(Received for publication, September 18, 1995; and in revised form, October 27, 1995)
From the
The structurally related immunophilins cyclophilin 40 (CyP-40)
and FKBP52 have been identified as components of the unactivated
estrogen receptor. Both immunophilins have a similar molecular
architecture that includes a C-terminal segment with a
tetratricopeptide repeat (TPR) domain predicted to mediate protein
interaction. hsp90 is a common cellular target for CyP-40 and FKBP52.
Deletion mutants of CyP-40 fused to glutathione S-transferase
were immobilized on glutathione-agarose and then used in a rapid hsp90
retention assay to define regions of the CyP-40 C terminus that are
important for hsp90 binding. Our evidence suggests that the TPR domain
is not sufficient for stable association of CyP-40 with hsp90 and
requires the participation of flanking acidic and basic residues
clustered at the N- and C-terminal ends, respectively. Both
microdomains are characterized by -helical structures with
segregated hydrophobic and charged residues. Corresponding regions were
identified in FKBP52. By preincubating myometrial cytosol with lysates
containing bacterially expressed FKBP52, we have shown that FKBP52
competes with CyP-40 for hsp90 binding. Our results raise the
possibility of a mutually exclusive association of CyP-40 and FKBP52
with hsp90. This would lead to separate immunophilin-hsp90-receptor
complexes and place the estrogen receptor under the control of distinct
immunophilin signaling pathways.
The immunophilin components of the unactivated estrogen
receptor, cyclophilin 40 (CyP-40), ()and FKBP52, share
significant sequence homology in their C-terminal regions (1) and represent separate classes of peptidylprolyl cis-trans-isomerases with binding specificities for the
immunosuppressants cyclosporin A and FK506, respectively(2) .
These immunophilins display a similar structural organization of their
functional domains characterized by an N-terminal region with
overlapping isomerase and ligand binding domains and a conserved
C-terminal segment that incorporates a 3-unit tetratricopeptide repeat
(TPR) domain terminated by a potential site for calmodulin
binding(1) . We have previously speculated that the TPR domain
may mediate the protein interaction properties of CyP-40 and
FKBP52(1) . This is consistent with evidence that similar
repeat units in members of the TPR gene family are involved in
functional association with target proteins(3) .
FKBP52 binds hsp90 within steroid receptor complexes (4) and also exists in association with hsp90 in the absence of receptor(5, 6) . The interaction of FKBP52 with hsp90 has been studied extensively(7) , and there is recent evidence that the TPR domain, localized in the C-terminal region of FKBP52, is fundamentally important for hsp90 binding(8) . The structural similarity between CyP-40 and FKBP52 has led several groups to propose that the immunophilins may have a similar or perhaps competing role in cellular function(8, 9, 10) . In this regard, a recent report describes the association of human CyP-40 with hsp90 and provides evidence that the C-terminal, FKBP52-like domain determines this interaction(11) .
We have previously described the bacterial overexpression of bovine CyP-40 (bCyp-40) as a glutathione S-transferase (GST) fusion protein and have shown the recombinant protein to be bioactive through a display of isomerase activity that is inhibitable by cyclosporin A (10) . In preliminary studies we have confirmed that, like FKBP52(12) , CyP-40 is a calmodulin-binding protein(10) . Here we describe the use of a rapid affinity chromatography-based method, with recombinant bCyP-40 and its derivatives immobilized as GST fusion proteins on glutathione-agarose, to define regions within CyP-40 critical for hsp90 interaction. Our results reveal that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and suggest that acidic and basic regions adjacent to the N- and C-terminal ends of the TPR domain, respectively, might be involved in hsp90 binding. Using the same CyP-40-hsp90 retention assay, we show for the first time that FKBP52 competes with CyP-40 for hsp90 binding. This raises the possibility that the immunophilins may bind hsp90 in a mutually exclusive fashion leading to the formation of separate CyP-40 and FKBP52-hsp90 complexes.
Figure 1:
Structure of GST-bCyP-40 fusion protein
and deletion mutants. The structural organization of CyP-40 functional
domains is shown schematically and includes the TPR domain (shaded) and putative calmodulin (CaM) binding site (black). The constructs GST-bCyP-40, 1-213, and
17-213 contain at their C terminii 6 amino acid residues
derived from expression vector sequence. Numbers refer to
amino acid positions. Within bCyP-40 cDNA, unique XhoI and DraI restriction sites occur at positions identified by amino
acids 16 and 213, respectively.
GST-bCyP-40 WT and the mutant expression plasmids GST-bCyP-40 91-370, GST-bCyP-40 185-370, GST-bCyP-40 1-352, and GST-bCyP-40 185-352 (Fig. 1) were generated from a bCyP-40 cDNA template by polymerase chain reaction (PCR) amplification using high fidelity Pfu DNA polymerase (Stratagene) and specific oligonucleotide primers containing built-in BamHI (5` end) and SmaI (3` end) restriction sites. The SmaI site in each was preceded by a TGA stop codon. After BamHI and SmaI digestion, the amplified fragments were ligated into pGEM-3Z. For GST-bCyP-40 WT and GST-bCyP-40 1-352, a XhoI to BclI excision allowed replacement with wild type DNA. A similar approach was used for GST-bCyP-40 91-370, GST-bCyP-40 185-370, and GST-bCyP-40 185-352 to replace the PflMI to BclI excised fragment with wild type sequence. This strategy minimized PCR-introduced mutations. Sequence fidelity of the remaining PCR-derived DNA was confirmed by automated sequence analysis (Applied Biosystems). Inserts released from those plasmids by BamHI/SmaI digestion were ligated into pGEX-2T.
Figure 2:
hsp90 binding properties of full-length
bCyP-40 and bCyP-40 derivatives with deleted N-terminal and C-terminal
domains. bCyP-40 and its derivatives 1-213 and 17-213
(see Fig. 1) were immobilized as GST fusion proteins on
glutathione-agarose. Fusion protein-charged gels (15-30 µl)
were incubated for 6 h at 4 °C with bovine myometrial cytosol (200
µl) prepared in 10 mM Tris, pH 7.3 buffer containing 100
mM KCl, 5 mM dithiothreitol, and 10% (v/v) glycerol.
After centrifugation, the gels were washed repeatedly with the same
buffer to remove unbound protein contaminants. Retained proteins (lanes 2, 4, and 6) were extracted from the
gels with SDS-PAGE sample buffer and were analyzed by SDS-PAGE on a
12.5% w/v polyacrylamide gel followed by Coomassie Blue staining.
Fusion protein-charged gels not exposed to cytosol were used as
controls (lanes 1, 3, and 5). Protein
molecular weight markers (Pharmacia) are shown on the left
side; BPB, bromphenol blue.
Figure 3:
Structure of the acidic domain in bCyP-40. A, the acidic domain of bCyP-40, spanning amino acid residues
185-225, is located at the N-terminal end of the 3-unit TPR
domain. Acidic and basic residues are highlighted by negative
and positive charges, respectively. Within this sequence exists an
-helical microdomain (indicated by the
-helix motif)
identified by residues 206-218. DraI digestion of
bCyP-40 cDNA disrupts this sequence at position 213 (arrowed).
The partial sequence of a corresponding region in FKBP52, immediately
adjacent to the TPR domain, is also illustrated. B, helical
wheel representations of amphiphilic microdomains identified by bCyP-40
residues 206-218 and FKBP52 residues
252-265.
A considerably longer (134 amino
acid residues) intervening sequence separates the ligand
binding/catalytic domain from the C-terminal TPR domain in
FKBP52(14) . Although bCyP-40 and FKBP52 do not share sequence
homology in this region, there are notable similarities. As in bCyP-40,
the intervening sequence in FKBP52 contains a net surplus of acidic
amino acid residues (29 acidic, 21 basic Arg, Lys, His)(14) .
Additionally, FKBP52 has a corresponding -helical microdomain with
a similar segregated distribution of hydrophobic and charged residues (Fig. 3B). These observations identify additional
structural similarities between the CyP-40 and FKBP52 gene products and
lend support to accumulating evidence that they may have similar or
competing modes of action in cellular processes.
Figure 4: Deletion mapping of the CyP-40 hsp90 binding domain. Wild type bCyP-40 and its deletion mutants were immobilized as GST fusion proteins on glutathione-agarose and assayed for hsp90 binding as described in Fig. 2. In each lane, the corresponding bCyP-40 construct is identified by the major protein band.
Figure 5: FKBP52 competes with CyP-40 for hsp90 binding. A, lysates (2 µl) from uninduced and IPTG-induced bacterial cultures overexpressing recombinant human FKBP52 were submitted to SDS-PAGE, and proteins were visualized by Coomassie Blue staining. B, induced FKBP52 lysate was added in 0-, 5-, 10-, 20-, 50-, and 100-µl aliquots to separate tubes containing bovine myometrial cytosol (200 µl), and the volumes were equalized to 400 µl with lysing buffer. After brief mixing, incubation was continued for 3 h at 4 °C. Glutathione-agarose (10 µl) containing immobilized GST-bCyP-40 fusion protein was added to each tube, and the mixtures were incubated with rotation for an additional 5 h at 4 °C. Gel-retained proteins were extracted with SDS-PAGE sample buffer and analyzed by SDS-PAGE with Coomassie Blue staining. A parallel study with 100 µl of uninduced lysate in cytosol was conducted as a control. C, separate tubes containing gel-immobilized GST-bCyP-40 (10 µl) were incubated with cytosol (200 µl) for 3 h at 4 °C, prior to the addition of 0 and 100-µl aliquots of uninduced lysate and 100 µl of induced FKBP52 lysate to individual tubes. After equalizing for volume (400 µl total), incubation was continued for 5 h at 4 °C. Gel-retained proteins were recovered and analyzed by SDS-PAGE as already described.
We have used a rapid, glutathione-agarose affinity
chromatography-based method with GST fusion proteins containing wild
type bCyP-40 and bCyP-40 deletion mutants, to identify regions within
the CyP-40 C terminus that are important for hsp90 binding. The
critical regions include acidic and basic peptides located at the N-
and C-terminal ends of the TPR domain, respectively. Both microdomains
are characterized by amphiphilic -helical structures suggesting
that hydrophobic and electrostatic interactions might contribute
significantly to hsp90 binding. We have previously proposed that the
3-unit TPR domain might mediate the protein interaction properties of
CyP-40(1) . Disruption of the acidic domain (as in construct
17-213) or complete removal of the C-terminal basic region
flanking the TPR domain (as in construct 1-352) leads to a sharp
reduction in hsp90 binding efficiency. The result suggests that for
CyP-40, an intact TPR domain is not sufficient for stable association
with hsp90, but is consistent with a requirement for multiple elements
present in the C-terminal half of the protein. In such a model it is
possible that the TPR domain, stabilized by internal contacts between
individual repeat units(21) , maintains an appropriate spacial
orientation of both charged regions relative to hsp90. Alternatively,
the charged domains might act coordinately enabling the TPR domain to
form a suitable binding conformation.
The importance of the TPR domain for the interaction of FKBP52 with hsp90 has been demonstrated clearly(8) . FKBP52 derivatives, altered within the third unit of the TPR domain, either by a 2-amino acid residue insertion or by a C-terminal truncation, failed to bind hsp90 (8) . The result supports a structural role for the TPR domain. The basic region located at the C-terminal end of the TPR domain is highly conserved between CyP-40 and FKBP52 and is a likely site for calmodulin interaction(1, 20) . Removal of this domain from FKBP52 appears not to disrupt hsp90 binding, and the complex remains stable during nondenaturing gel electrophoresis(8) . This contrasts with our observation of low level hsp90 retention by the corresponding bCyP-40 1-352 mutant. We have used an extensive washing procedure with buffer containing 100 mM KCl to minimize nonspecific protein interaction with gel-immobilized GST-bCyP-40 constructs. It is possible that the binding of the 1-352 derivative to hsp90 is sensitive to these washing conditions.
The nonhomologous, intervening sequences which separate
the N-terminal ligand/isomerase domain from the C-terminal TPR region
in CyP-40 and FKBP52 differ considerably in length (CyP-40, 41
residues; FKBP52, 134 residues), but display some common features. Both
are characterized by an excess of negatively charged amino acid
residues and contain similar amphiphilic -helical regions located
close to the TPR domain. We speculate that, as in CyP-40, the acidic
domain in FKBP52 may participate in hsp90 binding. Identification of
this common acidic region in CyP-40 and FKBP52 extends the remarkable
structural similarity between these proteins and suggests that they may
have closely related roles in cellular function.
We have shown for
the first time that FKBP52 competes with CyP-40 for hsp90 binding. Our
findings complement those reported by Owens-Grillo et al.(22) and suggest that CyP-40 and FKBP52 share an identical
interaction site within the hsp90 protein. The reversible nature of
this interaction is consistent with the presence of a dynamic system in
which the immunophilin component of immunophilin-hsp90 complexes might
be determined by the tissue distribution and the relative cellular
concentrations of CyP-40 and FKBP52(9) . The mutually exclusive
association of CyP-40 and FKBP52 with hsp90, leading to the formation
of separate immunophilin-hsp90-receptor complexes, would place steroid
receptors under the control of distinct immunophilin signaling
pathways. This raises the possibility of a modulating role for putative
endogenous immunophilin ligands in receptor activity. However, although
FK506 has been shown to enhance glucocorticoid and progesterone
receptor-mediated gene expression in intact
cells(23, 24) , in vitro studies indicate
that the drug does not influence hsp90 association with the
glucocorticoid receptor(25) . In our own studies, 20 µM cyclosporin A had no effect on hsp90 retention by gel-immobilized
GST-bCyP-40 fusion protein (not shown). Nevertheless, a model in which
the immunosuppressants, acting via CyP-40 and FKBP52, induce a
succession of conformational changes that result in altered hsp90 and
receptor function, cannot at this stage be discounted. This proposal is
supported by additional evidence that cyclosporin A can potentiate
glucocorticoid-(26) , progestin- (27) , and
estradiol-induced gene expression. ()
The major molecular chaperones hsp90 and hsp70 are key participants in steroid receptor signaling(28) , and the proper interaction of receptors with hsp90 is essential for efficient ligand binding and response(29, 30) . Recent evidence suggests that receptor-hsp90 complexes exist in dynamic equilibrium with hsp70 (31) and that the folding of receptors to a hormone-activable conformation also involves DnaJ and p23, together with additional components(9, 32, 33, 34) . The presence of p23 with hsp90 and the immunophilins, CyP-40 and FKBP52, as the major proteins in progesterone receptor complexes(9) , suggests that p23-hsp90-immunophilin heterocomplexes might represent important intermediates in receptor assembly and that they may act together to establish a poised receptor conformation that is optimally responsive to hormone. It has been suggested that DnaJ and hsp70 might also regulate the activities of a wider range of signaling proteins, all of which are targeted by hsp90(32) . Although the cellular functions of CyP-40 and FKBP52 have yet to be defined, a more general role for these immunophilins in modulating the cellular activity of hsp90 chaperone substrates is distinctly possible.
This report is dedicated to the memory of Edward J. Keogh, with gratitude for his leadership and support.