role of the immunophilins and p50cdc37 in the actions of
nuclear receptors and signaling protein kinases is unknown. The high molecular mass TPR domain immunophilins were discovered because they
were components of steroid receptor·hsp90 heterocomplexes, and it was
originally thought that their PPIase activity might be required for the
proper folding of the receptors and assembly of receptor·hsp90
heterocomplexes. However, it became clear that this is not their role
(see Ref. 1 for review). Another proposal is that the immunophilins and
p50cdc37 serve to target the movement of the receptors and
protein kinases in the appropriate anterograde or retrograde direction
to their sites of action in the nucleus and at the plasma membrane (20, 41). The ability of FKBP52 to interact directly with the GR and either
directly or indirectly with the microtubule-associated motor protein
dynein is consistent with the immunophilin performing such a role in
targeting of receptor movement.
Materials
Untreated rabbit reticulocyte lysate was from Green Hectares
(Oregon, WI). 125I-Conjugated goat anti-mouse and
anti-rabbit IgGs were from NEN Life Science Products. Horseradish
peroxidase-conjugated goat anti-mouse IgG, monoclonal nonimmune IgG and
IgM, preimmune rabbit serum, monoclonal anti-glutathione
S-transferase (GST) clone GST-2 ascites,
glutathione-cross-linked agarose, purified glutathione S-transferase, mouse monoclonal IgM (clone 70.1) against the
intermediate chain of dynein, and purified recombinant FKBP12 were from
Sigma. Actigel-ALD was from Sterogene Bioseparations Inc. (San Gabriel, CA), and Complete-Mini protease inhibitor mixture was from Roche Molecular Biochemicals. Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Pierce, and FK506 was from Alexis Biochemicals (San Diego, CA). Anti-cyclophilin 40 antibody (COOH-terminal peptide) and 3G3 anti-hsp90 monoclonal IgM were from Affinity Bioreagents (Golden, CO), and anti-FLAG M2 monoclonal IgG was from Kodak Scientific Imaging Systems. The UPJ56 antiserum against FKBP52 (42) was a gift
from Dr. Karen Leach (The Upjohn Co.), and the EC1 anti-FKBP52 monoclonal antibody was a gift from Dr. Lee Faber (Medical College of
Ohio, Toledo, OH). The rabbit antiserum against hsp70 and hsp90 (43)
was provided by Dr. Ettore Appella (NCI, National Institutes of
Health). Rabbit antiserum against PP5, FLAG-PP5, and the FLAG-tagged TPR domain of rat PP5, prepared as described previously (14), were
kindly provided by Dr. Michael Chinkers (University of South Alabama,
Mobile, AL). FLAG-p50cdc37 and antiserum to p50cdc37,
prepared as described previously (33), were a kind gift from Dr.
Nicholas Grammatikakis (Tufts University School of Medicine, Boston,
MA). The DS14F5 monoclonal antibody against Hop (hsp
organizer protein) (44) was kindly provided by
Dr. David Smith (University of Nebraska, Omaha, NE). The baculoviruses
expressing GST-hGR (45) and the two C-terminal truncations of the hGR,
GST-465* and GST-500* (45), were generously provided by Drs. Ganesan Srinivasan and E. Brad Thompson (University of Texas Medical Branch, Galveston, TX). The pGEX-2T plasmids encoding GST-rabbit FKBP52 and the
GST-rabbit FKBP52 PPIase domain (amino acids 19-262) were prepared as
described previously (46). The pGEX1 Methods
Cell Culture and Cytosol Preparation--
Sf9 cells were
harvested, washed once, suspended in 1 volume of HE buffer (10 mM Hepes, pH 7.4, and 1 mM EDTA) with 1 tablet of Complete-Mini protease inhibitor mixture/3 ml of buffer, and ruptured by Dounce homogenization. Homogenates were centrifuged for 15 min at 12,000 × g.
Immunoadsorption--
For immunoadsorption of immunophilins,
FLAG-PP5, or FLAG-p50cdc37, aliquots (150 µl) of rabbit
reticulocyte lysate were immunoadsorbed for 2 h at 4 °C to 10 µl of protein A-agarose prebound with the UPJ56 antiserum against
FKBP52 (2%), anti-CyP-40 (2%), or 6 µg of anti-FLAG M2 monoclonal
antibody. Immune pellets were washed three times by suspension in 1 ml
of TEG buffer (10 mM TES, pH 7.6, 50 mM NaCl, 4 mM EDTA, 10% (w/v) glycerol) with 20 mM sodium molybdate and centrifugation prior to gel electrophoresis.
For immunoadsorption of hsp90 and its associated proteins from rabbit
reticulocyte lysate, lysate was diluted 10-fold with HEG buffer (HE
buffer + 10% glycerol), and 100-µl aliquots were immunoadsorbed to
15-µl pellets of Actigel-ALD precomplexed with either nonimmune IgM
or 3G3 anti-hsp90 monoclonal IgM. Immune pellets were rotated with the
diluted lysate for 2 h at 4 °C and then washed three times by
suspension in 1 ml of HEG buffer prior to protein resolution by
SDS-polyacrylamide gel electrophoresis and Western blotting.
Western Blotting--
Immunoblots were probed with 0.1%
hsp70/hsp90 antiserum against hsp90; 0.1% UPJ56 antiserum or 1 µg/ml
EC1 monoclonal IgG against FKBP52; 0.1% PP5 antiserum; 0.1% antiserum
against p50cdc37; 0.1% anti-cyclophilin 40 antibody against
CyP-40; 0.1% DS14F5 against Hop; 0.1% anti-GST ascites against
GST-GR, GST-465*, and GST-500*; or 0.05% anti-dynein intermediate
chain ascites against cytoplasmic dynein. The immunoblots were then
incubated a second time with the appropriate
125I-conjugated counterantibody to visualize immunoreactive bands.
Expression of GST Fusions--
pGEX-2T plasmids expressing
GST-rabbit FKBP52 or GST-FKBP52 Pro19-Ser262
fragment were used to transform Escherichia coli strain
BL21(DE3). pGEX Binding of Purified FKBP52 to GST-hGR--
Sf9 cells were
infected with baculovirus encoding GST-hGR, GST-465*, or GST-500* at a
multiplicity of infection of 3; and after 48 h, cytosol was
prepared. Twenty-five µl of Sf9 cytosol or 15 µg of GST (as
a control) were immobilized on 15 µl of glutathione cross-linked to
agarose, and the mixtures were rotated for 2 h at 4 °C. The
glutathione pellets were washed three times by suspension in 1 ml of
phosphate-buffered saline and then two times with TEG buffer. The
pellets were then suspended in TEG buffer containing 0.5 M
NaCl and Complete-Mini protease inhibitor mixture and incubated at
30 °C for 2 h to dissociate the GR from insect hsp90. After the
incubation, the pellets were washed twice with 1 ml of radioimmune precipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and
0.1% SDS) and two times with TEG buffer. The washed pellets were
incubated with 25 µl of purified bacterially expressed FKBP52 or the
PPIase fragment of FKBP52 and adjusted to a final volume of 50 µl
with 10 mM Hepes, pH 7.5, 25 mM KCl, 2 mM dithiothreitol, and 0.02% Nonidet P-40. The mixtures
were incubated for 1 h on ice with suspension of the pellets by
shaking the tubes every 3 min. At the end of the incubation, pellets
were washed three times with 1 ml of HEG buffer, and proteins were
resolved by denaturing gel electrophoresis and Western blotting.
Glutaraldehyde Cross-linking of Purified Proteins--
Rabbit
hsp90 was purified from reticulocyte lysate by sequential
chromatography through DE52, hydroxylapatite, and ATP-agarose as
described previously (48). Purified hsp90 (15 µl) and purified FKBP52
were incubated for 1 h on ice with each other or separately in a
final volume of 30 µl containing 10 mM Hepes, pH 7.5, 25 mM KCl, 2 mM dithiothreitol, and 0.02% Nonidet
P-40. Cross-linking was carried out by adding 3.3 µl of 20 mM glutaraldehyde and incubating for 1 h at room
temperature. The reaction was terminated by adding 50 µl of 0.1 M Tris, pH 8.0, for 30 min; 60 µl of 4× sample buffer were added; samples were boiled for 5 min; and proteins were resolved on 7% SDS-polyacrylamide gels followed by Western blotting.
For cross-linking of GST-GR and FKBP52, GST-GR from 75 µl of
Sf9 cytosol was immobilized on glutathione-agarose (pellet
volume of ~50 µl) and stripped of hsp90 as described above. The
stripped receptor was preincubated for 1 h on ice with 5 µl of
purified FKBP52 in a final volume of 40 µl containing the
Hepes/dithiothreitol/Nonidet P-40 buffer. Cross-linking was carried out
by adding 1.75 µl of 1 mM glutaraldehyde and incubating
for 30 min at room temperature. The reaction was terminated, and
proteins were resolved as described above.
Binding of FKBP52 to hsp90--
As summarized above, the
immunophilins bind via TPR domains to a TPR acceptor site in the
C-terminal domain of hsp90, and the stoichiometry of steroid receptor
heterocomplexes is 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of immunophilin. To resolve whether there is one TPR acceptor
site/hsp90 dimer or whether there is one acceptor site on each monomer
with one being blocked when the receptor is bound to an hsp90 dimer, we performed the cross-linking experiment shown in Fig.
1. In this experiment, purified hsp90 and
purified FKBP52 were mixed together and cross-linked with
glutaraldehyde. Fig. 1 shows the two proteins after they were resolved
by SDS gel electrophoresis and Western blotting. Lanes 1-3
were probed with an antiserum that recognizes both hsp70 and hsp90, and
lanes 5-7 were probed with the UPJ56 antiserum against
FKBP52. Lane 4 was split, and each half was probed with the
respective antiserum. As shown in lane 2, when hsp90 alone
was treated with glutaraldehyde, the majority of the protein was
recovered as a 180-kDa hsp90 dimer. In contrast, little if any FKBP52
was cross-linked as a homodimer (lane 6). When the two
proteins were mixed together, glutaraldehyde treatment yielded a slow
migrating form that reacted with both the antibody against hsp90
(lane 3) and the antibody against FKBP52 (lane
5). This band was formed at the expense of some hsp90·hsp90
dimer, and it migrated as expected for an hsp90·hsp90·FKBP52
heterotrimer. This direct evidence strongly supports a model in which
there is one TPR acceptor site/hsp90 dimer.
hsp90 is a very abundant protein in the cytosol, and
immunoadsorption of immunophilins yields co-adsorption of
immunophilin·hsp90 heterocomplexes (20). The relative amount of a TPR
protein that is associated with hsp90 should depend on both its
affinity for the TPR acceptor site on the hsp90 dimer and its abundance
in the cytosol relative to other TPR proteins. The experiment of Fig.
2 was done to determine the relative
amounts of several TPR proteins that are bound to hsp90 in reticulocyte
lysate. hsp90 was immunoadsorbed from a small volume of rabbit
reticulocyte lysate with the 3G3 monoclonal antibody IgM, and the
amount of protein remaining in the lysate after adsorption with
nonimmune IgM (lane 1) or the 3G3 antibody (lane
2) is shown, as is the amount of protein recovered in the
nonimmune (lane 3) or 3G3 immune (lane 4)
pellets. Under these conditions, we immunoadsorbed essentially all of
the hsp90 protein and co-immunoadsorbed all of the Hop protein. Hop is
required for assembly of receptor·hsp90 heterocomplexes, and it
contains six tetratricopeptide repeats (see Ref. 1 for review). PP5
contains four TPRs in a TPR domain that binds very tightly to hsp90
(15, 33), and most of the PP5 in reticulocyte lysate is bound to hsp90.
Only a portion of the immunophilins FKBP52 and CyP-40, both of which
have three TPRs, is bound to hsp90 (Fig. 2). Of these two
immunophilins, CyP-40 is known to be more weakly bound in GR·hsp90
heterocomplexes (12), and less is associated with the washed hsp90
immune pellet (Fig. 2). Although p50cdc37 does not have TPRs,
its binding site is close to the TPR acceptor site on hsp90 (33), and
only a portion of the p50cdc37 in reticulocyte lysate is bound
to hsp90.
Binding of FKBP52 to the GR--
Fig.
3A shows that FKBP52 also
binds directly to the GR. In this experiment, GST-GR expressed in
Sf9 cells or GST alone was immobilized on glutathione-agarose
and stripped of associated insect hsp90 prior to incubation with
purified FKBP52. A small amount of insect hsp90 is visible in
lane 2 of the original autoradiogram that was not present in
the stripped samples of GST-GR in lanes 4 and 6.
As shown in lane 6, FKBP52 bound to GST-GR, but it did not
bind to GST alone (lane 5). GST-GR does not bind the
purified TPR domain fragment of PP5, and the PP5 TPR domain does not
compete for the binding of FKBP52 to GST-GR (data not shown) under
conditions in which it blocks FKBP52 binding to hsp90 (33). As shown in Fig. 3B, GST-GR did not bind the purified PPIase fragment of
FKBP52 (lane 6), and the FKBP52 PPIase domain did not
compete for the binding of FKBP52 to GST-GR (lane 8)
under conditions in which we will show that it blocks
co-immunoadsorption of dynein with FKBP52 (see Fig. 8).
In the experiment of Fig. 4, GST-GR that
was immobilized on glutathione-agarose and stripped of insect hsp90 was
mixed with purified FKBP52, and the mixture was cross-linked with
glutaraldehyde. The amount of full-length GST-GR was reduced when
FKBP52 and glutaraldehyde were present (cf. lanes 2 and
3), and a slow migrating band appeared that also reacted
with the UPJ56 antibody against FKBP52 (lane 4). This band
migrated at an apparent molecular mass predicted for a cross-linked
GST-GR·FKBP52 product. In a mistake in preparing the Sf9
cytosol used in this experiment, the protease inhibitor mixture was
omitted, and there was extensive cleavage of GST-GR to an ~76-kDa
fragment. Because the fragment is bound to glutathione-agarose, this
fragment must consist of GST plus an ~48-kDa amino-terminal segment
of the GR. Thus, it does not contain the hormone-binding domain, the
hinge region, and most of the DNA-binding domain. Because no
cross-linked 76-kDa GST-GR fragment·FKBP52 product was obtained, it
is reasonable to predict that FKBP52 binds to one or more of these
three missing GR domains.
In their early study of cross-linking of the untransformed GR
heteromer, Gehring and co-workers (49) identified a small peak of
cross-linked product that was the size of the receptor plus an
~50-kDa polypeptide. This cross-linked product was also obtained on
cross-linking of the nti (increased
nuclear transfer) mutant GR (49). The
nti mutant is composed of the DNA-binding, hinge, and
hormone-binding domains of the GR, and the 50-kDa component was later
identified as FKBP52 (36). To further localize a potential binding
site(s) for FKBP52 within this region, we assayed the binding of
purified FKBP52 in two mutants of the hGR, GST-500* and GST-465*. As
diagrammed in Fig. 5, GST-500* is a
carboxyl-terminal truncation of the GR lacking the hormone-binding
domain and the portion of the hinge region carboxyl-terminal to NL1
(nuclear localization signal 1 (50)) at amino acids 491-498 of the hGR (51), whereas GST-465* lacks
half of the carboxyl-terminal zinc finger and NL1 as well. As shown in
the Western blot of Fig. 5, FKBP52 bound to GST-500* (lane
3), although the binding was reduced compared with GST-GR (lane 2); and there is no binding of FKBP52 to GST-465*
(lane 4).
Binding of FKBP52 to Dynein--
We have reported previously that
immunoadsorption of FKBP52 from Chinese hamster ovary cell or chicken
brain cytosol with UPJ56 is accompanied by co-immunoadsorption of both
intermediate and heavy chains of cytoplasmic dynein; UPJ56 does not
itself recognize dynein; and the presence of dynein in UPJ56 immune
pellets is specific for the presence of FKBP52 (39). As shown in Fig. 6, immune adsorption of FKBP52 from
rabbit reticulocyte lysate was accompanied by co-adsorption of dynein,
again suggesting that dynein is bound, either directly or via other
proteins, to cytosolic FKBP52. Immune adsorption of CyP-40 usually (but
not always) yielded co-adsorption of trace amounts of dynein (Fig. 6),
suggesting a possible weak complex containing the two proteins. Immune
adsorption of either PP5 or p50cdc37 was not accompanied by
co-adsorption of dynein.
To determine the domain of FKBP52 involved in the interaction with
dynein, lysate from bacteria expressing the GST-FKBP52 Pro19-Ser262 fragment or lysate from control
bacteria expressing GST was mixed with reticulocyte lysate, and rabbit
FKBP52 was immunoadsorbed. The FKBP52 fragment encompasses the
N-terminal domain with high PPIase homology, and it possesses PPIase
activity (47). As shown in Fig.
7A, immunoadsorption of FKBP52
yielded co-immunoadsorption of both hsp90 and dynein (cf. lanes
1 and 2), and the binding of dynein (but not hsp90) was
competed by the PPIase fragment (lanes 4 and 6),
but not by FKBP12 (lane 7).
In Fig. 7B, either control Sf9 lysate or lysate from
Sf9 cells expressing the TPR domain of PP5 was added to
reticulocyte lysate prior to immunoadsorption of FKBP52.
Immunoadsorption of FKBP52 from reticulocyte alone yielded
co-adsorption of dynein and rabbit hsp90 (lane 2). In the
mixture of reticulocyte lysate and control Sf9 lysate, dynein
was still co-adsorbed, and co-adsorbed hsp90 was largely the more
rapidly migrating insect hsp90 (lane 3), which had exchanged
with rabbit hsp90. The presence of the PP5 TPR domain in the lysate
mixture did not affect co-adsorption of dynein under conditions in
which FKBP52 binding to hsp90 was blocked (lane 4). As shown
in Fig. 7C, dynein was co-adsorbed with FKBP52, regardless
of whether or not its PPIase domain was bound by FK506.
The FKBP52 Pro19-Ser262 fragment used to
compete for dynein co-adsorption in Fig. 7A contains FKBP52
domain I, which shares 49% sequence identity with FKBP12, followed by
a short hinge connector segment and domain II, which possesses much
less homology to FKBP12 (52). The experiment of Fig.
8A was performed to determine
what portion of the PPIase fragment was sufficient for competition for
dynein co-adsorption. Lysates from bacteria expressing GST-FKBP52 Met6-Gly148 (lane 5), which
encompasses domain I and the hinge, and GST-FKBP52 Gly32-Lys138 (lane 6), which
encompasses the core of domain I without the hinge, competed for dynein
co-adsorption with FKBP52. In contrast, bacterial lysate expressing a
comparable amount of GST-FKBP52 Gly149-Leu267
(lane 7), which encompasses domain II, yielded much less
competition. Thus, domain I alone is sufficient to compete for dynein
binding to FKBP52.
To rule out any contribution of GST in the fusion proteins, the entire
PPIase fragment (amino acids 19-262) and domain I plus the hinge
(amino acids 6-148) were separated from GST by thrombin cleavage and
added as the purified fragments to reticulocyte lysate. As shown in
Fig. 8 (B and C, lanes 4), both
fragments competed for dynein co-adsorption with FKBP52.
The cross-linking data in Fig. 1 support a model in which 1 molecule of FKBP52 is bound per dimer of hsp90. In the event that heterotetramers containing 2 molecules of hsp90 and 2 molecules of
FKBP52 were formed, they should be readily resolved from the heterotrimer on our gels. Because no slower migrating bands were detected, we conclude that there is one TPR acceptor site/dimer. hsp90
forms homodimers with very high affinity, and the dimerization site and
TPR acceptor site overlap each other in the C terminus of hsp90 (21,
53-55). Chen et al. (53) have shown that deletion of amino
acids 661-677 of chicken hsp90 eliminates dimerization while reducing
binding of FKBP52 to ~30% of that of wild-type hsp90. Thus, we
conclude that each hsp90 monomer contains a half-site that is
sufficient for low affinity TPR binding, with a single higher affinity
site being created by hsp90 dimerization.
It is clear that TPR proteins bind to hsp90 with different affinities,
ranging from the loosely bound CyP-40, which can be washed off hsp90
immune pellets with low salt buffers (12), to the very tightly bound
Hop, some of which binds to hsp90 despite the presence of extremely
high levels of competing TPR domain fragments of CyP-40 (20) or PP5
(33). In Fig. 2, we examined the proportion of each protein bound to
hsp90 in rabbit reticulocyte lysate because that is the system employed
for cell-free assembly of hsp90 heterocomplexes with receptors and
protein kinases. This assembly process follows a sequence in which Hop
is released by an unknown mechanism from an assembly intermediate
before an immunophilin is bound (56). The immunophilins, PP5, and
p50cdc37 then apparently compete with each other for binding to hsp90.
The native protein kinase·hsp90 heterocomplexes that are recovered
from cells contain predominantly p50cdc37, a composition that
may be selected because p50cdc37 binds directly to the kinase
as well as to hsp90 (29, 33). Similarly, the FKBP homolog
ARA9/AIP/XAP2, which is recovered selectively with AHR·hsp90
heterocomplexes (22-25), binds directly to the AHR as well as to hsp90
(57). It is likely that the native GR·hsp90 heterocomplexes, which
contain FKBP52 and no p50cdc37, are selected because FKBP52
binds to the GR as shown in Figs. 3-5. The region of FKBP52 that
determines its binding to the GR is not known, but the lack of
competition for binding by either the PP5 TPR domain or the FKBP52
PPIase domain suggests that neither region is involved. The
cross-linking data of Fig. 4 suggest that FKBP52 binds directly to the
GR, and the mutant GR data of Fig. 5 suggest that the 35-amino acid
segment between amino acids 465 and 500 of the hGR is sufficient for
FKBP52 binding. However, GST-500* binds less FKBP52 than GST-GR (Fig.
5), suggesting that the hormone-binding domain also contributes to
FKBP52 binding. Segment 465-500 of the GR that is sufficient for
FKBP52 contains NL1, and a second nuclear localization signal is
located within the hormone-binding domain (50). One possible way to
explain the observations of Fig. 5 is that FKBP52 binds to both NL1 and NL2; thus, binding is reduced from the wild-type GR in GST-500* and
eliminated in GST-465*.
Although the PPIase domain fragment of FKBP52 does not compete for
FKBP52 binding to the GR (Fig. 3B) or for FKBP52 binding to
hsp90 (Fig. 7A), it does compete for the co-adsorption of
cytoplasmic dynein with FKBP52 (Fig. 7A), suggesting that
this region of the immunophilin is responsible for either direct
binding to dynein or dynein binding via another protein. The
immunophilin CyP-40 may also engage in a similar interaction (Fig. 6),
but the interaction is so weak that we cannot be sure of it. Although
the PPIase domain appears to be involved in the dynein co-adsorption,
PPIase activity is not required. This is inferred from the fact that
the PPIase inhibitor FK506 does not affect the interaction (Fig.
7C). Although the PPIase domain (domain I) of FKBP52
competes for the co-adsorption of dynein with FKBP52 (Fig.
8A), FKBP12 does not compete (Fig. 7A). The lack
of an effect of FK506 or FKBP12 on the co-adsorption of dynein with
FKBP52 is not unique. Chambraud et al. (58) used PPIase
domain I of FKBP52 as bait in a yeast two-hybrid screen to identify a
peroxisomal enzyme as a potential FKBP-associated protein. In cell-free
experiments, it was shown that the enzyme did not bind FKBP12, and its
interaction with FKBP52 was not affected by FK506. Also, it should be
noted that FK506-bound FKBP52 or its PPIase domain does not inhibit
calcineurin activity in vitro (59), whereas FK506-bound
FKBP12 binds to and inhibits the calcineurin phosphatase (60). Thus,
there seem to be clear differences in the protein-protein interactions
of the FKBP52 PPIase domain and its homolog, FKBP12.
To eventually define the functions of the high molecular mass
components of receptor·hsp90 heterocomplexes and the function of the
p50cdc37 component of protein kinase·hsp90 heterocomplexes,
it is important to define the proteins with which these components
interact and the domains responsible for those interactions. We suggest
from this and previous work that FKBP52 has at least four regions
determining protein interactions. In the domain structure of FKBP52
suggested by the sequence of Callebaut et al. (52), domain I
has the highest homology (49%) to FKBP12. This domain expressed alone
has PPIase activity (47), and a proteolytic fragment comprising domain I binds FK506 (61). The competition data of Fig. 8 suggest that this
region of FKBP52 accounts for co-adsorption of dynein. Domain III
contains three TPRs, and both deletion of this TPR domain (16, 19) and
competition with TPR domain fragments (12, 15, 20, 33) block FKBP52
binding to hsp90. The C terminus of FKBP52 contains a predicted
calmodulin-binding site (52), and FKBP52 binds calmodulin-Sepharose in
a calcium-dependent manner (62). Additionally, we show here
that FKBP52 binds directly to the GR. The precise region of FKBP52 that
binds the GR is not yet known, but it appears to be separate from the
regions involved in binding to dynein or hsp90 because neither the
FKBP52 PPIase fragment nor the PP5 TPR fragment competes for GR binding.
We are indebted to Drs. Karen Leach, Ettore
Appella, Nicholas Grammatikakis, David Smith, Lee Faber, Michael
Chinkers, Ganesan Srinivasan, and E. Brad Thompson for providing the
antibodies and cDNAs used in this work.
*
This work was supported by National Institutes of Health
Grant CA28010 (to W. B. P.), a grant from the Ligue Nationale contre le Cancer (Comités des Yvelines et du Cher), and Association pour
la Recherche contre le Cancer Contract 9863 (to J.-M. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pharmacology, 1301 Medical Science Research Bldg. III, University of Michigan Medical School, Ann Arbor, MI 48109-0632. Tel.: 734-764-5414; Fax: 734-763-4450.
The abbreviations used are:
hsp, heat shock
protein;
TPR, tetratricopeptide repeat;
FKBP, FK506-binding protein;
CyP, cyclosporin A-binding protein;
PP5, protein phosphatase 5;
AHR, aryl hydrocarbon receptor;
GR, glucocorticoid receptor;
hGR, human
glucocorticoid receptor;
PPIase, peptidylprolyl isomerase;
GST, glutathione S-transferase;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid.
Different Regions of the Immunophilin FKBP52 Determine Its
Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic
Dynein*
,,,¶
Department of Pharmacology, University of
Michigan Medical School, Ann Arbor, Michigan 48109 and the
§ Faculté de Pharmacie, UMR 8612 CNRS, Pharmacologie
Cellulaire, 5 rue Jean-Baptiste Clément,
92296 Chatenay-Malabry Cedex, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T plasmids encoding GST-rabbit
FKBP52 domain I plus hinge (amino acids 6-148), GST-FKBP52 core domain
I (amino acids 31-138), and GST-FKBP52 domain II (amino acids
149-267) were prepared as described previously (47).
T plasmids expressing GST-FKBP52
Met6-Gly148, GST-FKBP52
Gly32-Lys138, or GST-FKBP52
Gly149-Leu267 were used to transform E. coli strain UT5600. Purification of the fusion proteins was
performed by binding GST-FKBP52 to GSH-agarose and incubation at
4 °C with thrombin, which cleaves at a site between the GST domain
and FKBP52.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (84K):
[in a new window]
Fig. 1.
Stoichiometry of hsp90·FKBP52
heterocomplex. Purified hsp90 and purified FKBP52 were mixed
together and cross-linked with glutaraldehyde as described under
"Methods." Proteins were resolved by SDS-polyacrylamide gel
electrophoresis and Western-blotted with the antiserum indicated under
each set of lanes. Lane 4 was cut in half so that it could
be probed with both antibodies. The noncross-linked and cross-linked
products are indicated to the right. Lanes 1 and
6, FKBP52 alone; lanes 2 and 7, hsp90
alone; lanes 3-5, hsp90 and FKBP52. The lowest
band in lanes 1, 3, and 4 is
bacterial DnaK in the purified FKBP52 preparation that is detected with
the antiserum against hsp70 and hsp90.
View larger version (59K):
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Fig. 2.
Relative amounts of hsp90-bound and free TPR
proteins and p50cdc37. Aliquots of rabbit reticulocyte
lysate were immunoadsorbed with nonimmune IgM or with 3G3 anti-hsp90
monoclonal IgM as described under "Methods." Proteins were resolved
by SDS-polyacrylamide gel electrophoresis and Western blotting.
Lane 1, supernatant from nonimmune pellet; lane
2, supernatant from absorption with 3G3; lane 3,
nonimmune pellet; lane 4, 3G3 immune pellet.
View larger version (38K):
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Fig. 3.
FKBP52 binds directly to the GR.
A, binding of purified FKBP52 to the hGR. Immobilized
GST-hGR was stripped of associated proteins and incubated with purified
FKBP52 as described under "Methods." hsp90 was detected with rabbit
antiserum against hsp70 and hsp90 that detects insect hsp90, of which a
small amount was visible in lane 2 of the original Western
blot. Lane 1, GST; lane 2, GST-hGR; lane
3, stripped GST; lane 4, stripped GST-hGR; lane
5, stripped GST incubated with purified FKBP52; lane 6,
stripped GST-hGR incubated with purified FKBP52. B, the
FKBP52 PPIase fragment does not compete for the binding of purified
FKBP52 to the hGR. Immobilized and stripped GST-hGR was incubated with
purified FKBP52 with or without the purified PPIase fragment of FKBP52
as described under "Methods." Lanes 1 and 2,
stripped GST and GST-hGR, respectively; lanes 3 and
4, stripped GST and GST-hGR incubated with purified FKBP52,
respectively; lanes 5 and 6, GST and GST-hGR
incubated with the FKBP52 PPIase fragment, respectively; lanes
7 and 8, GST and GST-hGR incubated with FKBP52 and its
PPIase fragment, respectively.
View larger version (58K):
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Fig. 4.
Cross-linking of the GR and FKBP52.
Immobilized GST-hGR was stripped of associated proteins, incubated with
FKBP52, and cross-linked with glutaraldehyde, and proteins were
resolved by SDS-polyacrylamide gel electrophoresis and Western
blotting. Lanes 1-3 were immunoblotted with anti-GST
antibody, and lane 4 was immunoblotted for FKBP52 with
UPJ56. The noncross-linked and cross-linked products are indicated to
the right. Lane 1 GST-GR alone; lane 2, GST-GR
alone treated with glutaraldehyde; lanes 3 and 4,
GST-GR and FKBP52 treated with glutaraldehyde. The Sf9 cytosol
from which GST-GR was immunoadsorbed in this experiment was prepared
without the protease inhibitor mixture, and it contains a lot of a
76-kDa GST-GR proteolytic fragment that binds to
glutathione-agarose (lanes 1-3).
View larger version (39K):
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Fig. 5.
FKBP52 binds to GST-500* mutant hGR, but not
to GST-465*. Immobilized GST-hGR, GST-500*, or GST-465* was
stripped of associated proteins and incubated with purified FKBP52 as
described under "Methods." Lane 1, GST; lane
2, GST-hGR; lane 3 GST-500*; lane 4,
GST-465*. In the diagrams of the fusions above the Western
blot, the GR hormone-binding domain (stippled), NL1
(solid), and the DNA-binding domain (hatched) are
indicated.
View larger version (16K):
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Fig. 6.
Co-adsorption of dynein with FKBP52 and in
trace amounts with CyP-40. Aliquots (150 µl) of rabbit
reticulocyte lysate were immunoadsorbed with UPJ56 antiserum against
FKBP52, antiserum against CyP-40, or preimmune rabbit serum. Other
150-µl aliquots of reticulocyte lysate were incubated for 30 min at
30 °C with 10 µl of Sf9 cytosol overexpressing FLAG-PP5 or
FLAG-p50cdc37 and then immunoadsorbed with nonimmune IgG or
anti-FLAG M2 monoclonal IgG. Lane 1, immunoadsorption with
nonimmune antibody; lane 2, immunoadsorption with the
antibody indicated at the top of each pair of lanes. Each immune pellet
was Western-blotted for the intermediate chain (IC) of
dynein and for the immunoadsorbed protein.
View larger version (24K):
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Fig. 7.
The FKBP52 PPIase fragment, but not FKBP12, a
TPR domain, or FK506, competes for the binding of dynein to
FKBP52. A, the PPIase fragment competes for the binding
of dynein to FKBP52. Aliquots (150 µl) of rabbit reticulocyte lysate
were incubated for 30 min at 30 °C with buffer, with control
bacterial lysate, with lysate from bacteria overexpressing the
GST-PPIase fragment, or with 13 µg of FKBP12. Samples were then
immunoadsorbed with preimmune rabbit serum (lane 1) or the
UPJ56 antiserum against FKBP52 (lanes 2-7). Lanes
1 and 2, nonimmune and immune pellets from reticulocyte
lysate, respectively; lanes 3 and 4, immune
pellets from reticulocyte lysate incubated with 5 µl of lysate from
control bacteria and from bacteria overexpressing the PPIase fragment
of FKBP52, respectively; lanes 5 and 6, same as
lanes 3 and 4, respectively, but with 10 µl of
bacterial lysate; lane 7, immune pellet from reticulocyte
lysate incubated with FKBP12. B, the TPR domain of rat PP5
does not affect the co-adsorption of dynein with FKBP52. Aliquots (100 µl) of rabbit reticulocyte lysate were incubated for 30 min at
30 °C without Sf9 lysate (lanes 1 and
2), with control Sf9 lysate (lane 3), or
with lysate from Sf9 cells expressing the FLAG-tagged TPR domain
of rat PP5 (lane 4). Samples were then immunoadsorbed with
preimmune serum or the UPJ56 antiserum against FKBP52. Lanes
1 and 2, nonimmune and immune pellets from reticulocyte
lysate, respectively; lanes 3 and 4, immune
pellets from reticulocyte lysate incubated with 60 µl of control
Sf9 lysate and lysate from Sf9 cells expressing the TPR
domain of rat PP5, respectively. C, FK506 does not affect
co-adsorption of dynein with FKBP52. Aliquots (150 µl) of rabbit
reticulocyte lysate were incubated with vehicle (lanes 1 and
2) or 125 nM FK506 (lanes 3 and
4) for 30 min at 30 °C and then immunoadsorbed with
preimmune rabbit serum (lanes 1 and 3) or the
UPJ56 antiserum against FKBP52 (lanes 2 and 4).
IC, intermediate chain of dynein.
View larger version (26K):
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Fig. 8.
Domain I of FKBP52 is sufficient to compete
for dynein binding to FKBP52. A, FKBP52 mutants that
contain the core PPIase sequence of domain I compete for the binding of
dynein to FKBP52. Aliquots (150 µl) of rabbit reticulocyte lysate
were incubated for 30 min at 30 °C with 15 µl of buffer, with 15 µl of control bacterial lysate, or with 15 µl of lysate from
bacteria overexpressing different GST-FKBP52 mutants. Samples were then
immunoadsorbed with preimmune rabbit serum (lane 1) or the
UPJ56 antiserum against FKBP52 (lanes 2-7). Lanes
1 and 2, nonimmune and immune pellets from reticulocyte
lysate, respectively; lane 3, immune pellet from
reticulocyte lysate incubated with 15 µl of lysate from control
bacteria; lanes 4-7, immune pellets from reticulocyte
lysate incubated with 15 µl of lysate from bacteria expressing
GST-PPIase (amino acids 19-262), GST-domain I plus hinge (amino acids
6-148), GST-core domain I (amino acids 32-138), and GST-domain II
(amino acids 149-267), respectively. B, the purified PPIase
fragment (amino acids 19-262) competes for the binding of dynein to
FKBP52. Aliquots (150 µl) of rabbit reticulocyte lysate were
incubated for 30 min at 30 °C without (lanes 1 and
2) or with 100 µl of buffer (lane 3) or 100 µl of purified PPIase fragment (3.2 mg/ml final concentration)
(lane 4). Samples were then immunoadsorbed with preimmune
serum (lane 1) or the UPJ56 antiserum against FKBP52
(lanes 2-4). C, purified FKBP52 domain I (amino
acids 6-148) competes for the binding of dynein to FKBP52. Aliquots
(150 µl) of rabbit reticulocyte lysate were incubated for 30 min at
30 °C without (lanes 1 and 2) or with 100 µl
of buffer (lane 3) or 100 µl of purified FKBP52 domain I
(3.2 mg/ml final concentration) (lane 4). Samples were then
immunoadsorbed with preimmune serum (lane 1) or the UPJ56
antiserum against FKBP52 (lanes 2-4). IC,
intermediate chain of dynein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Pratt, W. B.,
and Toft, D. O.
(1997)
Endocr. Rev.
18,
306-360 2.
Pratt, W. B.
(1997)
Annu. Rev. Pharmacol. Toxicol.
37,
297-326[CrossRef][Medline]
[Order article via Infotrieve]
3.
Sikorski, R. S.,
Boguski, M. S.,
Goebel, M.,
and Hieter, P.
(1990)
Cell
60,
307-317[CrossRef][Medline]
[Order article via Infotrieve]
4.
Yem, A. W.,
Tomasselli, A. G.,
Heinrikson, R. L.,
Zurcher-Neely, H.,
Ruff, V. A.,
Johnson, R. A.,
and Deibel, M. R., Jr.
(1992)
J. Biol. Chem.
267,
2868-2871 5.
Lebeau, M.-C.,
Massol, N.,
Herrick, J.,
Faber, L. E.,
Renoir, J.-M.,
Radanyi, C.,
and Baulieu, E.-E.
(1992)
J. Biol. Chem.
267,
4281-4284 6.
Tai, P. K.,
Albers, M. W.,
Chang, H.,
Faber, L. E.,
and Schreiber, S. L.
(1992)
Science
256,
1315-1318 7.
Peattie, D. A.,
Harding, M. W.,
Fleming, M. A.,
De Cenzo, M. T.,
Lippke, J. A.,
Livingston, D. J.,
and Benasutti, M.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
10974-10978 8.
Smith, D. F.,
Baggenstoss, B. A.,
Marion, T. N.,
and Rimerman, R. A.
(1993)
J. Biol. Chem.
268,
18365-18371 9.
Smith, D. F.,
Albers, M. W.,
Schreiber, S. L.,
Leach, K. L.,
and Deibel, M. R., Jr.
(1993)
J. Biol. Chem.
268,
24270-24273 10.
Nair, S. C.,
Rimerman, R. A.,
Toran, E. J.,
Chen, S.,
Prapapanich, V.,
Butts, R. N.,
and Smith, D. F.
(1997)
Mol. Cell. Biol.
17,
594-603[Abstract]
11.
Ratajczak, T.,
Carrello, A.,
Mark, P. J.,
Warner, B. J.,
Simpson, R. J.,
Moritz, R. L.,
and House, A. K.
(1993)
J. Biol. Chem.
268,
13187-13192 12.
Owens-Grillo, J. K.,
Hoffmann, K.,
Hutchison, K. A.,
Yem, A. W.,
Deibel, M. R., Jr.,
Handschumacher, R. E.,
and Pratt, W. B.
(1995)
J. Biol. Chem.
270,
20479-20484 13.
Renoir, J.-M.,
Mercier-Bodard, C.,
Hoffman, K.,
Le Bihan, S.,
Ning, Y. M.,
Sanchez, E. R.,
Handschumacher, R. E.,
and Baulieu, E.-E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
4977-4981 14.
Chen, M.-S.,
Silverstein, A. M.,
Pratt, W. B.,
and Chinkers, M.
(1996)
J. Biol. Chem.
271,
32315-32320 15.
Silverstein, A. M.,
Galigniana, M. D.,
Chen, M.-S.,
Owens-Grillo, J. K.,
Chinkers, M.,
and Pratt, W. B.
(1997)
J. Biol. Chem.
272,
16224-16230 16.
Radanyi, C.,
Chambraud, B.,
and Baulieu, E.-E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11197-11201 17.
Hoffmann, K.,
and Handschumacher, R. E.
(1995)
Biochem. J.
307,
5-8
18.
Ratajczak, T.,
and Carrello, A.
(1996)
J. Biol. Chem.
271,
2961-2965 19.
Barent, R. L.,
Nair, S. C.,
Carr, D. C.,
Ruan, Y.,
Rimerman, R. A.,
Fulton, J.,
Zhang, Y.,
and Smith, D. F.
(1998)
Mol. Endocrinol.
12,
342-354 20.
Owens-Grillo, J. K.,
Czar, M. J.,
Hutchison, K. A.,
Hoffmann, K.,
Perdew, G. H.,
and Pratt, W. B.
(1996)
J. Biol. Chem.
271,
13468-13475 21.
Young, J. C.,
Obermann, W. M. J.,
and Hartl, F. U.
(1998)
J. Biol. Chem.
273,
18007-18010 22.
Carver, L. A.,
and Bradfield, C. A.
(1997)
J. Biol. Chem.
272,
11452-11456 23.
Ma, Q.,
and Whitlock, J. P., Jr.
(1997)
J. Biol. Chem.
272,
8878-8884 24.
Meyer, B. K.,
Pray-Grant, M. G.,
Vanden Heuvel, J. P.,
and Perdew, G. H.
(1998)
Mol. Cell. Biol.
18,
978-988 25.
Carver, L. A.,
LaPres, J. J.,
Jain, S.,
Dunham, E. E.,
and Bradfield, C. A.
(1998)
J. Biol. Chem.
273,
33580-33587 26.
Brugge, J. S.
(1986)
Curr. Top. Microbiol. Immunol.
123,
1-22[Medline]
[Order article via Infotrieve]
27.
Hunter, T.,
and Poon, R. Y. C.
(1997)
Trends Cell Biol.
7,
157-161
28.
Grammatikakis, N.,
Grammatikakis, A.,
Yoneda, M., Yu, Q.,
Banerjee, S. D.,
and Toole, B. P.
(1995)
J. Biol. Chem.
270,
16198-16205 29.
Stepanova, L.,
Leng, X.,
Parker, S. B.,
and Harper, J. W.
(1996)
Genes Dev.
10,
1491-1502 30.
Perdew, G. H.,
Wiegand, H.,
Vanden Heuvel, J. P.,
Mitchell, C.,
and Singh, S. S.
(1997)
Biochemistry
36,
3600-3607[CrossRef][Medline]
[Order article via Infotrieve]
31.
Dey, B.,
Lightbody, J. J.,
and Bochelli, F.
(1996)
Mol. Biol. Cell
7,
1405-1417[Abstract]
32.
Cutforth, T.,
and Rubin, G. M.
(1994)
Cell
77,
1027-1036[CrossRef][Medline]
[Order article via Infotrieve]
33.
Silverstein, A. M.,
Grammatikakis, N.,
Cochran, B. H.,
Chinkers, M.,
and Pratt, W. B.
(1998)
J. Biol. Chem.
273,
20090-20095 34.
Whitelaw, M. L.,
Hutchison, K.,
and Perdew, G. H.
(1991)
J. Biol. Chem.
266,
16436-16440 35.
Stancato, L. F.,
Chow, Y.-H.,
Hutchison, K. A.,
Perdew, G. H.,
Jove, R.,
and Pratt, W. B.
(1993)
J. Biol. Chem.
268,
21711-21716 36.
Rexin, M.,
Busch, W.,
and Gehring, U.
(1991)
J. Biol. Chem.
266,
24601-24605 37.
Rehberger, P.,
Rexin, M.,
and Gehring, U.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
8001-8005 38.
Segnitz, B.,
and Gehring, U.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
2179-2183 39.
Czar, M. J.,
Owens-Grillo, J. K.,
Yem, A. W.,
Leach, K. L.,
Deibel, M. R.,
Welsh, M. J.,
and Pratt, W. B.
(1994)
Mol. Endocrinol.
8,
1731-1741 40.
Perrot-Applanat, M.,
Cibert, C.,
Geraud, G.,
Renoir, J.-M.,
and Baulieu, E.-E.
(1995)
J. Cell Sci.
108,
2037-2051[Abstract]
41.
Pratt, W. B.
(1993)
J. Biol. Chem.
268,
21455-21458 42.
Ruff, V. A.,
Yem, A. W.,
Munns, P. L.,
Adams, L. D.,
Reardon, I. M.,
Deibel, M. R., Jr.,
and Leach, K. L.
(1992)
J. Biol. Chem.
267,
21285-21288 43.
Erhart, J. C.,
Duthu, A.,
Ullrich, S.,
Appella, E.,
and May, P.
(1988)
Oncogene
3,
595-603[Medline]
[Order article via Infotrieve]
44.
Smith, D. F.,
Sullivan, W. P.,
Marion, T. N.,
Zaitsu, K.,
Madden, B.,
McCormick, D. J.,
and Toft, D. O.
(1993)
Mol. Cell. Biol.
13,
869-876 45.
Chen, H.,
Srinivasan, G.,
and Thompson, E. B.
(1997)
J. Biol. Chem.
272,
25873-25880 46.
Le Bihan, S.,
Renoir, J.-M.,
Radanyi, C.,
Chambraud, B.,
Joulin, V.,
Catelli, M.-G.,
and Baulieu, E.-E.
(1993)
Biochem. Biophys. Res. Commun.
195,
600-607[CrossRef][Medline]
[Order article via Infotrieve]
47.
Chambraud, B.,
Rouviere-Fourmy, N.,
Radanyi, C.,
Hsiao, K.,
Peattie, D. A.,
Livingston, D. J.,
and Baulieu, E.-E.
(1993)
Biochem. Biophys. Res. Commun.
196,
160-166[CrossRef][Medline]
[Order article via Infotrieve]
48.
Hutchison, K. A.,
Dittmar, K. D.,
Czar, M. J.,
and Pratt, W. B.
(1994)
J. Biol. Chem.
269,
5043-5049 49.
Rexin, M.,
Busch, W.,
Segnitz, B.,
and Gehring, U.
(1988)
FEBS Lett.
241,
234-238[CrossRef][Medline]
[Order article via Infotrieve]
50.
Picard, D.,
and Yamamoto, K. R.
(1987)
EMBO J.
6,
3333-3340[Medline]
[Order article via Infotrieve]
51.
Hollenberg, S. M.,
Weinberger, C.,
Ong, E. S.,
Cerelli, G.,
Oro, A.,
Lebo, R.,
Thompson, E. B.,
Rosenfeld, M. G.,
and Evans, R. M.
(1985)
Nature
318,
635-641[CrossRef][Medline]
[Order article via Infotrieve]
52.
Callebaut, I.,
Renoir, J.-M.,
Lebeau, M.-C.,
Massol, N.,
Burny, A.,
Baulieu, E.-E.,
and Mornon, J. P.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6270-6274 53.
Chen, S.,
Sullivan, W. P.,
Toft, D. O.,
and Smith, D. F.
(1998)
Cell Stress Chaperones
3,
118-129[CrossRef][Medline]
[Order article via Infotrieve]
54.
Carrello, A.,
Ingley, E.,
Minchin, R. F.,
Tsai, S.,
and Ratajczak, T.
(1999)
J. Biol. Chem.
274,
2682-2689 55.
Russell, L. C.,
Whitt, S. R.,
Chen, M.-S.,
and Chinkers, M.
(1999)
J. Biol. Chem.
274,
20060-20063 56.
Smith, D. F.,
Whitesell, L.,
Nair, S. C.,
Chen, S.,
Prapapanich, V.,
and Rimerman, R. A.
(1995)
Mol. Cell. Biol.
15,
6804-6812[Abstract]
57.
Meyer, B. K.,
and Perdew, G. H.
(1999)
Biochemistry
38,
8907-8917[CrossRef][Medline]
[Order article via Infotrieve]
58.
Chambraud, B.,
Radanyi, C.,
Camonis, J. H.,
Rajkowski, K.,
Schumacher, M.,
and Baulieu, E.-E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2104-2109 59.
Lebeau, M.-C.,
Myagkikh, I.,
Rouviere-Fourmy, N.,
Baulieu, E.-E.,
and Klee, C. B.
(1994)
Biochem. Biophys. Res. Commun.
203,
750-755[CrossRef][Medline]
[Order article via Infotrieve]
60.
Liu, J.,
Farmer, J. D.,
Lane, W. S.,
Friedman, J.,
Weissman, I.,
and Schreiber, S. L.
(1991)
Cell
66,
807-815[CrossRef][Medline]
[Order article via Infotrieve]
61.
Yem, A. W.,
Reardon, I. M.,
Leone, J. W.,
Heinrikson, R. L.,
and Deibel, M. R.
(1993)
Biochemistry
32,
12571-12576[CrossRef][Medline]
[Order article via Infotrieve]
62.
Massol, N.,
Lebeau, M.-C.,
Renoir, J.-M.,
Faber, L. E.,
and Baulieu, E.-E.
(1992)
Biochem. Biophys. Res. Commun.
187,
1330-1335[CrossRef][Medline]
[Order article via Infotrieve]
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