(Received for publication, January 18, 1995; and in revised form, May 30, 1995)
From the
We have recently shown that hsp56, the FK506-binding
immunophilin component of both the heat shock protein
(hsp90hsp70
hsp56) heterocomplex and the untransformed
glucocorticoid receptor heterocomplex, is bound directly to hsp90
(Czar, M.J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A.,
Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B.(1994) J. Biol. Chem. 269, 11155-11161). In this work, we show
that both untransformed glucocorticoid receptor and hsp90
heterocomplexes contain CyP-40, a 40-kDa immunophilin of the
cyclosporin A-binding class. CyP-40 is present in both native
glucocorticoid receptor heterocomplexes and receptor heterocomplexes
reconstituted with rabbit reticulocyte lysate, and the presence of
CyP-40 in the receptor heterocomplex is stabilized by molybdate.
Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of
both hsp56 and CyP-40, showing that both immunophilins are in native
heterocomplex with hsp90. However, immunoadsorption of hsp56 does not
yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not
exist in the same heterocomplex with hsp90. Both purified CyP-40 and
hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the
binding of hsp56, consistent with the presence of a common immunophilin
binding site on hsp90. Our data also suggest that there are at least
two types of untransformed glucocorticoid receptor-hsp90
heterocomplexes, one that contains hsp56 and another that contains
CyP-40. The role played by the immunophilins in steroid receptor action
is unknown, but it is clear that the peptidylprolyl isomerase activity
of immunophilins is not required for glucocorticoid receptor-hsp90
heterocomplex assembly and proper folding of the hormone binding domain
by the hsp90-associated protein folding system of reticulocyte lysate.
After cell rupture, untransformed steroid receptors are
recovered in the cytosolic fraction of hormone-free cells in
multiprotein complexes containing both heat shock protein (hsp) ()and immunophilin chaperones (for review see (1) and (2) ). The immunophilins are ubiquitous and
conserved proteins that bind immunosuppressant drugs, such as
cyclosporin A, FK506, and rapamycin ( (3) for review). All
members of the immunophilin protein family have peptidylprolyl
isomerase activity, and, like hsp70 and hsp90, they are thought to play
major roles in protein folding and trafficking in the cell.
The
first receptor-associated immunophilin was discovered when an antibody
directed against the partially purified, untransformed progesterone
receptor complex was found to react with a 59-kDa rabbit protein (4) that was shown to be associated with all untransformed
steroid receptor heterocomplexes although its function in such
complexes is unknown(5) . The human protein, with an apparent M of 56,000(6) , was found to be both a
heat shock protein (7) and a member of the FK506- and
rapamycin-binding class of immunophilins(8, 9) .
Rabbit(10) , human(11) , and mouse (12) cDNAs
for hsp56 were cloned, and because the human cDNA encodes an
FK506-binding protein with a predicted molecular mass of 52,000 the
protein is also called FKBP52(11) . Recently, the 50- and
54-kDa components of the untransformed chicken progesterone receptor
heterocomplex (13) were found to have significant sequence
identity with hsp56 (14) and to bind to an FK506 affinity
resin(15) . The 50-kDa FK506-binding protein is the avian
homolog of hsp56, but the 54-kDa protein is the homolog of a novel
55-kDa human FKBP(15) .
The immunophilins can be broadly
divided into two classes according to their ability to bind either
cyclosporin A (cyclophilins) or FK506 and rapamycin (FKBPs) to their
peptidylprolyl isomerase site. In addition to the FKBPs noted above, a
cyclophilin has been identified in the estrogen receptor heterocomplex.
Purification of the bovine estrogen receptor using an
estrogen-derivatized affinity resin yielded copurification of hsp90 and
an 40-kDa protein(16) . When the cDNA for the 40-kDa
protein was cloned(17) , it was found to be the same as a
40-kDa cyclosporin A-binding protein that was previously purified from
bovine brain (18) and cloned from a human library(19) .
This protein, known as cyclophilin-40 (CyP-40), contains a C-terminal
domain with significant sequence homology to an internal region of
hsp56(17, 19) . To date, CyP-40 has not been reported
in native hetercomplexes other than that of the bovine estrogen
receptor, although it has been reported in progesterone receptor
heterocomplexes reconstituted under cell-free conditions with rabbit
reticulocyte lysate(20) .
It has been shown that a portion
of the hsp90 and a portion of the hsp56 in cytosols exist together in a
multiprotein complex independent of the presence of steroid
receptors(6, 21, 22, 23, 24) .
Both cross-linking (25) and purified protein binding studies (26) have established that hsp56 binds directly to hsp90 in
this multiprotein complex. A 45-kDa yeast protein with N-terminal
homology to mammalian CyP-40 has been isolated in a multiprotein
complex with the yeast homolog of hsp90 (27) , and incubation
of a variety of tissue homogenates with a CyP-40/GST-fusion protein
bound to glutathione-agarose resulted in retention of hsp90, suggesting that CyP-40 also binds directly to hsp90. In this
work, we show that both native and cell-free reconstituted GR
heterocomplexes contain CyP-40. Like hsp56, a portion of the CyP-40 in
cytosol exists in multiprotein, hsp90-containing heterocomplexes, but
the FKBP and the cyclophilin do not exist simultaneously in the same
complex. Purified CyP-40 binds to purified hsp90 and competes for the
binding of purified hsp56, suggesting that there is a common
immunophilin binding site on hsp90. Our data also suggest that there
are at least two types of untransformed steroid receptor
heterocomplexes, one that contains hsp56 and another that contains
CyP-40.
Figure 1: CyP-40 is a component of the native mouse GR heterocomplex. A, GR was immunoadsorbed from aliquots of WCL2 cytosol (200 µl) with FiGR-agarose in the presence or absence of competing epitope peptide. The immunopellets were washed three times with 1 ml of buffer, and the proteins were resolved by SDS-PAGE followed by Western blotting. Lanes 1 and 2, immunopellets prepared in the presence (lane 1) or absence (lane 2) of competing FiGR epitope peptide and washed with HE buffer; lane 3, immunopellet prepared in the absence of competing epitope peptide and washed with HE buffer containing 20 mM molybdate; lanes 4 and 5, immunopellets prepared in the presence (lane 4) or absence (lane 5) of competing epitope peptide and washed with TEG buffer; lane 6, immunopellet prepared in the absence of competing epitope peptide and washed with TEG buffer containing 20 mM molybdate. B, GR was immunoadsorbed from aliquots (500 µl) of L cell cytosol as above. Lane 1, 40 µl of whole cytosol; lanes 2 and 3, immunopellets prepared in the presence (lane 2) or absence (lane 3) of competing epitope peptide.
Figure 6: CyP-40 competition of hsp56 binding to hsp90. A, CyP-40 competes for binding of partially purified rabbit brain hsp56 to purified hsp90. Rabbit brain cytosol was chromatographed on a column of DE52, and fractions containing hsp56 were pooled as described under ``Methods.'' Aliquots (25 µl) of purified rabbit hsp90 (1 mg/ml) were immunoadsorbed to 5-µl pellets of Actigel precoupled with 8D3 antibody. Pellets were washed twice with 1 ml of Hepes, pH 7.35, and suspended in Hepes plus 50 mM KCl and 0.1% Nonidet P-40 in a final volume of 100 µl including 20 µl of DE52-purified hsp56 and 20 or 40 µg of purified human CyP-40 as noted. Incubations were rotated for 1 h at 4 °C and washed twice with 1 ml of Hepes, and proteins were resolved by SDS-PAGE and Western blotting. Lane 1, 2 µl of DE52-purified hsp56; lane 2, 4 µg of purified CyP-40; lane 3, 2 µg of hsp90; lane 4, Actigel-8D3 pellet without hsp90 incubated with hsp56; lane 5, Actigel-8D3 pellet with hsp90 incubated with hsp56; lanes 6 and 7, hsp90-bound Actigel-8D3 pellets incubated with hsp56 in the presence of 20 µg (lane 6) or 40 µg (lane 7) of purified CyP-40. B, purified human CyP-40 competes for binding of purified calf hsp56 to rabbit hsp90. Aliquots of purified hsp90 were immunoadsorbed to 8D3-precoupled Actigel pellets, and washed pellets were suspended in Hepes plus 25 mM KCl and 0.02% Nonidet P-40 (0.1% Nonidet P-40 in the incubations of lanes 2 and 3) in a final volume of 50 µl including 0.4 µg of purified calf hsp56 plus or minus 20 µg purified human CyP-40. Incubations were rotated 1 h at 4 °C, and pellet-associated proteins were resolved by Western blotting. Lane 1, 0.1 µg of purified calf hsp56; lane 2 Actigel-8D3 pellet without hsp90 plus 20 µg of CyP-40; lane 3, Actigel-8D3 pellet with hsp90 plus 20 µg of CyP-40; lane 4, 8D3 pellet with hsp90 plus hsp56; lane 5, 8D3 pellet with hsp90 plus hsp56 plus 20 µg of purified CyP-40.
The purified human CyP-40 used in this work
is a mutant in which His-141 is replaced by Trp. The preparation of the
cDNA and its expression in E. coli are described elsewhere. ()The protein was purified on a cyclosporin A affinity
matrix as described by Kieffer et al. (18) . After
thorough washing, the protein was eluted with pH 3.0 phosphate buffer
(50 mM) and immediately neutralized with pH 7.8 Tris buffer
(200 mM). The eluates were concentrated by ultrafiltration
(Centricon 30) to 2 mg/ml, and the purified CyP-40 was stored at 4
°C.
Unliganded receptors that have been stripped free of hsp90 are
reassociated with hsp90 when immunopellet-bound GR is incubated with
rabbit reticulocyte lysate(33) . Formation of the GR-hsp90
complex in reticulocyte lysate is due to an
ATP/Mg-dependent and K
-dependent (34) protein folding mechanism that requires
hsp70(35) . When the GR-hsp90 complex is formed by reticulocyte
lysate, hsp56 is also present in the newly formed
heterocomplex(35) . As shown in Fig. 2, when the
immunoadsorbed GR (lane 2) is stripped of receptor-associated
proteins (lane 3) and incubated with reticulocyte lysate (lane 5), the mouse receptor enters a heterocomplex (or
heterocomplexes) containing some rabbit CyP-40 in addition to hsp90 and
hsp56. This complex is functional in the sense that the GR has been
returned to the steroid binding conformation (compare lanes 3 and 5 in the bar graph in Fig. 2).
Figure 2:
Rabbit reticulocyte lysate mediates
reconstitution of GR complexes with both CyP-40 and hsp56. GR was
immunoadsorbed to 10% FiGR-agarose from replicate aliquots of WCL2 cell
cytosol in the presence (nonimmune) or absence (immune) of the epitope
peptide, and the immunopellets were stripped of hsp90, hsp56, and
CyP-40 with 0.5 M NaCl. The salt-stripped immunopellets were
incubated with rabbit reticulocyte lysate and an ATP-generating system.
Receptor, hsp90, hsp70, hsp56, and CyP-40 were assayed in each sample
by SDS-PAGE and Western blotting. Duplicate pellets were incubated with
50 nM [H]triamcinolone acetonide to
determine steroid binding activity (bargraph). Lane 1, untreated nonimmune pellet; lane 2, untreated
immune pellet; lane 3, salt-stripped immune pellet; lane
4, stripped nonimmune pellet incubated with reticulocyte lysate; lane 5, stripped immune pellet incubated with reticulocyte
lysate.
Figure 3: CyP-40 is coimmunoadsorbed with hsp90. hsp90 was immunoadsorbed from aliquots (400 µl) of rabbit reticulocyte lysate (A) or WCL2 cytosol (B) with the 8D3 monoclonal antibody or nonimmune mouse IgM. After washing the immunopellets, the proteins were resolved by SDS-PAGE and Western blotting with AC88 for hsp90, anti-hsp 72/73 for hsp70, anti-CyP-40, or antibody against hsp56. The rabbit hsp56 (darkbandabove the IgG heavy chain in lane 2 of panelA) was probed with the EC1 antibody, and the hamster hsp56 in panelB was resolved by two-dimensional gel analysis of replicate immunopellets prior to probing with the UPJ56 antiserum. Lane 1, nonimmune pellet; lane 2, immune pellet.
Figure 4: CyP-40 is not in the same hsp90 heterocomplex as hsp56. Aliquots of rabbit reticulocyte lysate (200 µl) were immunoadsorbed to protein A-agarose with 2.5% UPJ56 antiserum or with preimmune rabbit serum. The immunopellets were washed in HE buffer and resolved by SDS-PAGE followed by Western blotting. Lane 1, 10 µl of whole lysate; lane 2, preimmune pellet; lane 3, immune pellet.
If CyP-40 is in a different hsp90
heterocomplex than hsp56, then there may be two classes of
untransformed GR-hsp90 heterocomplexes as well, one containing the FKBP
hsp56 and another containing the cyclophilin CyP-40. This possibility
was tested in the experiment of Fig. 5. In this experiment WCL2
cytosol was immunoadsorbed with sufficient UPJ56 antiserum to remove
all of the hsp56 (compare preimmune supernatant in lane 3 with
UPJ56 supernatant in lane 4). As expected, some GR but no
CyP-40 is coimmunoadsorbed with the hsp56 (lane 2). When the
now hsp56-free supernatant from UPJ56 immunoadsorption (lane
4) was immunoadsorbed with the BuGR antibody against the GR, hsp90
and CyP-40 were coadsorbed (lane 6). These observations are
consistent with the notion that there are at least two different GR
heterocomplexes, depending upon the immunophilin that is bound. GR in
the UPJ56 immunopellet condition of lane 2 binds steroid
(9,400 cpm [H]triamcinolone acetonide versus 146,700 cpm for the condition of lane 6).
Figure 5:
CyP-40 and hsp56 are in different GR
heterocomplexes. Aliquots (200 µl) of WCL2 cytosol were
immunoadsorbed with UPJ56 antiserum or preimmune serum. An aliquot of
each immunoadsorption supernatant was retained for Western blotting,
and the remainder was immunoadsorbed with FiGR-agarose to isolate the
GR heterocomplex. Lane 1, preimmune immunopellet; lane
2, UPJ56 immunopellet; lane 3, 50 µl of preimmune
supernatant; lane 4, UPJ56 supernatant; lane 5, UPJ56
supernatant immunoadsorbed with FiGR in the presence of competing
epitope peptide; lane 6, UPJ56 supernatant immunoadsorbed with
FiGR without peptide. To blot hsp56, which runs with the heavy chain of
the immunoadsorbing antibody, three different development procedures
were used. Lanes1 and 2 were probed with
the EC1 monoclonal antibody, which does not react with the rabbit UPJ56
heavy chain. Lanes 3 and 4 were probed with UPJ56 and
developed with horseradish peroxidase-conjugated counterantibody. Lanes 5 and 6 were probed with UPJ56 and developed
with I-conjugated goat anti-rabbit counterantibody, which
does not react with the FiGR monoclonal antibody heavy
chain.
To demonstrate that no other proteins are required for CyP-40 and hsp56 binding to hsp90, we performed the experiment shown in Fig. 6B with human CyP-40, calf hsp56, and rabbit hsp90, all purified to near homogeneity. CyP-40 (compare lanes 2 and 3) binds directly to hsp90, and an excess of CyP-40 competes for the binding of hsp56 (compare lanes 4 and 5), an observation that is consistent with the two immunophilins binding to the same site on hsp90. Because of the very limited amount of purified hsp56 available, we were not able to test whether excess hsp56 would compete for binding of CyP-40. The binding of hsp56 and CyP-40 to hsp90 is not affected by the presence of 1 µM FK506 or cyclosporin A (data not shown).
Figure 7:
Evidence that prolylisomerase activity of
immunophilins is not required for reconstitution of GR heterocomplexes
with hsp90. GR immunoadsorbed to 10% FiGR-agarose was salt-stripped of
associated proteins, and the immune pellets were incubated with either
whole rabbit reticulocyte lysate or lysate that was preabsorbed with a
protein A-agarose-preimmune serum pellet or preadsorbed with a protein
A-agarose-UPJ56 pellet to deplete hsp56. All samples contained an
ATP-generating system, and receptor and hsp90 in each sample were
resolved by SDS-PAGE and Western blotting. One half of each pellet was
incubated with [H]triamcinolone acetonide to
determine steroid binding activity (bargraph). Lane 1, stripped receptor; lane 2, stripped receptor
plus whole lysate; lane 3, stripped receptor plus lysate
extracted with preimmune serum; lane 4, stripped receptor plus
UPJ56-extracted lysate; lane 5, stripped receptor plus
UPJ56-extracted lysate and 1 µM cyclosporin A; lane
6, stripped receptor plus whole lysate and both cyclosporin A and
FK506 at 1 µM. The Western blot under the graph
shows the hsp56 in 5 µl of whole lysate (lane 2),
preimmune extracted lysate (lane 3), and UPJ56-extracted
lysate (lane 4).
CyP-40, which was first found to copurify with the native
bovine estrogen receptor-hsp90 complex(16, 17) , has
now been identified in progesterone receptor heterocomplexes
reconstituted by reticulocyte lysate (20) and in both native (Fig. 1) and reconstituted (Fig. 2) GR heterocomplexes.
CyP-40 is also present in native GR heterocomplexes immunoadsorbed from
L cell cytosol (Fig. 1B); thus, the association is not
unique to the overexpressed receptor. The stoichiometry of CyP-40 with
respect to the steroid receptor in these complexes is unknown.
Molybdate stabilizes the presence of hsp56 in steroid receptor
heterocomplexes (26) and it stabilizes the presence of p50 in
pp60(36) and Raf (23) heterocomplexes
with hsp90. Given the likelihood that hsp56 and CyP-40 may bind in the
same manner to a common site on hsp90, it is perhaps not surprising
that molybdate inhibits salt-mediated dissociation of CyP-40 from the
GR heterocomplex (Fig. 1). Molybdate is thought to bind directly
to the hsp90 component of these heterocomplexes(37) , and the
metal oxyanion has been shown to induce a conformational change in
purified hsp90 (38) that may account for its stabilizing
effect.
A portion of the hsp56 is known to exist in a native
cytosolic heterocomplex with hsp90 independent of the association of
hsp90 with steroid receptors or protein kinases. This has been shown by
copurification of the two proteins with antibodies directed against
either hsp56 (6) or hsp90 (21) and by copurification of
hsp90 with hsp56 on an FK506 affinity matrix(9) . In that
purified hsp56 and purified hsp90 bind to each other(26) , the
association of the two hsps reflects a direct protein-protein
interaction. Because passage of cytosol through a matrix with
immobilized CyP-40 yields coretention of hsp90, a direct
interaction between the cyclophilin and hsp90 is also likely. The
coimmunoadsorption of CyP-40 with the 8D3 monoclonal antibody against
hsp90 shown in Fig. 3confirms the presence of CyP-40 in native
cytosolic hsp heterocomplexes.
While this work was in progress, Johnson and Toft (24) reported that immunoadsorption of rabbit reticulocyte lysate with the EC1 monoclonal antibody against hsp56 did not yield coimmunoadsorption of CyP-40, suggesting that the immunophilins exist in different complexes with hsp90. The experiment of Fig. 4supports the conclusion that the two immunophilins are in different native heterocomplexes with hsp90, and the data of Fig. 5are consistent with the conclusion that there are different GR-hsp90 heterocomplexes depending upon the immunophilin that is bound to hsp90.
The existence of a direct protein-protein
interaction between CyP-40 and hsp90 is demonstrated by the complex
formation with the purified proteins in Fig. 6B. The
fact that purified CyP-40 competes for the binding of hsp56 to hsp90 (Fig. 6, A and B), is consistent with the
existence of a common immunophilin binding site on hsp90. The
C-terminal 150 amino acids of CyP-40 share 30.7% identify with an
internal region of hsp56(19) . This region of homology contains
three repetitive sequence motifs of 34 amino acids called
tetratricopeptide repeat domains or TPR domains (17) , and it
is known that the TPR domains of hsp56 are required for its binding to
hsp90(39) . The N-terminal and C-terminal halves of CyP-40 have
been expressed independently, and the hsp90 binding site was localized
to the C-terminal half containing the TPR domains. TPR
domains are thought to be involved in protein-protein
interactions(40, 41, 42) , and it is likely
that they are responsible for immunophilin binding to hsp90.
The role of hsp56 and CyP-40 in steroid receptor function is unknown. It has been suggested that the immunophilins are required for proper receptor folding and heterocomplex assembly with hsp90(9, 10, 17, 24) . The data of Fig. 7suggest that this is not the case. It is possible that, when hsp56 is removed from reticulocyte lysate, any role it plays in receptor folding and heterocomplex assembly with hsp90 is subsumed by CyP-40 and/or other immunophilins. However, the failure of high concentrations of FK506 and cyclosporin A to affect GR folding to the high affinity steroid-binding state and receptor association with hsp90 (Fig. 7) argues strongly against any requirement for a cyclosporin A or FK506-inhibited peptidylprolyl isomerase activity in the process. It is possible that an unknown third class of isomerase exists in reticulocyte lysate that could substitute for the activity of the cyclosporin A- and FK506-binding classes of isomerase.
It has
also been suggested that hsp56 may play a role in nuclear localization
of the steroid receptors(43) , and the great majority of hsp56
in the cell is located in the nucleus where it is found by confocal
imaging to colocalize in the same nonrandom, mottled pattern as the
GR(44) . Curiously, both the GR and hsp56 are excluded from
nucleoli(44) , but CyP-40 is localized to nucleoli by indirect
immunofluorescence, ()a localization that makes any
potential role for CyP-40 in steroid receptor function even more
cryptic.